In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation.

In this study, we examined the involvement of the phosphatidylinositol 3-kinase (PI3-K) and p70S6 kinase signal transduction pathway in the interleukin-1(IL-1)-mediated proliferation and cytokine production by normal and leukemic myeloid cells. Total AML blast populations, early progenitor (CD34(+)/CD36(-)) cells, and more differentiated (CD34(-)/CD36(+)) cells were treated with the PI3-K inhibitor Ly294002 and p70S6K inhibitor rapamycin. The effects on proliferation, IL-6 protein secretion, and intracellular signaling cascades were determined and compared with normal CD34(+) cells and monocytes. The function of the PI3-K pathway was dependent on the differentiation state of the AML cell population. In immature blasts, the IL-1-induced proliferation was strongly inhibited by Ly294002 and rapamycin, without a distinct effect on IL-6 protein production. In contrast, in mature monocytic blast cells inhibition of the PI3-K signaling route had a stimulatory effect on IL-6 protein secretion. Interestingly, these findings were not specifically linked to the malignant counterpart but were also observed with normal CD34(+) sorted cells vs mature monocytes. Evidence is provided that the Ly294002-induced increase in IL-6 protein secretion is linked to the cAMP dependent signaling pathway and not to changes in the phosphorylation of ERK or p38. However, although the enhanced IL-6 protein secretion is cAMP dependent, it was not found to be mediated by protein kinase A (PKA) or by the GTP-ase Rap1. This study indicates that inhibition of the PI3-K signaling pathway has an inhibitory effect on cell proliferation but a stimulatory effect on IL-6 expression mediated by a cAMP-dependent but PKA-independent route.

Differential effects of interleukin-3 and interleukin-1 on the proliferation and interleukin-6 protein secretion of acute myeloid leukemic cells; the involvement of ERK, p38 and STAT5.

In the present study we examined whether the p38 and extracellular signal-regulated kinase (ERK) signal transduction pathways are involved in the interleukin-3 (IL-3)- or interleukin-1 (IL-1)-mediated proliferation and cytokine production of acute myeloid leukemic (AML) cells. The IL-3- and IL-1-mediated proliferation were both inhibited by the specific p38 and MEK1 inhibitors SB203580 and PD98059, respectively. Specificity of these inhibitors was demonstrated by in vitro kinase assays. Furthermore, we examined whether STAT5 (signal transducer and activator of transcription) activity is modulated by the p38 and ERK signal transduction pathways, since STAT5 activation has been linked to proliferation. We provide evidence that the p38 kinase pathway, but not the ERK pathway, is to a certain degree involved in the modulation of STAT5 transactivation since SB203580 and overexpression of an inactive MKK3 mutant inhibited the IL-3-induced STAT5 reporter transactivation. In addition, the p38 and ERK pathways are also involved in cytokine production. The IL-1-enhanced IL-6 protein secretion was strongly reduced by SB203580 and PD98059. Despite the fact that IL-3 did induce p38 and ERK kinase activity, it was not able to enhance IL-6 protein secretion, which coincided with the inability of IL-3 to induce NFkappaB (nuclear factor kappaB) activation and IkappaB (inhibitory protein kappaB) degradation. This study demonstrates that the p38 and ERK pathways play a functional role in cell proliferation and IL-6 secretion of AML cells which are dependent on the activated cytokine receptors.

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.

Interleukin-15 differentially enhances the expression of interferon-gamma and interleukin-4 in activated human (CD4+) T lymphocytes.

In this study interleukin (IL)-15 was examined for its ability to modulate the expression of interferon-gamma (IFN-gamma) and IL-4 in activated human T lymphocytes. The effect of IL-15 was compared with IL-2 and IL-7, cytokines all known to use the IL-2 receptor gammaC chain. The results demonstrate that the extent of upregulation of IFN-gamma and IL-4 mRNA was dependent on the applied cytokine (IL-2>IL-15>IL-7) and on the stimulatory signal. IFN-gamma and IL-4 mRNAs were upregulated by IL-15 in concanavalin A- (twofold) and anti-CD3 plus anti-CD28- (fivefold) stimulated T lymphocytes. IFN-gamma mRNA accumulation, but not IL-4 mRNA, was additively upregulated by IL-15 plus IL-7 (ninefold) in anti-CD3 stimulated T lymphocytes, and bypassed the requirement of CD28 signalling. Fluorescence-activated cell sorting (FACS) experiments demonstrated that IFN-gamma mRNA was upregulated by IL-15 in both CD4+ and CD8+ T lymphocytes, whereas IL-4 mRNA accumulation predominantly occurred in CD4+ cells. Preincubation of highly purified CD4+ T lymphocytes during 7 days with IL-15 and/or IL-7, followed by activation, also showed enhanced IL-4 protein secretion, but predominantly upregulated IFN-gamma protein. The net effect was a dramatically increased IFN-gamma/IL-4 ratio. Taken together, IL-15 and IL-7 can act as costimulatory signals, which may favour a T helper 1 (Th1) immune response, particularly in the absence of sufficient CD28 costimulation.

Beta-adrenoceptor-mediated inhibition of IFN-gamma, IL-3, and GM-CSF mRNA accumulation in activated human T lymphocytes is solely mediated by the beta2-adrenoceptor subtype.

Cytokine gene expression in T lymphocytes is a strictly regulated process, involving both stimulatory and inhibitory signals. beta-Adrenoceptor (betaAR) agonists are widely used in the treatment of asthma and are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In this study, the characterization of betaAR subtype(s) (beta1, beta2, and beta3) involved in the regulation of interleukin (IL)-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) mRNA accumulation was studied by using various betaAR agonists and antagonists. Concanavalin A (Con A)-induced IFN-gamma, GM-CSF, and IL-3 mRNAs are dose-dependently inhibited by the nonselective betaAR agonist isoproterenol and by the selective beta2AR agonist fenoterol. IL-4 mRNA accumulation was not susceptible to betaAR stimulation. The observed inhibition on IFN-gamma, GM-CSF, and IL-3 mRNA was blocked by the selective beta2AR antagonist ICI 118,551 (10(-6) M) and by timolol (10(-6) M), a nonselective antagonist. The selective beta1AR antagonist atenolol (0.3 x 10(-6) M) did not have any effect. Secretion of GM-CSF protein in the presence of increasing concentrations of isoproterenol followed a similar pattern as observed for GM-CSF mRNA. In addition, the betaAR-mediated inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation and GM-CSF protein secretion were related to the accumulation of intracellular cyclic adenosine monophosphate (cAMP) levels. Although beta3AR mRNA was detectable in Con A-activated T lymphocytes, we could not demonstrate a functional activity in the regulation of cytokine expression: the beta3AR agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, and did not significantly affect cellular cAMP levels. These data demonstrate that beta-agonist-induced inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation is solely mediated by beta2-adrenoceptors.

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 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.

The effects of IL-1 and IL-4 on the Epo-independent erythroid progenitor in polycythaemia vera.

Human recombinant interleukin-1 (IL-1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL-1 was noticed on the normal, erythropoietin (Epo) dependent, erythroid burst-forming unit (BFU-E) using peripheral blood or bone marrow. In contrast, the Epo-independent BFU-E from peripheral blood of PV patients could be stimulated significantly. This enhancing effect of IL-1 was not only observed with unsorted but also with sorted CD34+ cells. In addition, it was shown that IL-1 indirectly stimulated the Epo-independent BFU-E because anti-GM-CSF could abrogate the supportive effects of IL-1. In contrast to the Epo-independent BFU-E, the Epo-dependent erythroid colony formation from PV patients could not be augmented by IL-1. Finally, we studied the effects of IL-4 on the Epo-independent BFU-E, because IL-4 can affect the erythroid colony formation and modulate the effects of IL-1. IL-4 suppressed the Epo-independent BFU-E. This effect could be counteracted by the addition of IL-1 to the culture medium. However, the suppressive effect of IL-4 was not related to a decline in spontaneous release of IL-1, because an anti-IL-1 antibody did not modify the spontaneous erythroid colony formation. These data indicate that IL-1 and IL-4 exert separate influences on the Epo-independent erythroid colony formation in PV.

Effect of ultrafilterable platinum concentration on cisplatin and carboplatin cytotoxicity in human tumor and bone marrow cells in vitro.

The importance of the ultrafilterable platinum (fPt) fraction of cisplatin (CDDP) and carboplatin (CBDCA) for cytotoxicity and myelotoxicity was studied in vitro. By incubating CDDP or CBDCA with fetal calf serum (FCS) various fractions of fPt were prepared and determined by atomic absorption spectroscopy. A relation of % fPt fraction and incubation time (h) of 87e-0.1123t (r = -0.99) and 101e-0.0087t (r = -0.99) were determined for CDDP and CBDCA, respectively. Cytotoxicity in the human small cell lung carcinoma cell line GLC4 and fPt fraction were closely related for CDDP (r = 0.99) and for CBDCA r = 0.97). However, at a similar fPt fraction the concentrations inhibiting cell survival by 50% (IC50) of CBDCA exceeded that of CDDP by a factor of 10-18 with 4 h exposure and a factor of 5 with continuous exposure. Tested in the range of peak concentrations in plasma of patients and at a clinically relevant fPt fraction of 10%, CDDP was not toxic for human bone marrow cells in the CFU-GM assay, whereas it was toxic at fPt fractions of 50% and 90%. However, CBDCA was myelotoxic at a (clinically relevant) fPt fraction of 50%, and also at 75% and 90%. The use of different fPt fractions, produced by the incubation method described in this study, permits the study of platinum drugs in vitro while approximating in vivo conditions might be used to evaluate myelotoxicity of new platinum drugs prospectively. For CDDP and CBDCA the fraction fPt determines cytotoxicity on tumor cells, and their different fPt fraction in patients account at least partly for their difference in myelotoxicity.

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.

Differential regulation of M-CSF and IL-6 gene expression in monocytic cells.

Using the human monocytic cell line Mono Mac 6 we studied the involvement of Ca2+, protein kinase A (PKA), and protein kinase C (PKC) dependent pathways in the regulation of M-CSF and IL-6 gene expression. The results demonstrate that on activation with the calcium ionophore A23187 both M-CSF and IL-6 mRNA are induced after 3 and 6 h respectively. Co-stimulation with A23187 plus PMA resulted in an up-regulation of M-CSF mRNA and a down-regulation of IL-6 mRNA. Conversely co-stimulation with A23187 plus DBcAMP resulted in a down-regulation of M-CSF mRNA and an up-regulation of IL-6 mRNA. Nuclear run-on and mRNA half-life studies showed that the effects on the M-CSF expression were related to changes at transcriptional and post-transcriptional level. In contrast, the effects on the IL-6 gene expression seems to be mediated at post-transcriptional level. With regard to the secretion of the IL-6 protein it was shown that it closely follows the accumulation of IL-6 mRNA. Taken together, the data show that several intracellular signalling pathways control strictly the cytokine expression in monocytic cells which gives the cells the opportunity to respond variably to external activation signals.

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.

Recombinant human mast cell growth factor supports erythroid colony formation in polycythemia vera in the presence and absence of erythropoietin and serum.

The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.

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.

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.

The release of endotoxin from antibiotic-treated Escherichia coli and the production of tumour necrosis factor by human monocytes.

The influence of antibiotic-induced release of endotoxin from in-vitro grown Escherichia coli on the production of tumour necrosis factor-alpha (TNF) by human monocytes was studied. Antibiotics tested were: cefuroxime (7.5 and 75 mg/L); ceftazidime (10 and 100 mg/L); aztreonam (10 and 100 mg/L); imipenem (10 and 100 mg/L); and tobramycin (8 mg/L). The effect of the combination of cefuroxime plus tobramycin, and the effect of taurolidine, an endotoxin-binding agent, on TNF production was also tested. After incubation for 4 h, all antibiotic-treated cultures (high-dose) induced a similar rise in extracellular TNF production when compared to the controls. However, after incubation for 24 h, a significant rise in TNF production was noticed in the cefuroxime and aztreonam-treated cultures (6440 and 5969 ng/L, respectively) compared to the ceftazidime and imipenem-treated cultures (846 and 381 ng/L, respectively). The cefuroxime-induced release of TNF could be reduced by addition of tobramycin (from 6440 to 1615 ng/L). Similar differences in TNF production were noticed in cell-associated TNF. Dose-response curves did not demonstrate differences in TNF production in aztreonam or imipenem-treated cultures. However, for both cefuroxime and ceftazidime-treated cultures, low-dose treatment resulted in significantly higher production of TNF. The differences in TNF production between these antibiotics could be explained by the production of filaments following treatment with cefuroxime, aztreonam and low-dose ceftazidime, resulting in late bacterial lysis with high levels of endotoxin, whereas treatment with imipenem or high-dose ceftazidime resulted in the formation of spheroplasts, resulting in early lysis of the bacteria and much lower levels of endotoxin. The addition of taurolidine to either imipenem or aztreonam-treated cultures prevented a rise in TNF production as a result of nearly complete neutralization of the released endotoxin. It was concluded that the observed differences in TNF production by human monocytes in vitro were related to differences in the mechanisms and amount of antibiotic-induced release of endotoxin.

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.

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.