Mt Esselink

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-κB (NF-κB) 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-κB, 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‐γ (IFN‐γ) 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 γC chain. The results demonstrate that the extent of upregulation of IFN‐γ and IL‐4 mRNA was dependent on the applied cytokine (IL‐2>IL‐15>IL‐7) and on the stimulatory signal. IFN‐γ and IL‐4 mRNAs were upregulated by IL‐15 in concanavalin A‐ (twofold) and anti‐CD3 plus anti‐CD28‐ (fivefold) stimulated T lymphocytes. IFN‐γ 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‐γ 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‐γ protein. The net effect was a dramatically increased IFN‐γ/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.

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

Summary 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 downregulation 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.

Interleukin-4 prevents the induction of G-CSF mRNA in human adherent monocytes in response to endotoxin and IL-1 stimulation

Human recombinant interleukin‐4 (IL‐4) was studied for its effects on the expression of granulocyte‐colony stimulating factor (G‐CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL‐1), IL‐4 (15 ng/ml) did not induce G‐CSF transcripts in monocytes but suppressed the endotoxin‐induced G‐CSF expression when added simultaneously. Sequential treatment of monocytes with IL‐4 followed by endotoxin suppressed G‐CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL‐4 dose. Comparable results were obtained with IL‐1, IL‐1 (50 U/ml) induced G‐CSF expression in human adherent monocytes which could be counteracted by IL‐4 pretreatment. In addition, it was shown that the induction of G‐CSF mRNA by the calcium‐ionophore A23187 or by c‐AMP elevating agents could be blocked by IL‐4. These suppressive effects of IL‐4 were not related to changes in the half‐life of G‐CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL‐4 had comparable effects on the G‐CSF secretion of endotoxin and IL‐1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL‐4 down‐regulates the expression of G‐CSF gene and secretion of proteins in human activated monocytes.

INTERLEUKIN-4 SUPPRESSES THE INTERLEUKIN-3 DEPENDENT ERYTHROID COLONY FORMATION FROM NORMAL HUMAN BONE-MARROW CELLS

Human recombinant interleukin 4 (IL‐4) was studied for its effects on the erythroid burst forming unit (BFU‐E) from human bone marrow cells. IL‐4 alone neither supports nor suppresses the erythropoietin (Epo)‐dependent colony formation. Different results were obtained when IL‐4 was combined with interleukin‐3 (IL‐3) in the presence of Epo. IL‐4 suppressed the IL‐3 supported erythroid colony formation in all cases (an increase of 58 ± 8% with IL‐3 versus an increase of 14 ± 7% with IL‐3 plus IL‐4, n= 8). This antagonizing effect was dependent on the continuous presence of IL‐4 in the culture medium, but was independent of adherent cells. B‐, T‐cells, or the presence of serum in the culture medium. Finally, the effects of IL‐4 and IL‐3 were studied on the ‘Epo‐independent’BFU‐E by adding Epo on day 3. A decline of the IL‐3 supported BFU‐E was observed in the presence of IL‐4 but the degree of reduction was equivalent to the results obtained when Epo was supplied at day 0. These findings indicate that IL‐4 acts as suppressive growth factor for the IL‐3 supported erythroid colony formation from human bone marrow cells.

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

Summary. 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.