Jan A. M. Raaijmakers

Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1

ABSTRACT Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

Upregulation of formyl-peptide and interleukin-8—induced eosinophil chemotaxis in patients with allergic asthma

BACKGROUND The cytokine granulocyte-macrophage colony-stimulating factors, interleukin-3 and interleukin-5, are important modulators of eosinophilia and eosinophil function. In particular, eosinophil chemotaxis is very sensitive to cytokine priming. METHODS We evaluated chemotactic responses of eosinophils from patients with allergic asthma. These cells exhibited a primed phenotype as deduced from enhanced responses toward formyl-methionyl-leucyl-phenylalanine and platelet-activating factor and a decreased responsiveness toward granulocyte-macrophage colony-stimulating factor. Bronchoprovocation of patients with allergic asthma with allergen was performed as a possible means to enhance in vivo priming. RESULTS Indeed, eosinophils isolated 3 hours after allergen challenge exhibited a more pronounced primed phenotype, which was reflected by an induction of responsiveness towards interleukin-8. Eosinophil responses induced by platelet-activating factor, formyl-methionyl-leucyl-phenylalanine, complement fragment C5a, interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor were not significantly altered after allergen challenge. CONCLUSION These data provide further evidence that eosinophils are already primed in the peripheral blood of individuals with allergic asthma. This is most likely due to the presence of circulating cytokines in the peripheral blood of those individuals. This in vivo priming results in selective upregulation and downregulation of responses toward various chemotaxins, which may be released in the lungs during allergic inflammation.

Association of RACK1 and PKCβ with the common β-chain of the IL-5/IL-3/GM-CSF receptor

Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific α-chain and a signal transducing β-chain (βc). While, the role of phosphotyrosine residues in the βc as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with βc, we screened a yeast two-hybrid library with the intracellular domain of βc. We found that RACK1, a molecule associating with activated PKC, PLCγ and Src kinases, associated with the membrane proximal region of βc in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of βc and PKCβ was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors.

Activation of the STAT3/Acute Phase Response Factor Transcription Factor by Interleukin-5

The receptor for interleukin-5 (IL-5R) is composed of a unique α chain (IL-5Rα) expressed on eosinophils and basophils, associated with a βc subunit, which is shared by the receptors for IL-3 and granulocyte macrophage-colony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1α or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the α and βc subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a βc receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the βc receptor is not essential for STAT3 activation.

Mechanisms involved in eosinophil migration. Platelet-activating factor-induced chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals.

Eosinophils play an important role in the pathogenesis of allergic diseases such as allergic asthma. Eosinophil migration in vitro can be divided into directed migration, or Chemotaxis, and random migration, or chemokinesis. Here, we studied intracellular signals involved in eosinophil migration in vitro induced by platelet‐activating factor (PAF) and interleukin‐5 (IL‐5), applying a Boyden chamber assay. Migration induced by PAF (10‐11‐10‐6 M) largely consisted of Chemotaxis with some chemokinesis, whereas IL‐5 (10‐12‐10‐8 M) induced chemokinesis only. Eosinophils were depleted from intracellular and extracellular Ca2+ to study the role of Ca2+ as a second messenger. Ca2+ depletion did not change PAF‐induced Chemotaxis, however, IL‐5‐induced chemokinesis was inhibited. Interestingly, PAF, but not IL‐5, induced changes in [Ca2+]i. This rise originated mainly from internal stores. Inhibition of protein kinase A by H‐89 and protein kinase C by GF 109203X had no effect on both forms of eosinophil migration. Addition of the protein kinase inhibitor staurosporine significantly inhibited IL‐5‐induced chemokinesis. Inhibition of tyrosine kinases by herbimycin A completely blocked IL‐5‐induced chemokinesis. PAF and IL‐5‐induced actin polymerization was studied to compare migratory responses with a migration‐associated intracellular response. Ca2+ depletion significantly enhanced PAF‐induced (10‐8 M) actin polymerization, whereas IL‐5‐induced actin polymerization was not influenced. Addition of staurosporine led to an increase in F‐actin. Subsequent addition of PAF or IL‐5 resulted in an additive increase in F‐actin content. In summary, both forms of eosinophil migration are protein kinase A and protein kinase C independent. In contrast to PAF‐induced Chemotaxis, IL‐5‐induced chemokinesis was found to be completely Ca2+ and tyrosine kinase dependent.

Cytokine priming of the respiratory burst in human eosinophils is Ca2+ independent and accompanied by induction of tyrosine kinase activity.

We report that pretreatment of human eosinophils with GM‐CSF, IL‐3, or IL‐5 enhanced the respiratory burst induced by opsonized particles. In order to gain more insight into the intracellular mechanism(s) involved in cytokine priming, the role of [Ca2+], and tyrosine kinases was studied. Optimal priming concentrations of GM‐CSF, IL‐3, and IL‐5 did not induce a rise in [Ca2+]i, and Ca2+‐depleted eosinophils ([Ca2+]i < 20 nM) were still primed after preincubation with these cytokines. GM‐CSF, IL‐3, and IL‐5 induced phosphorylation of two proteins (102 and 122 kd) on tyrosine residues, as deduced from Western blot analysis with an antiphospho‐ tyrosine monoclonal antibody (4G10). This cytokine‐ stimulated tyrosine phosphorylation was not inhibited under Ca2+‐depleted conditions. In conclusion, this study demonstrates that GM‐CSF, IL‐3, and IL‐5 priming of the opsonized particle‐induced respiratory burst in human eosinophils is completely Ca2+ independent. Moreover the tyrosine phosphorylation of a 102‐kd and a 122‐kd protein is Ca2+ independent, suggesting that this event might be involved in cytokine priming.

Monitoring of neutrophil priming in whole blood by antibodies isolated from a synthetic phage antibody library

Neutrophil activation is a multistep process. In vitro activation of neutrophils with semiphysiological activators is optimal only after preactivation or priming with cytokines, chemotaxins, and/or bacterial products. Until now, no antibodies have been developed that can distinguish between resting and (cytokine) primed neutrophils with a sufficient dynamic range necessary for screening clinical samples. We have isolated two human phage antibodies, designated MoPhab A17 and A27, from a synthetic bacteriophage antibody library. These phage antibodies recognize epitopes that are upregulated on neutrophils present in whole blood treated with low priming concentrations of cytokines, such as GM‐CSF and TNF‐α. This induction was time‐ and concentration‐dependent and optimal at concentrations that are sufficient for priming functional responses in neutrophils: GM‐CSF (10 pM) and TNF‐α (100 IU/ml). PMNs, isolated from the peripheral blood of chronic obstructive pulmonary disease (COPD) patients with a clinical exacerbation, exhibited a partial in vivo primed phenotype. These antibodies promise to be an ideal tool to monitor disease activity in whole blood of patients with inflammatory diseases.

Differential Activation of Functionally Distinct STAT5 Proteins by IL-5 and GM-CSF During Eosinophil and Neutrophil Differentiation from Human CD34+ Hematopoietic Stem Cells

Interleukin‐5 (IL‐5) and granulocyte macrophage‐colony stimulating factor (GM‐CSF) are important cytokines for the proliferation, differentiation, and activation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predominantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT proteins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34+ hematopoietic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL‐5 (eosinophil lineage) and GM‐CSF (neutrophil lineage). However, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in heterodimer DNA‐binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells.

Regulation and function of protein kinase B and MAP kinase activation by the IL-5/GM-CSF/IL-3 receptor.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) regulate proliferation, differentiation and apoptosis of target cells. Receptors for these cytokines consist of a cytokine-specific α subunit and a common shared βc subunit. Tyrosine phosphorylation of the βc is thought to play a critical role in mediating signal transduction events. We have examined the effect of mutation of βc tyrosines on the activation of multiple signal transduction pathways. Activation of protein kinase B (PKB) required JAK2 and was inhibited by dominant-negative phosphatidylinositol 3-kinase (P13K). Overexpression of JAK2 was sufficient to activate both protein kinase B (PKB) and extracellular regulated kinase-1 (ERK1). Tyrosine 577 and 612 were found to be critical for the activation of PKB and ERK1, but not activation of STAT transcription factors. Activation of both PKB and ERK have been implicated in the regulation of proliferation and apoptosis. We generated GM-CSFR stable cell lines expressing receptor mutants to evaluate their effect on these processes. Activation of both PKB and ERK was perturbed, while STAT activation remained unaffected. Tyrosines 577 and 612 were necessary for optimal proliferation, however, mutation of these tyrosine residues did not affect GM-CSF mediated rescue from apoptosis. These data demonstrate that while phosphorylation of βc tyrosine residues 577 and 612 are important for optimal cell proliferation, rescue from apoptosis can be mediated by alternative signalling routes apparently independent of PKB or ERK activation.

Lineage-specific activation of STAT3 by interferon-gamma in human neutrophils.

Binding of interferon‐γ (IFN‐γ) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1α. Selective activation of STAT1α at the IFN‐γ receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN‐γ receptor α‐chain and the SH2 domain of STAT1α. We demonstrate that, in addition to STAT1α, STAT3 is also activated by IFN‐γ in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL‐60 cells, although the STAT3 protein was expressed in these cells. The cell type‐specific activation of STAT3 by IFN‐γ was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell‐specific manner, which might result in cell‐specific gene transcription. J. Leukoc. Biol. 65: 391–396; 1999.