Mice deficient for the IL-3/GM-CSF/IL-5 beta receptors]

Candida species are increasingly important fungal pathogens. The reaction of rat alveolar macrophages (AM) to Candida albicans was compared with that to C. glabrata and C. krusei. Phagocytosis of C. glabrata was similar to that of C. albicans, but significantly slower for C. krusei due to reduced attachment. After opsonization, attachment of C. albicans and C. krusei to AM was significantly increased and there was no significant difference between the two species. The oxidative metabolism of AM with candida species was two to three times higher than that of the resting AM both during and 24 h after the phagocytosis. All three species showed a considerable fraction (5-10%) of phagolysosomes with pH > or = 6.5 after 3 h and a smaller percentage (1%) after 24 h.     

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.     

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.     

Growth and differentiation of eosinophils--special reference to IL-5 and its receptor

IL-5 is a 46 KDa glycoprotein secreted from activated T cells, and plays an important role both in the growth and production of eosinophils. Murine eosinophils have been reported to express high and low affinity receptors. The IL-5 receptor is composed of two membrane protein subunits, alpha and beta subunits. Both alpha and beta chain of mouse and human IL-5 receptor have been identified and sequenced. The alpha subunit is a 60 KDa protein which binds to IL-5 with low affinity. The beta subunit is a 130 KDa protein which does not bind to IL-5 by itself, but forms the high affinity IL-5 receptor in the presence of the alpha subunit. On the other hand, only a high affinity receptor population has been identified on human eosinophils. Affinity cross-linking experiments resulted in the identification of human IL-5 receptor on eosinophils with a molecular mass of 55-60 KDa.     

Regulation of growth region cartilage proliferation and differentiation by perichondrium

Pharmacokinetic and dosimetric parameters of the hypoxic tissue imaging agent iodoazomycin arabinoside (123I-IAZA) have been investigated in human volunteers. In conjunction with this study it was necessary to develop an assay for low levels of the radiolabelled compound in blood and urine. A combination of high-performance liquid chromatography (HPLC) and gamma counting produced a highly selective, sensitive and rapid assay for the analysis of 123/125I-IAZA in human and animal blood and urine samples. Conventional HPLC assays for the tracer quantities of this radioactive agent in blood have not been reported previously. The addition of non-radiolabelled IAZA to the blood and urine samples containing radiolabelled IAZA allowed the pharmaceutical to serve as its own internal standard. This reverse isotope dilution approach permitted identification of the appropriate HPLC peak by UV detection, followed by highly sensitive quantification of the radiolabelled species by gamma counting. Blood samples were prepared for HPLC by a solid-phase extraction without the loss of IAZA from serum, with an extraction efficiency of 99.7 +/- 7.1% from human serum. Urine samples could be analyzed directly by HPLC, without the solid-phase extraction step. The detection limit in biological fluids depends on the specific activity of radiolabelled 123/125I-IAZA. In this study it was possible to detect serum concentrations of 123I-IAZA as low as 7.46 pg (21 fmol) per ml. The radiometric detection limit for 123I-IAZA in this assay was 10.8 Bq ml-1 of serum.     

Persistence of pulmonary pathology and abnormal lung function in IL-3/GM-CSF/IL-5 beta c receptor-deficient mice despite correction of alveolar proteinosis after BMT

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor (beta cR KO) develop lung disease similar to that seen in human pulmonary alveolar proteinosis (PAP) which includes lymphocytic infiltration around airways and vessels and the progressive accumulation of surfactant and macrophages within the alveolar space. We investigated bone marrow transplantation (BMT) as a curative treatment of PAP in beta cR KO mice by semiquantitative histologic analysis and evaluation of pulmonary function. BMT from wild-type (WT) donors into lethally irradiated beta cR KO recipients (WT --> KO) led to the complete resolution of alveolar protein accumulation and to normalization of BAL fluid cellularity and macrophage morphology. However, detailed microscopic analysis of lung tissue revealed the persistence of significant cellular infiltrates in WT --> KO recipients which were equivalent to those seen in KO --> KO animals. Evaluation of pulmonary function demonstrated that only dynamic compliance (Cdyn) and not airway conductance (G[L]) was significantly improved in the WT --> KO group compared to KO --> KO animals and that both of these measurements remained significantly abnormal when compared to WT --> WT controls. We conclude, that although BMT for PAP reverses alveolar macrophage and protein accumulation, it does not decrease the interstitial inflammatory component of this disease. The importance of this residual pathology is demonstrated by the incomplete correction of alveolar function (Cdyn) and lack of improvement in increased airway resistance (G[L]). These findings may have important implications with regard to the extent that BMT can be considered a potential curative procedure for this clinical disorder.     

Stimulation of blood mononuclear cells of atopic children with the relevant allergen induces the release of eosinophil chemotaxins such as IL-3, IL-5, and GM-CSF

Even among comprehensive local public mental health systems, there remain large gaps in continuity of care following discharge from inpatient settings. The authors describe a modification of the assertive community treatment (ACT) program model that links inpatients to ongoing community-based care, and provide preliminary evidence of its effectiveness as a component in a rationally organized comprehensive system of care. Given the recent trend toward managed Medicaid arrangements, there will be increased pressure to reduce clients' length of stay in ACT programs. State mental health authorities are cautioned to resist allowing managed care contractors to radically change the conditions under which ACT programs operate until there is greater evidence of the effectiveness of alternative approaches.     

Regulation of hippocampal synaptic plasticity by the tyrosine kinase receptor, REK7/EphA5, and its ligand, AL-1/Ephrin-A5

The Eph-related tyrosine kinase receptor, REK7/EphA5, mediates the effects of AL-1/Ephrin-A5 and related ligands and is involved in the guidance of retinal, cortical, and hippocampal axons during development. The continued expression of REK7/EphA5 in the adult brain, in particular in areas associated with a high degree of synaptic plasticity such as the hippocampus, raises the question of its function in the mature nervous system. In this report we examined the role of REK7/EphA5 in synaptic remodeling by asking if agents that either block or activate REK7/EphA5 affect synaptic strength in hippocampal slices from adult mouse brain. We show that a REK7/EphA5 antagonist, soluble REK7/EphA5-IgG, impairs the induction of long-term potentiation (LTP) without affecting other synaptic parameters such as normal synaptic transmission or paired-pulse facilitation. In contrast, perfusion with AL-1/Ephrin-A5-IgG, an activator of REK7/EphA5, induces a sustained increase in normal synaptic transmission that partially mimics LTP. The sustained elevation of normal synaptic transmission could be attributable to a long-lasting binding of the AL-1/Ephrin-A5-IgG to the endogenous REK7/EphA5 receptor, as revealed by immunohistochemistry. Furthermore, maximal electrical induction of LTP occludes the potentiating effects of subsequent treatment with AL-1/Ephrin-A5-IgG. Taken together these results implicate REK7/EphA5 in the regulation of synaptic plasticity in the mature hippocampus and suggest that REK7/EphA5 activation is recruited in the LTP induced by tetanization.     

Regulation of the IL-12 receptor beta2 subunit by soluble antigen and IL-12 in vivo

Sympathetic innervation of lymphoid tissues is localized to specific tissue compartments, but little is known of the "factors" that are important in establishing this pattern during development. Numerous studies have shown interactions of nerve growth factor (NGF) with the immune system, which may include modulation of immune innervation. We previously have shown that NGF transgenic mice, which overexpress NGF in skin and not immune tissues, have a dramatic hyperinnervation of splenic marginal zone and peripheral lymph node medulla and capsule. The purpose of the current studies was to determine if the presence of elevated NGF would alter immune system development and the process of sympathetic ingrowth. The results show that the splenic innervation in NGF transgenics gradually diverged from controls during the first two postnatal weeks, with the greatest change occurring between postnatal days 13 and 16 when the splenic organization was reaching the adult pattern. In contrast, the peripheral lymph nodes were hyperinnervated at an earlier age. mesenteric lymph nodes never diverged from the normal pattern. NGF levels in transgenic spleen were much higher than controls at postnatal days 1 and 2, when little innervation was present, and declined as the tissue matured, possibly because of NGF uptake by the ingrowing sympathetic fibers. This suggests that immune tissues are capable of concentrating NGF, which in turn may modulate the level of innervation by the sympathetic nervous system.     

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.     

Allergen-induced release of GM-CSF and IL-8 in vitro by nasal polyp tissue from atopic subjects prolongs eosinophil survival

Eosinophilia is a feature of nasal polyposis. The aim of this study was to determine the role of cytokines and allergen in maintaining the eosinophilic infiltrate in this condition. Polyp fragments from house dust mite (HDM)-sensitive atopic individuals and nonatopic individuals were cultured in the presence of HDM, or phytohaemagglutinin (PHA) or culture medium alone. Culture supernatants were assayed for interleukins (IL) 3, 5, and 8 and granulocyte macrophage colony stimulating factor (GM-CSF), and eosinophil survival enhancing activity (ESEA) in vitro. Significant ESEA was produced spontaneously. When polyp tissue from atopics, but not from nonatopics, was stimulated with allergen for 2 days there was a further increase in ESEA associated with a median 12 and fourfold increase in IL-8 and GM-CSF, respectively. This increased ESEA was markedly reduced with anti-GM-CSF and, to a lesser extent, anti-IL-8 blocking antibodies. When stimulated with PHA, polyp tissue from atopic subjects also produced increased ESEA, implicating possible T-cell involvement. This was associated with a small (twofold), but significant, increase in IL-8 and a less consistent increase in GM-CSF. However, anti-IL-8 or anti-GM-CSF blocking antibodies failed to reduce the ESEA in these supernatants, suggesting involvement of other mechanisms. This study suggests that in sensitized individuals, allergen may contribute to polyp eosinophilia by stimulating the production of granulocyte/macrophage colony stimulating factor and interleukin 8.     

Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.     

Degranulation of human basophils by picomolar concentrations of IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor

PURPOSE: To determine capillary blood flow measurements in eyes with branch retinal vein occlusion using a scanning laser Doppler flowmeter. METHODS: Retinal capillary blood flow in branch retinal vein occlusion areas and corresponding ipsilateral nonbranch retinal vein occlusion areas, 11 equivalent areas of the contralateral fellow eye of 12 consecutive untreated branch retinal vein occlusion patients, and 16 eyes of 11 age-matched normal control subjects were measured with scanning laser Doppler flowmetry. A template consisting of eight squares, each with a field of 100 x 100 microm (10 x 10 pixel) with space interval of 500 microm equidistant horizontally and vertically was used to obtain blood flow measurements in all subjects. Mean blood volume, flow, and velocity were obtained by averaging the mean values measured in each field. We avoided measurement over large retinal vessels to prevent the aliasing artifact of blood cells from moving faster than the sampling frequency. RESULTS: Branch retinal vein occlusion areas have significantly decreased microvascular blood volume (P = .0009), flow (P = .02), and velocity (P = .016) compared with ipsilateral nonbranch retinal vein occlusion areas in the same eye. Branch retinal vein occlusion areas also have decreased blood volume (P = .001), flow (P = .0042), and velocity (P = .0044) compared with areas of contralateral fellow eyes of branch retinal vein occlusion subjects. Branch retinal vein occlusion areas have significantly decreased blood volume (P = .0012), flow (P = .008), and velocity (P = .02) compared with age-matched normal areas. CONCLUSION: Average retinal blood volume, flow, and velocity in areas of branch retinal vein occlusion are significantly lower than in healthy retinas. The ability to noninvasively measure hemodynamic changes in the retinal capillary bed may be relevant to development of new therapies for retinovascular disease.     

A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells

Activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been implicated in the induction of apoptosis in a variety of systems [1] [2] [3] [4] [5] [6] [7] [8]. BAF3 cells are pre-B cells that undergo apoptosis following IL-3 withdrawal or ceramide treatment [9] [10]. JNK/SAPK in BAF3 cells is stimulated by ceramide and also during cell proliferation in response to IL-3 [11], but its role in the apoptotic response is not clear. We have devised a method of selectively inhibiting JNK/SAPK activity using a dual-specificity threonine/tyrosine phosphatase, M3/6. Expression of this phosphatase in BAF3 cells prevented ceramide stimulation of JNK/SAPK activity but did not affect apoptosis following IL-3 withdrawal or ceramide treatment. IL-3-stimulated proliferation of BAF3 cells expressing the phosphatase was, however, inhibited. Hence JNK/SAPK activation is likely to be involved in the proliferative response of these cells but is not required for apoptosis. Selective ablation by dual-specificity phosphatases should be a general method for determining the functions of specific mitogen-activated kinase pathways.     

Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806-3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.