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Biomedical subjects

A F Lopez

Publications and source records attributed to A F Lopez.

At least 19 recordsLinked to original sources
PubMed2001

GM-CSF binding to its receptor induces oligomerisation of the common beta-subunit.

The stoichiometry of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor complex is still unresolved. We have utilised a sensitive, functional assay for receptor homodimerisation to show that GM-CSF induces dimerisation of the common signalling subunit, hbeta(c). We generated a chimeric cytokine receptor in which the extracellular and transmembrane domains of hbeta(c)are fused to the cytoplasmic domain of erythropoietin receptor (EPO-R). Given that to induce EPO-R activation and mitogenic signalling there is a requirement for formation of a specific homodimeric complex, we reasoned that the cytoplasmic domain of EPO-R could be utilised as a highly sensitive reporter for functional homodimer formation. We show that, in the presence of a cytoplasmically truncated GM-CSF alpha-subunit, the hbetac-EPO receptor chimera transduces a mitogenic signal in BaF-B03 in response to GM-CSF. This is consistent with formation of a hbeta(c)homodimer following GM-CSF binding and implies that ligand stimulation induces formation of a higher order complex that contains the hbeta(c)homodimer.

Animals↗
PubMed2001

Structural and functional hot spots in cytokine receptors.

The activation of cytokine receptors is a stepwise process that depends on their specific interaction with cognate cytokines, the formation of oligomeric receptor complexes, and the initiation of cytoplasmic phosphorylation events. The recent determination of the structure of extracellular domains of several cytokine receptors allows comparison of their cytokine-binding surfaces. This comparison reveals a common structural framework that supports considerable diversity and adaptability of the binding surfaces that determine both the specificity and the orientation of subunits in the active receptor complex. These regions of the cytokine receptors have been targeted for the development of specific agonists and antagonists. The physical coupling of signaling intermediates to the intracellular domains of their receptors plays a major role in determining biological responses to cytokines. In this review, we focus principally on the receptors for cytokines of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family and, where appropriate, compare them with related cytokine receptors. Several paradigms are beginning to emerge that focus on the ability of the extracellular portion of the cytokine receptor to recognize the appropriate cytokine and on a phosphorylated motif in the intracellular region of the GM-CSF receptor that couples to a specific signaling pathway.

Amino Acid Motifs↗
PubMed2001

New approaches in the treatment of asthma.

Asthma is a common and complex inflammatory disease of the airways that remains incurable. Current forms of therapy are long term and may exhibit associated side-effect problems. Major participants in the development of an asthma phenotype include the triggering stimuli such as the allergens themselves, cells such as T cells, epithelial cells and mast cells that produce a variety of cytokines including IL-5, GM-CSF, IL-3, IL-4 and IL-13 and chemokines such as eotaxin. Significantly, the eosinophil, a specialized blood cell type, is invariably associated with this disease. The eosinophil has long been incriminated in the pathology of asthma due to its ability to release preformed and unique toxic substances as well as newly formed pro-inflammatory mediators. The regulation of eosinophil production and function is carried out by soluble peptides or factors. Of these IL-5, GM-CSF and IL-3 are of paramount importance as they control eosinophil functional activity and are the only known eosinophilopoietic factors. In addition they regulate the eosinophil life span by inhibiting apoptosis. While one therapeutic approach in asthma is directed at inhibiting single eosinophil products such as leukotrienes or single eosinophil regulators such as IL-5, we believe that the simultaneous inhibition of more than one component is preferable. This may be particularly important with eosinophil regulators in that not only IL-5, but also GM-CSF has been repeatedly implicated in clinical studies of asthma. The fact that GM-CSF is produced by many cells in the body and in copious amounts by lung epithelial cells highlights this need further. Our approach takes advantage of the fact that the IL-5 and GM-CSF receptors (as well as IL-3 receptors) utilize a shared subunit to bind, with high affinity, to these cytokines and the same common subunit mediates signal transduction culminating in all the biological activities mentioned. By generating the monoclonal antibody BION-1 to the cytokine binding region of the common subunit (betac) we have shown that the approach of inhibiting IL-5, GM-CSF and IL-3 binding and the resulting stimulation of eosinophil production and function with a single agent is feasible. Furthermore we have used BION-1 as a tool to crystallize and define the structure of the cytokine binding domain of betac. This knowledge and this approach may lead to the generation of novel therapeutics for the treatment of asthma.

Animals↗
PubMed2000

Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist.

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)

Antibodies, Monoclonal↗
PubMed2000

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5.

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.

Amino Acid Motifs↗
PubMed2000

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity.

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (hbetac) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3Ralpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type hbetac exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type hbetac, despite failing to undergo IL-3-stimulated hbetac tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and hbetac tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

Animals↗
PubMed2000

Site-specific serine phosphorylation of the IL-3 receptor is required for hemopoietic cell survival.

In the hemopoietic compartment, IL-3, GM-CSF, and IL-5 receptors are major transducers of survival signals; however, the receptor-proximal events that determine this vital function have not been defined. We have found that IL-3 stimulation induces phosphorylation of Ser-585 of beta(c). This promotes the association of phospho-Ser-585 of beta(c) with 14-3-3 and the p85 subunit of PI 3-K. Mutation of Ser-585 specifically impairs the PI 3-K signaling pathway and reduces cell survival in response to IL-3. These results define a distinct IL-3 receptor-mediated survival pathway regulated by site-specific receptor serine phosphorylation and 14-3-3 binding and suggest that this novel mode of signaling may be utilized by disparate transmembrane receptors that have as a common theme the transduction of survival signals.

14-3-3 Proteins↗
PubMed1999

Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF.

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

14-3-3 Proteins↗
PubMed1999

Simultaneous antagonism of interleukin-5, granulocyte-macrophage colony-stimulating factor, and interleukin-3 stimulation of human eosinophils by targetting the common cytokine binding site of their receptors.

Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific alpha chain and a shared subunit (beta(c)). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor alpha chains is the first step in receptor activation, it is the recruitment of beta(c) that allows high-affinity binding and signal transduction to proceed. Thus, beta(c) is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of beta(c). BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of (125)I-IL-5, (125)I-GM-CSF, and (125)I-IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of beta(c). Interestingly, epitope analysis using several beta(c) mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of beta(c), suggesting that ligand contact with beta(c) is a prerequisite for recruitment of beta(c), receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

Animals↗
PubMed1999

The functional basis of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 receptor activation, basic and clinical implications.

The cytokines granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 have overlapping activities on cells expressing their receptors. This is explained by their sharing a receptor signal transduction subunit, beta c. This communal signaling subunit is also required for high affinity binding of all three cytokines. Therapeutic approaches attempting to interfere or modulate haemopoietic cells using cytokines or their analogues can in some instances be limited due to functional redundancy amongst cytokines using shared receptor signaling subunits. Therefore, a better approach would be to develop therapeutics against the shared subunit. Studies examining the GM-CSF, IL-3 and IL-5 receptors have identified the key events leading to functional receptor activation. With this knowledge, it is now possible to identify new targets for the development of a new class of antagonist that blocks the biological activity of all the cytokines utilizing beta c. This approach may be extended to other receptor systems such as IL-4 and IL-13 where receptor activation is dependent on a common signaling and binding subunit.

Animals↗
PubMed1998

Effect of GM-CSF on HIV-1 replication in monocytes/macrophages in vivo and in vitro: a review.

Cells of macrophage lineage constitute the main cellular target of Human Immunodeficiency Virus type 1 (HIV-1). Replication of HIV-1 in monocyte/macrophages is generally augmented by factors promoting their differentiation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key regular of the differentiation of cells of macrophage lineage. The effects of GM-CSF on HIV-1 replication in vitro are still controversial. Most of the published studies suggest that GM-CSF upregulates HIV-1 expression in both primary cultured macrophages and promonocytic cell lines. There have also been reports demonstrating that GM-CSF does not affect HIV-1 replication in cells of macrophage lineage or that GM-CSF can actually suppress HIV-1 expression. In vivo, GM-CSF administrated to HIV-positive patients at any stage of disease, without any antiretroviral therapy, appears to increase HIV-1 activity. The possible mechanism by which GM-CSF might affect HIV-1 replication in macrophages remains unclear.

Animals↗
PubMed1998

Identification of a Cys motif in the common beta chain of the interleukin 3, granulocyte-macrophage colony-stimulating factor, and interleukin 5 receptors essential for disulfide-linked receptor heterodimerization and activation of all three receptors.

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific alpha chains with the common beta subunit (betac) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in betac to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in betac but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 betac mutants retained their ability to form non-disulfide-linked dimers with IL-3Ralpha, substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all betac mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that betac mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of betac in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of betac, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of betac. Sequence alignment of betac with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse beta chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

Amino Acid Sequence↗
PubMed1998

Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors.

The process of ligand binding leading to receptor activation is an ordered and sequential one. High-affinity binding of GM-CSF, interleukin 3 (IL-3), and IL-5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src-homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM-CSF/IL-3/IL-5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM-CSF, IL-3, or IL-5 receptors, and C) how the presence of novel phosphotyrosine-independent signaling motifs within a specific cytoplasmic domain of betaC may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all-encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM-CSF/IL-3/IL-5 receptor subfamily from other cytokine receptors.

Animals↗
PubMed1998

IL-3, IL-5, granulocyte-macrophage colony-stimulating factor receptor alpha-subunit, and common beta-subunit expression by peripheral leukocytes and blood dendritic cells.

BACKGROUND: IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors consist of cytokine-specific alpha-subunits, which associate with a shared signalling common beta-subunit (beta(c)) to form a high-affinity complex. The expression of IL-3, IL-5, and GM-CSF is upregulated in atopic inflammation, and these cytokines are thought to contribute to pathology through mechanisms that include eosinophil activation. OBJECTIVE: We sought to examine the distribution of receptor expression between cells relevant to allergic inflammation from individual subjects and to compare atopic and nonatopic individuals. METHODS: Peripheral blood was obtained from atopic and nonatopic volunteers. Cytokine-receptor expression was examined by flow cytometry with monoclonal antibodies specific for alpha-subunits and beta(c) in combination with phenotypic markers for eosinophils, basophils, neutrophils, dendritic cells, monocytes, and T cells. RESULTS: Using a ligand-independent system, we confirmed the cellular distribution of IL-5Ralpha, IL-3Ralpha, and GM-CSFRalpha. IL-3Ralpha and GM-CSFRalpha were detected on high-affinity IgE receptor blood dendritic cells. Beta(c) expression was detected on basophils, eosinophils, neutrophils, and, at low levels, on monocytes and dendritic cells. There was intense staining of basophils for IL-3Ralpha relative to IL-5Ralpha, GM-CSFRalpha, and beta(c), whereas eosinophil-staining intensity was similar for IL-3Ralpha, IL-5Ralpha, GM-CSFRalpha, and beta(c). There were no significant differences between atopic and nonatopic subjects in cytokine-receptor staining. CONCLUSION: IL-3Ralpha and GM-CSRalpha are shown on a newly defined population of Fc(epsilon)RI-high dendritic cells. The intense staining of basophils for IL-3Ralpha, relative to that of IL-5Ralpha and GM-CSFRalpha, is in contrast to eosinophils from the same subjects and may explain the higher sensitivity of basophils to IL-3 compared with IL-5 and GM-CSF. We found no evidence for downregulation of receptor expression in atopic compared with nonatopic subjects, suggesting that these receptors remain accessible as potential targets for therapeutic intervention in atopic allergic disease.

Adolescent↗
PubMed1997

Inhibition of granulocyte-macrophage colony-stimulating factor prevents dissemination and induces remission of juvenile myelomonocytic leukemia in engrafted immunodeficient mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor alpha (TNFalpha) have been implicated in the pathogenesis of the fatal childhood disease termed juvenile myelomonocytic leukemia (JMML). We used a severe combined immunodeficient/nonobese diabetic (SCID/NOD) mouse model of JMML and examined the effect of inhibiting these cytokines in vivo with the human GM-CSF antagonist and apoptotic agent E21R and the anti-TNFalpha monoclonal antibody (MoAb) cA2 on JMML cell growth and dissemination in vivo. We show here that JMML cells repopulated to high levels in the absence of exogeneous growth factors. Administration of E21R at the time of transplantation or 4 weeks after profoundly reduced JMML cell load in the mouse bone marrow. In contrast, MoAb cA2 had no effect on its own, but synergized with E21R in virtually eliminating JMML cells from the mouse bone marrow. In the spleen and peripheral blood, E21R eliminated JMML cells, while MoAb cA2 had no effect. Importantly, studies of mice engrafted simultaneously with cells from both normal donors and from JMML patients showed that E21R preferentially eliminated leukemic cells. This is the first time a specific GM-CSF inhibitor has been used in vivo, and the results suggest that GM-CSF plays a major role in the pathogenesis of JMML. E21R might offer a novel and specific approach for the treatment of this aggressive leukemia in man.

Adolescent↗
PubMed1997

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor exists as a preformed receptor complex that can be activated by GM-CSF, interleukin-3, or interleukin-5.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor alpha chain (GMR alpha) and to the common beta chain of the GM-CSF, IL-3, and IL-5 receptors (beta(c)) immunoprecipitated both GMR alpha and beta(c) from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMR alpha and beta(c), as shown by the ability of soluble beta(c) to associate with membrane-anchored GMR alpha or soluble GMR alpha. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMR alpha-associated beta(c). These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

Animals↗
PubMed1997

Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro.

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-alpha (TNF-alpha), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-alpha inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4, and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-alpha was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-alpha neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-alpha to accelerate apoptosis was lost if neutrophils were primed with 1 micromol/L PAF or aged for 6 hours before TNF-alpha addition. The TNFR55-selective TNF-alpha mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-alpha. Although the TNFR75-selective mutant (D143F) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-alpha-stimulated apoptosis. Hence, TNF-alpha has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.

Antibodies, Monoclonal↗