Cassandra C. Paul

Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at ∼7 nM GM-CSF, and the EC50 was ∼0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B4, and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T421/S424 and T389 phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.

The Aml14 and Aml14.3D10 Cell Lines: A Long-Overdue Model for the Study of Eosinophils and More

Over the past three decades, a number of myeloid cell lines have been established and have proven useful for the study of various aspects of normal and disordered hematopoiesis. However, one myeloid lineage for which a useful cell line model has been sorely lacking is the eosinophil. We review the characteristics of the recently developed AML14 and AML14.3D10 cell lines and summarize how they have been used to obtain important new information relevant to eosinophil biology. Observations regarding the apparent ability of the AML14.3D10 cell line to “switch” lineages and to produce and use GM‐CSF in an autocrine fashion are also reviewed.

Evidence That Scabies Mites (Acari: Sarcoptidae) Influence Production of Interleukin-10 and the Function of T-Regulatory Cells (Tr1) in Humans

Abstract We performed experiments to determine whether an extract of Sarcoptes scabiei (De Geer) influenced cytokine expression by human T-lymphocytes. Peripheral blood mononuclear cells from five sensitized donors and four donors without sensitization to scabies mites were challenged with a T-cell mitogen alone, with scabies extract (SS) alone, or with mitogen and SS together. Supernatants were analyzed for the cytokines interferon-γ (IFNγ), interleukin (IL)-2, IL-4, and IL-10. No IL-2 or IL-4 was produced in response to scabies extract. Cells from both naïve and sensitized donors produced large amounts of IFNγ and IL-10. The lack of IL-4 but high levels of IL-10 suggests that IL-10 was likely secreted by type 1 T-regulatory cells, which were activated by something in the scabies extract. IL-10 has anti-inflammatory and immune-suppressive effects. It may play a key role in depressing the inflammatory and immune responses in humans so that clinical symptoms are not seen until 4–6 wk after a person becomes infested with scabies mites.

Cytokine induction of granule protein synthesis in an eosinophil‐inducible human myeloid cell line, AML14

Study of eosinophil growth and differentiation has been hampered by the difficulty of obtaining adequate numbers of highly purified eosinophil progenitors or mature eosinophils for analysis. The AML14 myeloid leukemia cell line has the unusual ability to exhibit eosinophilic differentiation in response to stimulation by combinations of the eosinophil‐active cytokines interleukin‐3 (IL‐3), granulocyte‐macrophage colony‐stimulating factor, and IL‐5. We now demonstrate that AML14 cells can be stimulated by a combination of these cytokines to produce mRNA encoding all the eosinophil granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), eosinophil‐derived neurotoxin (EDN), and the Charcot‐Leyden crystal (CLC) protein (eosinophil lysophospholipase). The production of the mature proteins was demonstrated by Western blotting, and ultrastructural analysis demonstrated the presence of immature secondary granules in cells that had been induced to differentiate to eosinophils. These findings demonstrate the utility of the AML14 cell line as a model for the study of cytokine induction of eosinophil growth and differentiation. J. Leukoc. Biol. 56: 74–79; 1994.

Autocrine activation of the IL‐3/GM‐CSF/IL‐5 signaling pathway in leukemic cells

The AML14.3D10 human myeloid leukemic cell line expresses receptors for granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin‐5 (IL‐5), but not IL‐3. We have found that this cell line produces GM‐CSF in amounts up to 113 pg/ml in culture supernatants. Deprivation of endogenous GM‐CSF by addition of neutralizing anti‐GM‐CSF antibody strongly inhibits proliferation of the cells, suggesting a GM‐CSF autocrine growth mechanism. To examine whether endogenously produced GM‐CSF activates intracellular GM‐CSF/IL‐3/IL‐5‐related signal transduction pathways, we performed anti‐phosphotyrosine immunoblotting of cell lysates of AML14.3D10 cells before and after deprivation of endogenous GM‐CSF. We found constitutive tyrosine‐phosphorylation of a number of proteins in AML14.3D10 that could not be detectably increased by the addition of exogenous GM‐CSF, IL‐3, or IL‐5. However, GM‐CSF‐deprived cells demonstrated a marked increase in phosphorylation of proteins of identical molecular mass following addition of GM‐CSF and IL‐5, but not IL‐3, consistent with the receptor expression of the cells and the known use of the same signaling pathways by the three cytokines. This suggests that AML14.3D10 cells use endogenously produced GM‐CSF to activate signal transduction pathways, interfering with activation by exogenous cytokine until the endogenous stimulation is removed. We then assessed the activation of the β‐subunit common to the GM‐CSF/IL‐3/IL‐5 receptors (βc), JAK2 and p53/56 lyn, known to be involved in the common signaling pathways of the three cytokines. We found that phosphorylation of βc and JAK2 in response to GM‐CSF and IL‐5 could be markedly enhanced by depriving cells of endogenous GM‐CSF. Constitutive hyperphosphorylation of lyn was found in AML14.3D10 cells, and no further activation of lyn in response to cytokine was demonstrable in GM‐CSF‐deprived cells, suggesting that lyn is activated in this cell line by a mechanism other than GM‐CSF. These studies represent the first demonstration of autocrine activation of intracellular cytokine signaling pathways by malignant hematopoietic cells. Because the addition of anti‐GM‐CSF to cell cultures improved responsiveness of intracellular signal transducing molecules to exogenous GM‐CSF and IL‐5, it can be inferred that endogenously produced GM‐CSF exerts its effects by secretion and binding to surface GM‐CSF receptors, although an intracellular component to signaling cannot be excluded. These observations provide further information regarding an autocrine contribution to leukemic cell growth, and establish a new model for study of these events. Am. J. Hematol. 56:79–85, 1997.

Human cell line that differentiates to all myeloid lineages and expresses neutrophil secondary granule genes

The aim of this study was to characterize a human leukemic cell line that appears capable of spontaneous differentiation to all myeloid lineages. The MPD cell line was derived using standard tissue culture techniques from the peripheral blood of a patient with an aggressive nonchronic myelogenous leukemia myeloproliferative disorder. Immunophenotyping, cytogenetic analysis, reverse transcriptase polymerase chain reaction, Northern blotting, immunoblotting, and colony assays were used to characterize the line and to assess its ability to express lineage-specific genes representative of advanced differentiation.Light microscopic morphologic analysis of the MPD cell line suggests that it has the unique property of spontaneous differentiation to mature-appearing neutrophils, macrophages, eosinophils, and basophils in proportions that approximate those found in normal bone marrow or peripheral blood. It was demonstrated that this cell line is capable of producing lineage-specific mRNA and granule proteins of at least two myeloid lineages, neutrophil and eosinophil, including neutrophil secondary granule proteins, which are not expressed in other available human cell lines. MPD cells were found to be capable of producing differentiated myeloid colonies (neutrophil, eosinophil, macrophge, mixed) in semisolid medium. The ability of MPD cells to express genetic programs associated with advanced differentiation of multiple myeloid lineages will make it a valuable tool for the study of the processes underlying lineage commitment and the regulation of expression of lineage-specific genes.

Interleukin-5 Receptor α Subunit Gene Regulation in Human Eosinophil Development: Identification of a Unique Cis-Element that acts lie an Enhacer in Regulating Activity of the IL-5Rα Promoter

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. IL-5, produced primarily by activated T cells [1] and mast cells [2], is integral to both the differentiation and functional maturation of the human eosinophil lineage [3-6]. The development and maturation of eosinophils in the bone marrow, and their post-mitotic functional activation in tissues occurs in response to a number of cytokines in addition to IL-5, including GM-CSF and IL-3 [7-9]. In humans, both IL-3 and GM-CSF have activities on other hematopoietic lineages, whereas IL-5 is eosinophil-specific and plays a crucial role in regulating the differentiation and development of the eosinophil lineage [3]. Although IL-3 and GM-CSF participate in the proliferation and committment of progenitors to the eosinophil lineage, IL-5 is both necessary and sufficient for eosinophil development to proceed[3, 10]. In humans, the high affinity receptor for IL-5 is apparently restricted to eosinophils and hematopoietically related basophils [11]; in contrast to murine B cells, the activity of IL-5 on human B cells is controversial [3, 12] and is still being delineated [13]. Thus, the expression of the high-affinity receptor for IL-5 is an important prerequisite and very early lineage-specific event in the hematopoietic program for these granuloctyes. Overexpression of IL-5 is observed in many eosinophil-associated diseases [14-16] and IL-5 transgenic mice develop profound eosinophilia [4, 5], indicating that IL-5 plays critical roles in promoting the production and function of eosinophils in vivo Of note, IL-5 is also active in vitro both in the production of eosinophils from bone marrow, umbilical cord and peripheral blood progenitors, as well as in the priming, activation and enhanced survival of mature eosinophils.