Leo Koenderman

Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1

Cell death is regulated mainly through an evolutionarily conserved form of cell suicide termed apoptosis [1]. Deregulation of apoptosis has been associated with cancer, autoimmune diseases and degenerative disorders. Many cells, particularly those of the hematopoietic system, have a default program of cell death and survival that is dependent on the constant supply of survival signals. The Bcl-2 family, which has both pro- and anti-apoptotic members, plays a critical role in regulating cell survival [2]. One family member, the Bcl-2 interacting mediator of cell death (Bim), contains only a protein-interaction motif known as the BH3 domain, allowing it to bind pro-survival Bcl-2 molecules, neutralizing their function [3]. Disruption of the bim gene results in resistance to apoptosis following cytokine withdrawal in leukocytes, indicating that regulation of the pro-apoptotic activity of Bim is critical for maintenance of the default apoptotic program [4]. Here, we report that withdrawal of cytokine results in upregulation of Bim expression concomitant with induction of the apoptotic program in lymphocytes. Activation of the forkhead transcription factor FKHR-L1, previously implicated in regulation of apoptosis in T lymphocytes [5], was sufficient to induce Bim expression. We propose a mechanism by which cytokines promote lymphocyte survival by inhibition of FKHR-L1, preventing Bim expression.

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.

FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B–enhanced cell survival through maintenance of mitochondrial integrity

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim−/− mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB–mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days

Neutrophils are essential effector cells of the innate immune response and are indispensable for host defense. Apart from their antimicrobial functions, neutrophils inform and shape subsequent immunity. This immune modulatory functionality might however be considered limited because of their generally accepted short lifespan (< 1 day). In contrast to the previously reported short lifespans acquired by ex vivo labeling or manipulation, we show that in vivo labeling in humans with the use of (2)H(2)O under homeostatic conditions showed an average circulatory neutrophil lifespan of 5.4 days. This lifespan is at least 10 times longer than previously reported and might lead to reappraisal of novel neutrophil functions in health and disease.

A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1

Suppression of immune responses is necessary to limit damage to host tissue during inflammation, but it can be detrimental in specific immune responses, such as sepsis and antitumor immunity. Recently, immature myeloid cells have been implicated in the suppression of immune responses in mouse models of cancer, infectious disease, bone marrow transplantation, and autoimmune disease. Here, we report the identification of a subset of mature human neutrophils (CD11cbright/CD62Ldim/CD11bbright/CD16bright) as what we believe to be a unique circulating population of myeloid cells, capable of suppressing human T cell proliferation. These cells were observed in humans in vivo during acute systemic inflammation induced by endotoxin challenge or by severe injury. Local release of hydrogen peroxide from the neutrophils into the immunological synapse between the neutrophils and T cells mediated the suppression of T cell proliferation and required neutrophil expression of the integrin Mac-1 (αMβ2). Our data demonstrate that suppression of T cell function can be accomplished by a subset of human neutrophils that can be systemically induced in response to acute inflammation. Identification of the pivotal role of neutrophil Mac-1 and ROS in this process provides a potential target for modulating immune responses in humans.

REGULATION OF PROLIFERATION, DIFFERENTIATION AND SURVIVAL BY THE IL-3/IL-5/GM-CSF RECEPTOR FAMILY

The receptors for the I1-3/IL-5/GM-CSF cytokine family are composed of a heterodimeric complex of a cytokine-specific alpha chain and a common beta chain (betac). Binding of IL-3/IL-5/GM-CSF to their respective receptors rapidly induces activation of multiple intracellular signalling pathways, including the Ras-Raf-ERK, the JAK/STAT, the phosphatidylinositol 3-kinase PKB, and the JNK/SAPK and p38 signalling pathways. This review focuses on recent advancements in understanding how these different signalling pathways are activated by IL-3/IL-5/GM-CSF receptors, and how the individual pathways contribute to the pleiotropic effects of IL-3/IL-5/GM-CSF on their target cells, including proliferation, differentiation, survival, and effector functions.

The role of STATs in myeloid differentiation and leukemia

Myeloid differentiation is a highly regulated process governed by various cytokines, such as EPO, TPO, G-CSF, IL-3, IL-5 and GM-CSF. These cytokines act in part through activation of the STAT transcription factor family. In particular, various isoforms of STAT3 and STAT5 are activated during myeloid differentiation in a cell-type and maturation-state dependent fashion. In vitro studies have shown that STAT proteins are essential for cytokine-regulated processes such as cellular proliferation, differentiation as well as survival. Similarly, various STAT knock-outs have highlighted the role of STATs in myeloid differentiation in vivo. STATs also appear to play an important role in various myeloid malignancies, which are characterized by arrested maturation and cytokine-independent proliferation of myeloid progenitors. Constitutive activation of STAT3 and/or STAT5 resulting in enhanced transcription of anti-apoptotic- cell-cycle progression genes is likely to contribute to the pathogenesis of various myeloid leukemias.

Systemic inflammation in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by a chronic inflammation in the pulmonary tissue. The disease is associated with a switch from a selflimiting inflammatory response, mainly initiated by smoke inhalation, to a chronic persistent inflammatory response after prolonged interaction with cigarette smoke. The extent of the inflammatory reaction is correlated with the severity of the disease. Chronic inflammation in the pulmonary tissue is also associated with systemic effects. These effects range from cytokineinduced priming of peripheral leukocytes, to muscle wasting induced by cytokines such as tumour necrosis factor‐α. Despite a general consensus that chronic inflammation is a characteristic phenomenon of the disease, surprisingly little is known regarding the underlying pathogenetic mechanisms. A clear communication is present between the disease mechanisms in the pulmonary compartment and peripheral tissues, leading to the concept of COPD as a systemic inflammatory disease. This communication can be mediated by: 1) leakage of reactive oxygen species and stressinduced cytokines directly into the peripheral blood, 2) (pre)activation of peripheral blood leukocytes that can result in aberrant homing and activation of inflammatory cells in distant tissues, and 3) the liberation of proinflammatory mediators by leukocytes and/or stromal cells present in the pulmonary tissues during progression of the disease. The current authors hypothesise that the occurrence of a chronic inflammatory response after prolonged interaction of the pulmonary tissue with cigarette smoke causes aberrant homing of leukocytes to the tissue and delayed apoptosis. This leads to the autonomous characteristic of the inflammatory response in patients with chronic obstructive pulmonary disease.

An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37° followed by a one‐step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 ± 4% at 89 ± 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml.

Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences

Neutrophils are essential effector cells in the host defense against invading pathogens. Recently, novel neutrophil functions have emerged in addition to their classical anti-microbial role. One of these functions is the suppression of T cell responses. In this respect, neutrophils share similarities with granulocytic myeloid-derived suppressor cells (G-MDSCs). In this review, we will discuss the similarities and differences between neutrophils and G-MDSCs. Various types of G-MDSCs have been described, ranging from immature to mature cells shaping the immune response by different immune suppressive mechanisms. However, all types of G-MDSCs share distinct features of neutrophils, such as surface markers and morphology. We propose that G-MDSCs are heterogeneous and represent novel phenotypes of neutrophils, capable of suppressing the immune response. In this review, we will attempt to clarify the differences and similarities between neutrophils and G-MDSCs and attempt to facilitate further research.