Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Effects of IL-10 and IL-4 on LPS-induced transcription factors (AP-1, NF-IL6 and NF-kappa B) which are involved in IL-6 regulation.

Interleukin-10 (IL-10), like IL-4, is known to inhibit cytokine expression in activated human monocytes. We showed that both IL-10 and IL-4 inhibit LPS-induced IL-6 mRNA and protein expression by inhibiting the transcription rate of the IL-6 gene. The strong inhibition of the IL-6 transcription rate prompted us to study the effect of IL-10 and IL-4 on the expression of transcription factors. We questioned whether or not IL-10 and IL-4 affected the expression of transcription factors that are known to be involved in the control of the IL-6 transcription rate, namely activator protein-1 (AP-1), nuclear factor IL-6 (NF-IL6), and nuclear factor kappa B (NF-kappaB). In electrophoretic mobility shift assays (EMSAs) we showed that IL-10 and IL-4 inhibited LPS-induced AP-1 binding activity. The inhibiting effect of IL-4 was slightly more pronounced than that of IL-10. Downregulation of LPS-induced AP-1 was accompanied, and thus possibly explained, by a reduced expression at mRNA level of the two major components of the AP-1 complex, namely c-fos and c-jun as determined by Northern experiments. Binding activity of NF-IL6 was also strongly inhibited by IL-4 whereas IL-10 showed no effect. NF-IL6 mRNA levels were not affected by IL-10 or IL-4, suggesting that IL-4 affects binding activity of preexisting NF-IL6. Neither IL-10 nor IL-4 inhibited LPS-induced NF-kappa B binding activity. In agreement with this finding, Northern experiments where p65 and p105 mRNA levels were determined, demonstrated that expression of these components of the NF-kappa B transcription factor were not affected by IL-10 or IL-4. Furthermore, neither IL-10 nor IL-4 showed any effect on I-kappa B mRNA expression as determined by Northern experiments. Thus, IL-10 and IL-4 similarly affect IL-6 expression. However, for IL-4 this was accompanied with a reduction of AP-1 and NF-IL6 binding activity whereas IL-10 only inhibited AP-1 binding activity.

Interleukin-4-mediated inhibition of C-Fos mRNA expression: role of the lipoxygenase directed pathway.

Interleukin-4 inhibits several monocyte functions like A23187-induced expression of cytokines and c-fos and c-jun proto-oncogene mRNA expression. In an attempt to elucidate the mechanism by which this inhibitive effect is mediated, we compared the effect of IL-4 on A23187-induced c-fos and c-jun mRNA expression in conjunction with inhibitors that selectively inhibit the cyclooxygenase dependent (indomethacin) and lipoxygenase dependent (NDGA) pathway of arachidonic acid (AA) metabolism. NDGA inhibited A23187-induced c-fos mRNA expression by a similar magnitude as IL-4, whereas the effect of indomethacin was only minor. A23187-induced c-jun mRNA expression was not affected by indomethacin and only slightly inhibited by NDGA. These results indicate that in human monocytes c-fos mRNA expression is at least partly controlled by the lipoxygenase directed pathway of AA metabolism, whereas the cyclooxygenase dependent pathway is not involved in the regulation of proto-oncogene expression. This was supported by the finding that leukotriene B4 (LTB4) and 5'-hydroperoxyeicosatetraenoic acid (5'-HPTETE), which are two lipoxygenase metabolites, strongly induced c-fos mRNA, whereas c-jun mRNA expression was slightly affected. However, the inhibitive effect of IL-4 could not be ascribed to a reduced production of LTB4 suggesting that the mode of IL-4 action lies behind the conversion of AA to 5'-HPETE and LTB4.

The spontaneous expression of interleukin-1 beta and interleukin-6 is associated with spontaneous expression of AP-1 and NF-kappa B transcription factor in acute myeloblastic leukemia cells.

The nature of the spontaneous expression of cytokines that is observed in blasts of some AML patients is unclear. We studied whether or not the spontaneous expression of IL-1 beta and IL-6 is due to an increased transcription rate of the cytokine gene and associated with a spontaneous expression of two transcription factors that play an important role in IL-1 beta and IL-6 gene transcription, namely activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B). In eight of the 19 AML patients a spontaneous expression of IL-1 beta mRNA was observed, whereas IL-6 mRNA was expressed in seven of the cases. Expression of IL-6 mRNA correlated nicely with the secretion of IL-6 protein. Nuclear run-on experiments showed that spontaneous expression of IL-1 beta and IL-6 was at least partly due to an increased transcription rate of the respective genes compared to the results from healthy unstimulated monocytes. Electrophoretic mobility shift assays demonstrated that especially spontaneous expression of NF-kappa B is associated with spontaneous cytokine expression. However, the spontaneous expression of transcription factors is not due to the endogenous secretion of IL-1 since the addition of anti-IL-1 monoclonal antibody did not affect the expression of NF-kappa B. Finally, supershift experiments were performed that demonstrated that the NF-kappa B consists of the p50 and the p65 subunits. In summary, these results demonstrate that the spontaneous expression of cytokines is frequently associated with an increased transcription rate and a spontaneous expression of transcription factors.

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation.

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-kappaB, might determine the final cellular response.

Regulation of p100 (NFKB2) expression in human monocytes in response to inflammatory mediators and lymphokines.

The transcription factor NF-kappaB plays an important role in the regulated expression of cytokines in human monocytes. A p100 subunit of NF-kappaB has IkappaB-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient transfection assays we demonstrated that p100 has an inhibitory effect on the NF-kappaB-dependent IL-6 promoter activity. In view of this finding, we studied the regulation of the p100 subunit in human monocytes in response to LPS, the inflammatory cytokines IL-1beta and TNF-alpha and lymphokines. The results demonstrate that LPS, IL-1beta, and TNF-alpha induce p100 expression at mRNA and protein level while IFN-gamma, IL-3 and IL-4/IL-10 have no effect. The induction of p100 expression was shown to be mediated by a two-fold increase in the p100 transcription rate and a two-fold increase in p100 mRNA stability. Furthermore the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-kappaB is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregulate p100 transcription. We therefore examined the composition of NF-kappaB induced by LPS vs the different lymphokines. LPS-induced NF-kappaB showed a distinct p65 supershift whereas the composition of NF-kappaB induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the transcription factor NF-kappaB is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-kappaB predominantly consisting of p50 homodimers.

Interferon-gamma enhances the LPS-induced G-CSF gene expression in human adherent monocytes, which is regulated at transcriptional and posttranscriptional levels.

Human adherent monocytes were studied with regard to the expression of granulocyte colony-stimulating factor (G-CSF) at mRNA and protein levels in response to lipopolysaccharide (LPS) and gamma-interferon (IFN-gamma) stimulation. Monocytes did not express G-CSF transcripts in response to IFN-gamma treatment. In contrast, monocytes exposed to IFN-gamma plus LPS showed a dose-dependent increase in G-CSF mRNA accumulation and protein secretion compared to LPS-stimulated monocytes. The augmented G-CSF mRNA expression in response to IFN-gamma plus LPS was the result of a slight increase in the G-CSF transcription rate (2.2-fold) and a more than 6-fold increase in the G-CSF mRNA half-life (20 minutes vs. > 120 minutes). In addition, it was shown that the effects of IFN-gamma on LPS-induced G-CSF protein secretion could be mimicked by the calcium ionophore A23187, suggesting that the Ca(2+)-dependent pathway might be triggered after binding of the ligand to the receptor. Finally, it was observed that the potentiating effects of IFN-gamma on LPS-induced G-CSF secretion could be blocked by interleukin-4 (IL-4). These data indicate that two cytokines produced by activated T cells have opposite effects on G-CSF production by human activated monocytes.

Interleukin-4 mRNA and protein in activated human T cells are enhanced by interleukin-7.

We studied the effect of the stroma-derived cytokine interleukin-7 (IL-7) on the expression of IL-4 in human T cells at mRNA and protein level. The results demonstrate that IL-7 did not induce IL-4 mRNA in resting T cells. However, concanavalin A (con A)-induced IL-4 mRNA expression was enhanced by costimulation with con A plus IL-7. Nuclear run-on analysis revealed that IL-7 did not affect the transcription rate of the IL-4 gene. The half-life of con A-induced IL-4 transcripts, however, was increased upon con A plus IL-7 treatment, indicating that the effect of IL-7 is mediated at posttranscriptional level. In accordance with the mRNA results, IL-4 protein was not detected in supernatants of unstimulated T cells or T cells exposed to IL-7. In contrast, IL-7 augmented the con A-induced secretion of IL-4 protein significantly. In addition, it was noticed that anti-IL-1 beta and anti-tumor necrosis factor-alpha (anti-TNF-alpha) did not abolish the effect of IL-7 on the con A-induced IL-4 secretion, indicating that the IL-7 effect is not mediated by the release of these cytokines. These results indicate that a stroma-derived factor can affect IL-4 expression in activated human T cells.

Divergent effects of IL-10 and IL-4 on the proliferation and growth factor secretion by acute myeloblastic leukemic cells.

The effects of interleukin-10 (IL-10) and IL-4 were studied on the spontaneous and IL-1, IL-3, and Granulocyte-Colony Stimulating Factor (G-CSF) supported proliferation of acute myeloid leukemic cells. IL-10 inhibited the spontaneous proliferation in 1 out of 12 (1/12) cases while the IL-1 stimulated the tritiated thymidine (3H-TdR) uptake was suppressed in 2/12 cases as a result of IL-10 administration. In the presence of G-CSF, IL-10 affected 3H-TdR uptake in 2/12 and no distinct changes were observed in the presence of IL-3. In contrast IL-4 alone stimulated (3H-TdR) uptake with a factor two or more in 7/12 cases. In the presence of IL-1 and G-CSF a further enhancement was noted in 2 and 5 cases respectively. In 2/12 cases IL-4 inhibited the spontaneous or IL-1 and G-CSF supported proliferation. To study whether the changes in 3H-TdR uptake are related to the endogenous secretion of G-CSF and GM-CSF, AML blast cells (n = 5) were cultured in medium supplemented with IL-1 or IL-3 in the absence and presence of IL-10 and IL-4. IL-10 did not inhibit the spontaneous secretion of G-CSF or GM-CSF but suppressed the IL-1 induced GM-CSF secretion in 2/5 cases. These moderate effects were observed despite the strong inhibition of IL-10 on the IL-6 secretion by human activated monocytes. In contrast to IL-10, IL-4 also inhibited the spontaneous (3/5) and cytokine induced (5/5) secretion of G-CSF and GM-CSF (4/5) protein in the cases in which an enhancement of the 3H-TdR uptake was noticed. In summary the data indicate that the proliferative effects of IL-4 are in some cases uncoupled from the endogenous secretion of cytokines. In addition IL-10 affects the AML cells in a limited number of cases despite the similarity in effects between IL-4 and IL-10 in suppressing cytokine secretion from activated human monocytes.

The regulation of interleukin 5 and interleukin 3 gene expression in human T cells.

Interleukin 5 (IL-5) and Interleukin 3 (IL-3) mRNA levels in human peripheral blood T cells were compared by semi-quantitative polymerase chain reaction (PCR) analysis. Unstimulated T Cells did not express IL-5 and IL-3 mRNA. IL-5 and IL-3 mRNA expression were similarly induced by the lectin concanavalin A (Con A). The protein kinase C (PKC) activator phorbol myristate acetate (PMA) triggered both IL-3 and IL-5 mRNA expression, whereby IL-5 and IL-3 mRNA expression was observed after 9 and 3 h treatment, respectively. Stimulation with calcium ionophore A23187 induced IL-3 mRNA expression, whereas it failed to induce IL-5 mRNA. In contrast to IL-3 mRNA, the expression of IL-5 mRNA was dependent on de novo protein synthesis, since cycloheximide (CHX) blocked the Con A plus PMA induced IL-5 mRNA expression. In contrast, cyclosporin A (CsA) inhibited but failed to completely block the expression of IL-3 and IL-5 mRNA. mRNA studies in T cell subsets revealed that the expression of IL-5 mRNA was restricted to the CD4 positive T cell subset in response to Con A plus PMA stimulation. On the other hand, IL-3 mRNA expression was noticed in both the CD4 and the CD8 positive T cell subset. These data indicate that the selective expression of IL-5 by human T cells can either be explained by activation of a selective intracellular signalling pathway or by selective activation of a T cell subset. Alternatively, both processes could be involved.

Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components.

In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 ('reactivating kinase') in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser(727) phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant-negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6-induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress-activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant-negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser(727) phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser(727) phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser(727) phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac-1, MEKK and SEK-1/MKK-4 as signal transduction components.

Differential binding activity of the transcription factor LIL-STAT in immature and differentiated normal and leukemic myeloid cells.

Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients, suggesting involvement of constitutive Stat activity in the events of leukemogenesis. In the present study, blasts of nine AML cases were investigated for the constitutive binding activity of the recently identified transcription factor LIL-Stat (LPS- and IL-1-inducible Stat). Band-shift assays were performed using the LPS-and IL-1-responsive element (LILRE) oligonucleotide, a gamma interferon activation site-like site that is present in the human IL-1beta promoter. Constitutive LIL-Stat binding activity was observed in three leukemic cell lines and in seven out of nine AML cases. Transient transfection studies with a reporter plasmid containing three sequential LIL-Stat binding sites showed distinct transcriptional activity of LIL-Stat only in those AML blasts that constitutively expressed LIL-Stat. In CD34+ cells LIL-Stat also constitutively bound to its consensus sequence. However, when these cells were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and stem cell factor (SCF) for differentiation along the monocytic lineage, the LIL-Stat binding activity disappeared totally. In agreement with these findings neither mature monocytes nor granulocytes showed constitutive or inducible LIL-Stat binding activity. We conclude that the LIL-Stat transcription factor is constitutively activated in undifferentiated and leukemic hematopoietic cells, but not in mature cells. This may suggest a role for this transcription factor in the process of differentiation.

Interleukin-4 inhibits the lipopolysaccharide-induced expression of c-jun and c-fos messenger RNA and activator protein-1 binding activity in human monocytes.

We studied the effect of interleukin-4 (IL-4) on the lipopolysaccharide (LPS) induction of two immediate early genes c-fos and c-jun. These genes encode proteins that form the dimeric complex activator protein-1 (AP-1), which is active as a transcriptional factor. Maximal accumulation of either c-fos and c-jun messenger RNA (mRNA) occurred 30 minutes after LPS addition. When cells were treated with IL-4 for 5 hours before LPS activation, both the c-fos and the c-jun mRNA expression was decreased. The inhibition of c-fos and c-jun expression by IL-4 in LPS-treated cells was shown to be due to a lower transcription rate of the c-fos and c-jun genes. IL-4 did not affect the stability of the c-fos and c-jun transcripts. Finally, using electrophoretic mobility shift assays, evidence was obtained that IL-4 inhibits LPS-induced expression of AP-1 protein. These data indicate that IL-4 suppresses the induction of transcription factors in human activated monocytes.

Transcriptional and posttranscriptional regulation of the interleukin-4 and interleukin-3 genes in human T cells.

Human T cells were studied with regard to the regulation of interleukin-4 (IL-4) and IL-3 gene expression. IL-4 and IL-3 mRNA were undetectable in unstimulated T cells. On activation with the lectin concanavalin A (Con A), both IL-4 and IL-3 mRNA were expressed. Accumulation of IL-4 mRNA peaked after 6 to 12 hours, whereas IL-3 mRNA levels peaked after 3 to 6 hours of stimulation with Con A. Nuclear run-on assays showed a low constitutive transcription for both genes. The transcription rates were increased by Con A resulting in a peak for IL-4 after 1 hour (30% increase) and for IL-3 after 3 hours (40% increase) of Con A treatment. mRNA stability studies demonstrated that on activation with Con A both messages decayed with a half-life of approximately 90 minutes. No IL-4 or IL-3 mRNA expression was induced by the protein kinase C activator phorbol myristate acetate (PMA). However, PMA augmented the Con A-induced IL-4 and IL-3 mRNA accumulation. This was shown to be mediated at posttranscriptional level by a large increase in the stability of both messages (t 1/2 > 3 hours). The transcription rate of both genes was also enhanced by Con A+PMA and reached peak levels for IL-4 after 1 hour (90% increase) and for IL-3 after 3 hours (70% increase) of stimulation. Furthermore, it appeared that the induction of IL-4 mRNA was dependent on protein synthesis because cycloheximide (CHX) blocked the Con A- and Con A+PMA-induced expression of IL-4 mRNA. In contrast, CHX inhibited, but failed to completely block, the Con A- and Con A+PMA-induced IL-3 mRNA expression, whereas the expression of both genes was completely blocked by cyclosporine A. With regard to the secretion of IL-4 protein it was shown that it closely follows the accumulation of IL-4 mRNA. Taken together, the data show that expression of the IL-4 and IL-3 genes in human T cells is controlled by different activation pathways that affect the gene regulation at transcriptional and posttranscriptional levels.

IL-7 enhances the expression of IL-3 and granulocyte-macrophage-CSF mRNA in activated human T cells by post-transcriptional mechanisms.

The stromal derived growth factor IL-7 was studied for its ability to modulate cytokine expression in human T cells. IL-7 alone did not induce IL-3 or granulocyte-macrophage-CSF (GM-CSF) mRNA. However, IL-7 enhanced the Con A-induced IL-3 and GM-CSF mRNA accumulation in a dose-dependent way. mRNA stability studies revealed that the effect of IL-7 was caused by post-transcriptional stabilization of the IL-3 and GM-CSF transcripts. Upon Con A treatment, the IL-3 and GM-CSF mRNA decayed with a t1/2 of approximately 90 and 50 min, respectively. Costimulation with Con A plus IL-7 stabilized both transcripts to t1/2 of greater than 2 h for IL-3 mRNA and 90 min for GM-CSF mRNA. Using nuclear run-on assays, we showed that the transcription rate of both genes was not affected by IL-7. Furthermore, it appeared that the effect of IL-7 was independent on protein synthesis, because cycloheximide did not abolish the promotive effect of IL-7. Finally, it was shown that in accordance with the mRNA results IL-7 enhanced the secretion of GM-CSF protein in Con A-activated T cells. After 12 h of stimulation T cells cultured in the presence of Con A secreted 575 +/- 309 pg GM-CSF/ml (x +/- SD, n = 5), which increased to 1425 +/- 758 pg/ml in the presence of Con A plus IL-7 (p < 0.01). In summary, these data demonstrate that IL-7 augments the expression and secretion of CSF in activated human T cells.

Interleukin-4 (IL-4) receptor expression on human T cells is affected by different intracellular signaling pathways and by IL-4 at transcriptional and posttranscriptional level.

Interleukin-4 (IL-4) modulates the survival, proliferation, and differentiation of a variety of hematopoietic cells. The effects are mediated through a single class of high-affinity receptors for IL-4. To understand the biologic effects of IL-4 on human T cells, we studied the regulation of IL-4 receptor (IL-4R) gene expression. We showed that IL-4R mRNA accumulation in human T cells is enhanced fourfold after activation of different secondary signaling pathways by concanavalin A (Con A), phorbol myristate acetate (PMA), the calcium ionophore A23187, and combinations of these factors. This could be ascribed to an increase in the IL-4R transcription rate and to stabilization of IL-4R mRNA resulting in a half-life of 80 to 90 minutes (v 35 to 40 minutes in resting T cells). IL-4 did enhance the IL-4R mRNA accumulation by a factor 10, which was caused by an increase in the IL-4R transcription rate and prolonging the half-life of IL-4R transcripts to 140 to 160 minutes. Finally, it was shown that A23187 induced IL-4R mRNA expression is a protein synthesis-dependent process. In contrast, Con A-, PMA-, Con A + PMA-, and Con A + A23187-induced expression of IL-4R mRNA is protein-synthesis independent. Cyclosporine A inhibited the A23187- and Con A + A23187-induced IL-4R mRNA accumulation, whereas Con A-, PMA-, and Con A + PMA-induced IL-4R mRNA expression was not affected by this drug. These data indicate that expression of IL-4 receptors on human T cells can be modulated by different intracellular signaling pathways at both transcriptional and posttranscriptional levels.

Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes.

In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun.

G(AnH)MTetra, a naturally occurring 1,6-anhydro muramyl dipeptide, induces granulocyte colony-stimulating factor expression in human monocytes: a molecular analysis.

N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.