A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands

The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.     

Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils

The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL-3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.     

Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.     

Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific alpha chain and a beta common chain (beta c) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. Beta c plays an essential role in the transduction of extra cellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including beta c) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the beta c cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-beta c chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of beta c and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of beta c (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between beta c and shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with beta c. These observations provide an explanation for the previously described requirement for Tyr577 of beta c for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.     

Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.     

Effects of PIXY321, a granulocyte-macrophage colony-stimulating factor/interleukin 3 fusion protein, on human tumor colony-forming units taken directly from patients

PIXY321, a granulocyte-macrophage colony-stimulating factor/interleukin 3 (GM-CSF/IL-3) genetically engineered hybrid, has shown greater biological activity in stimulating committed myeloid progenitors than either GM-CSF or IL-3 in vitro, in vivo, and in patients treated with high-dose chemotherapy. However, one concern is that PIXY321 may stimulate the proliferation of malignant cells which have functional GM-CSF or IL-3 receptors. Therefore, using a human tumor cloning assay, we have tested the effects of several concentrations of PIXY321 ranging from 0.1 to 100 ng/ml on tumor cells taken directly from 98 patients with solid tumors and Hodgkin's or non-Hodgkin's lymphomas. Of the 34 evaluable specimens, including 15 breast cancers, 5 ovarian cancers, 5 lung cancers, and 9 lymphomas, none showed stimulation of tumor growth. Interestingly, a significant inhibition of the tumor proliferation was seen in one breast cancer and in one large cell immunoblastic non-Hodgkin's lymphoma after continuous exposure of PIXY321. In conclusion, the use of PIXY321 to reduce myelosuppression after high-dose chemotherapy appears unlikely to result in stimulation of the growth of malignant cells in patients with lymphoma or cancers of the breast, lung, and ovary.     

The cytokine network of wallerian degeneration: IL-10 and GM-CSF

Wallerian degeneration (WD) is the inflammatory response of peripheral nerves to injury. Evidence is provided that granulocyte macrophage colony stimulating factor (GM-CSF) contributes to the initiation and progression of WD by activating macrophages and Schwann, whereas IL-10 down-regulates WD by inhibiting GM-CSF production. A significant role of activated macrophages and Schwann for future regeneration is myelin removal by phagocytosis and degradation. We studied the timing and magnitude of GM-CSF and IL-10 production, macrophage and Schwann activation, and myelin degradation in C57BL/6NHSD and C57BL/6-WLD/OLA/NHSD mice that display normal rapid-WD and abnormal slow-WD, respectively. We observed the following events in rapid-WD. The onset of GM-CSF production is within 5 h after injury. Production is steadily augmented during the first 3 days, but is attenuated thereafter. The onset of production of the macrophage and Schwann activation marker Galectin-3/MAC-2 succeeds that of GM-CSF. Galectin-3/MAC-2 production is up-regulated during the first 6 days, but is down-regulated thereafter. The onset of myelin degradation succeeds that of Galectin-3/MAC-2, and is almost complete within 1 week. IL-10 production displays two phases. An immediate low followed by a high that begins on the fourth day, reaching highest levels on the seventh. The timing and magnitude of GM-CSF production thus enable the rapid activation of macrophages and Schwann that consequently phagocytose and degrade myelin. The timing and magnitude of IL-10 production suggest a role in down-regulating WD after myelin is removed. In contrast, slow-WD nerves produce low inefficient levels of GM-CSF and IL-10 throughout. Therefore, deficient IL-10 levels cannot account for inefficient GM-CSF production, whereas deficient GM-CSF levels may account, in part, for slow-WD.     

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.     

Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and stem cells in culture: transduction of the negative signals by betac and betaIL-3 proteins of IL-3 receptor and absence of negative regulation by granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit betac. However, in the mouse, there is an additional IL-3 signaling protein, betaIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from betac- and betaIL-3-knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin-) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin-Sca-1-positive, c-kit-positive marrow cells from 5-FU-treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with "compromised" marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors.     

Understanding the dendritic cell lineage through a study of cytokine receptors

Dendritic cells form a system of antigen presenting cells that are specialized to stimulate T lymphocytes, including quiescent T cells. The lineage of dendritic cells is not fully characterized, although prior studies have shown that growth and differentiation are controlled by cytokines, particularly granulocyte/macrophage colony-stimulating factor (GM-CSF). To further elucidate the nature and control of the dendritic cell lineage, we have studied the expression of specific cytokine receptors. Sufficient numbers of dendritic cells were purified from spleen and skin to do quantitative binding studies with radiolabeled M-CSF, GM-CSF, and interleukin 1 (IL-1). To verify the nonlymphoid nature of dendritic cells, we made an initial search for rearrangements in T cell receptor and immunoglobulin genes and none were found. M-CSF binding sites, a property of mononuclear phagocytes, also were absent. In contrast, GM-CSF receptors were abundant on mature dendritic cells, with approximately 3,000 binding sites/cell with a single Kd of 500-1,000 pM. Substantial numbers of high affinity (< 100 pM) IL-1 binding sites were identified as well; cultured epidermal dendritic cells (i.e., epidermal Langerhans cells) had 500/cell and spleen dendritic cells approximately 70/cell. Cross-linking approaches showed the 80-kD species that is expected of high-affinity type 1 IL-1 receptor. Anti-type 1 IL-1 receptor (R) mAbs also visualized these receptors by flow cytometry on freshly isolated epidermal dendritic cells. These results provide new evidence that dendritic cells represent a differentiation pathway distinct from lymphocytes and monocytes. Together with recent findings on the effects of IL-1 and GM-CSF on epidermal dendritic cells in situ (see Results and Discussion), the data lead to a proposal whereby IL-1 signals IL-1R to upregulate GM-CSF receptors and thereby, the observed responsiveness of dendritic cells to GM-CSF for growth, viability, and function.     

Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis

The pathogenesis of acquired pulmonary alveolar proteinosis (PAP), a rare lung disease characterized by excessive surfactant accumulation within the alveolar space, remains obscure. Gene-targeted mice lacking the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) or the signal-transducing beta-common chain of the GM-CSF receptor have impaired surfactant clearance and pulmonary pathology resembling human PAP. We therefore investigated the hematopoietic effects of GM-CSF in patients with PAP. The hematologic response of 5 infants with congenital PAP to 5 microgram/kg/d was of normal magnitude. By contrast, despite normal expression of GM-CSF receptor alpha- and beta-common chains on peripheral blood myelomonocytic cells (n = 6) and normal binding affinity of bone marrow mononuclear cells for GM-CSF (n = 3), each of the 12 patients with acquired PAP treated displayed impaired responses to GM-CSF; 5 microgram/kg/d produced only minor eosinophilia, and doses of 7.5 to 20 microgram/kg were required to induce >/=1.5-fold neutrophil increments in the 3 patients who underwent dose-escalation. However, neutrophilic responses to 5 microgram/kg granulocyte colony-stimulating factor (G-CSF) were normal (n = 4). In vitro, the proportion of hematopoietic progenitors responsive to GM-CSF (16.1% +/- 8.9%; P = .042) or interleukin-3 (IL-3; 19.3% +/- 7.7%; P = .063), both of which utilize the beta-common chain of the GM-CSF receptor complex, were reduced among patients with acquired PAP (n = 4) compared with normal bone marrow donor controls (47.2% +/- 25.9% and 40.9% +/- 18.6%, respectively). In the one individual who had complete resolution of lung disease during the period of study, this was temporally associated with correction of this defective in vitro response to GM-CSF and IL-3 on serial assessment. These data establish that patients with acquired PAP have an associated impaired responsiveness to GM-CSF that is potentially pathogenic in the development of their lung disease. Based on these observations, we propose a model of the pathogenesis of acquired PAP that suggests the disease arises as a consequence of an acquired clonal disorder within the hematopoietic progenitor cell compartment.     

Functional analysis of mature hematopoietic cells from mice lacking the betac chain of the granulocyte-macrophage colony-stimulating factor receptor

Mice with a null mutation of the betac chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors (betac-null mice) develop an alveolar proteinosis-like lung disease. The pathogenesis of this disease is uncertain and, although a defect in alveolar macrophage function has been postulated, no previous analysis of mature hematopoietic cells in mice with alveolar proteinosis has been reported. Therefore, we undertook a functional analysis of the mature hematopoietic cell compartment in betac-null mice. In addition, we reexamined the roles of the GM-CSF receptor chain and the betac chain in signaling by GM-CSF. Neutrophils and macrophages from betac-null mice were capable of normal survival and phagocytosis in the absence of stimulus and of similar levels of nitric oxide production in response to interferon-gamma and lipopolysaccharide. GM-CSF-mediated augmentation of survival, phagocytosis, and hydrogen-ion production were absent in neutrophils from betac-null mice. Interestingly, we were unable to show any ability of the GM-CSF receptor -chain alone to mediate glucose transport in these cells. In keeping with the betac-null mice lung pathology, examination of lavage fluid from the lungs of betac-null mice showed increased cellularity. This was caused by an increase in the number of lymphocytes, neutrophils, and macrophages. Large foamy cells in the lavage fluid from betac-null mice were identified as macrophages using immunohistochemistry. Functional analysis showed that these betac-null alveolar macrophages were capable of phagocytosis but uptake of colloidal carbon and cellular adhesion were reduced. In summary, mature hematopoietic cells with a null mutation of the betac receptor were unable to perform GM-CSF-mediated hematopoietic cell functions including glucose transport, but responded normally to a range of other ligands.     

BAG-1 and Bcl-2 in IL-2 signaling

Some cytokines can prolong cell survival in hematolymphoid cells and thus may be crucial for regulation of hematolymphoid cell numbers. It has been shown that mitogenic cytokines can induce not only cellular proliferation but also cellular survival by inhibiting apoptosis in hematolymphoid cells. The signals transduced by these cytokines eventually go to the nucleus and induce expression of their specific target genes. In this context, the induction of anti-apoptotic molecules such as Bcl-2 oncoprotein and BAG-1 protein seems to be a key event for the anti-apoptotic function of cytokines. In T lymphocytes, the interaction of interleukin-2 (IL-2) with its receptor (IL-2R) induces both cellular proliferation and cellular survival. The IL-2R consists of three subunits, i.e., IL-2Ralpha, IL-2R(beta)c, and IL-2R(gamma)c chains. Structure-function analysis of the IL-2R(beta)c chain has revealed that there are at least two functional domains within the subunit. The serine-rich (S) region but not the acidic (A) region within the (beta)c chain is responsible for the mitogenic signaling of IL-2R. The S region is also crucial for the cellular survival signaling, which include the induction of anti-apoptotic gene expressions bcl-2 and bag-l. However, the cellular survival signaling is segregated from the mitogenic signaling in independence from the Jak-family protein kinase activation and rapamycin sensitivity. Segregation of the two signaling pathways of a cytokine receptor has also been shown in receptors of the other mitogenic cytokines. Current topics regarding signal transductions of cytokine receptors responsible for the suppression of apoptosis are discussed in this review.     

Dexamethasone plus retinoids decrease IL-6/IL-6 receptor and induce apoptosis in myeloma cells

Interleukin 6 (IL-6) is the most important known growth factor for multiple myeloma, and IL-6 signalling pathways are potential targets for therapy. We hypothesized that interfering with the IL-6 signalling pathway at more than one level would be more effective than a single block in inhibiting proliferation of myeloma cells. Accumulating data support the concept that glucocorticoids down-regulate IL-6, whereas retinoic acid derivatives (RA) down-regulate IL-6R in myeloma. We found that all-trans RA (ATRA), 13-cis-RA and 9-cis-RA each similarly inhibited growth of RPMI 8226 myeloma cells and that addition of dexamethasone (DEX) added to RA growth inhibition. The major effects of retinoids were to reduce the proliferative fraction and induce apoptosis whereas DEX increased the apoptotic fraction. When combined, apoptosis was enhanced. Effects of RA + DEX were also least able to be overcome by exogenous IL-6. RA decreased IL-6R levels and addition of DEX to RA delayed recovery of IL-6R levels compared with RA alone. Since RPMI 8226 cells have undetectable IL-6, we investigated U266B1 cells and found that RA and DEX decreased both IL-6 secretion and IL-6 RNA levels. Mechanistically, IL-6R down-regulation by RA was enhanced by DEX, whereas IL-6 protein and RNA levels were reduced by DEX and by RA. In summary, combinations of RA + DEX were not only more effective in inhibiting myeloma cells growth by the dual mechanisms of decreasing proliferative fraction and increasing apoptotic fraction, but were also less able to be overcome by IL-6.     

The kinase domain of Jak2 mediates induction of bcl-2 and delays cell death in hematopoietic cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 stimulate DNA synthesis and proliferation and inhibit apoptosis in hematopoietic cells. Multiple signal pathways are activated by binding of these ligands to their receptors, which share a common beta subunit. Janus protein kinase 2 (Jak2) binds to the membrane proximal domain of the beta chain and is phosphorylated on receptor ligation. To explore the role of Jak2 in the regulation of specific signal transduction pathways, we constructed fusion proteins with a CD16 external domain, a CD7 transmembrane region, and a Jak2 cytoplasmic domain. This cytoplasmic domain consisted either of wild type Jak2 (CD16/Jak2-W) or Jak2 mutations with deletions of (a) the amino terminus (CD16/Jak2-N), (b) kinase-like domain (CD16/Jak2-B), (c) kinase domain (CD16/Jak2-C), or (d) amino-terminal and kinase-like domains, leaving the kinase domain (CD16/Jak-K) intact. In contrast to the CD16/Jak2-W fusion protein, which requires cross-linking for activation, CD16/Jak2-N, CD16/Jak2-B, and CD16/Jak2-K were constitutively phosphorylated, and they stimulated Shc phosphorylation and increased binding of STAT to DNA in Ba/F3 cells. Cell lines derived from IL-3-dependent Ba/F3 cells stably transfected with CD16/Jak2-W, CD16/Jak2-N, or CD16/Jak2-B mammalian expression vectors died at a rate similar to that of the parental cells on IL-3 deprivation. In contrast, CD16/Jak2-K cell lines exhibited increased expression of bcl-2 and pim-1 mRNA and maintained their viability when compared with control cell lines. Thus, activation of tyrosine phosphorylation by creating a CD16/Jak2-K fusion is sufficient to activate pathways that prevent cell death.     

Treatment with interleukin-4 prolongs allogeneic neonatal heart graft survival by inducing T helper 2 responses

BACKGROUND: Eosinophils are a prominent feature of chronic hyperplastic sinusitis with nasal polyposis (CHS/NP). Our previous studies showed that their presence was associated with the expression of GM-CSF and RANTES mRNA. In allergic NP, increased expression of IL-5 was also found. OBJECTIVE: We wished to examine cytokine immunoreactivity for IL-5, GM-CSF and RANTES mRNA in allergic and non-allergic NP and compare immunoreactivity with expression of cytokine mRNA by in situ hybridization. Methods NP were obtained from five allergic and eight non-allergic subjects with CHS/ NP. Middle turbinate tissue from eight normal subjects were used as controls. Cell-associated cytokine mRNA was detected by in situ hybridization (ISH). Cytokine immunoreactive cells were enumerated by immunostaining. Colocalization immunostaining was also performed to identify specific cell types producing IL-5. RESULTS: Immunostaining for GM-CSF, IL-5 and RANTES protein was increased in both allergic and non-allergic NP compared with control middle turbinates. Allergic polyps contained greater numbers of IL-5 immunoreactive cells (P = 0.01), whereas non-allergic polyps contained greater numbers of GM-CSF immunoreactive cells (P = 0.04). Immunostaining was primarily associated with inflammatory cells, but immunostaining for RANTES and, to a lesser extent GM-CSF, was also seen in the epithelium. The density of immunoreactive cells was variably correlated with cytokine mRNA+ cells (GM-CSF: R=0.56, P=0.05; IL-5: R=0.76, P=0.003; and RANTES: R=0.89, P=0.0005). Colocalization immunostaining revealed that the majority of IL-5 immunoreactive cells in both allergic and non-allergic NP were T lymphocytes. However, allergic NP contained greater numbers of IL-5+/CD3+ T lymphocytes and IL-5+ mast cells, whereas non-allergic NP contained greater numbers of IL-5+ eosinophils. CONCLUSION: We conclude that GM-CSF, IL-5 and RANTES are produced in increased amounts in both allergic and non-allergic NP. Distinguishing features of non-allergic NP include fewer numbers of CD3 T lymphocytes, fewer IL-5+/CD3+ T lymphocytes and greater numbers of IL-5+ eosinophils. These differences may suggest different mechanisms of eosinophil accumulation and activation in allergic vs non-allergic NP.     

Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells

Cytokine-mediated signaling pathways were studied in mouse dendritic cells (DC) by analysis of the activation pattern of STAT factors. Electrophoretic mobility shift assays were performed to detect STAT isoform-specific complexes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) simultaneously induced complexes containing STAT1, STAT3, STAT5A, STAT5B and STAT6. In non-DC, a similar broad activation pattern of STAT factors by GM-CSF or other cytokines has not been observed so far. By comparison, in peritoneal macrophages, GM-CSF induced a complex with the properties of a truncated form of STAT5. Other cytokines tested on DC either failed to induce STAT factors [interleukin (IL)-1 beta, IL-2, IL-15], or activated the same STAT factors as observed in peritoneal macrophages (IL-4, IFN-gamma). Our results implicate a specific effect of GM-CSF on STAT signaling in DC which might account for the cell type-specific effect of this cytokine on development and function.