Purification of morphologically and functionally intact human basophils to near homogeneity

Haisch, Karin; Gibbs, Bernhard F.; Körber, H.; Ernst, Martin; Grage-Griebenow, Evelin; Schlaak, M; Haas, Helmut

doi:10.1016/s0022-1759(99)00059-9

Cited by 44 publications

(40 citation statements)

References 17 publications

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“…Basophils were purified using a three-step procedure involving Ficoll density centrifugation followed by elutriation and negative selection using magnetic cell sorting (MACS basophil isolation kit; Miltenyi Biotec). We have previously reported this technique for basophil enrichment and observed that there are no functional differences between purified and unpurified basophils using this negative selection procedure [19]. Basophils obtained were 95.6 ± 0.6% pure, as determined by Alcian blue staining, with a mean yield of 5.2 ± 0.4 × 10 6 basophils per donor and more than 96% viable cells, as judged by Trypan blue exclusion.…”

Section: Methodsmentioning

confidence: 94%

Differential Role for Mitogen-Activated Protein Kinases in IgE-Dependent Signaling in Human Peripheral Blood Basophils: In Contrast to p38 MAPK, c-Jun N-Terminal Kinase Is Poorly Expressed and Does Not Appear to Control Mediator Release

Gibbs

Wolff

Zillikens

et al. 2005

Int Arch Allergy Immunol

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Background: Exposure of human basophils to allergens results in a rapid secretion of histamine, LTC₄, IL-4 and IL-13, which dominate both the symptomology of allergic diseases and support the underlying Th2/IgE predominance associated with these reactions. The IgE-dependent release of these mediators in basophils crucially involves PI 3-kinase and the subsequent activation of p38 MAPK and ERK1&2. Here, we investigated the role of the third major member of the mitogen activated kinase family, namely the c-Jun amino terminal kinase (JNK), which is rapidly activated following IgE receptor cross-linking in murine mast cells. Methods: Human basophils were highly purified by magnetic cell sorting. The activities of various intracellular signaling components, in basophils that had been stimulated under various conditions, were assessed by Western blotting. Mediator secretions were also determined using either spectrofluorometric analysis (histamine) or ELISA (LTC₄, IL-4 and IL-13). Results: Our results show that while JNK is moderately expressed in human basophils, it is not consistently phosphorylated upon anti-IgE stimulation. Phosphorylation of the transcription factor c-Jun, a downstream target of JNK, was also undetected in contrast to p38 MAPK and ERK1&2, which were clearly activated following anti-IgE stimulation of the cells. Additionally, inhibitors of the JNK pathway failed to prevent basophil mediator release and had no effect on the phosphorylation of p38 MAPK or ERK1&2 at concentrations which were specific for JNK blockade. Conclusions: These data suggest major differences in utilizing various members of the mitogen-activated kinase family in the signal transduction cascade of IgE-receptor-bearing cells.

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Section: Methodsmentioning

confidence: 94%

Differential Role for Mitogen-Activated Protein Kinases in IgE-Dependent Signaling in Human Peripheral Blood Basophils: In Contrast to p38 MAPK, c-Jun N-Terminal Kinase Is Poorly Expressed and Does Not Appear to Control Mediator Release

Gibbs

Wolff

Zillikens

et al. 2005

Int Arch Allergy Immunol

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“…Human basophils were obtained from peripheral blood of healthy donors by using a three-step purification protocol as described before (21). Yield and viability were determined by trypan blue exclusion, and basophil purity was determined by staining cytospin preparations with May-Grünwald stain.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-4-Inducing Principle from Schistosoma mansoni Eggs Contains a Functional C-Terminal Nuclear Localization Signal Necessary for Nuclear Translocation in Mammalian Cells but Not for Its Uptake

et al. 2011

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Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively by the eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates human basophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potential nuclear localization signal (NLS) at the C terminus of IPSE/alpha-1. Here we show that this sequence (125-PKRRRTY-131) is both necessary and sufficient for nuclear localization of IPSE or IPSE-enhanced green fluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclear in the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear and cytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered the protein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescence microscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein added exogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. A mutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrate CHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1 was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium-and temperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. In contrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intact NLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclear functions in host cells.

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“…Human basophils were purified by a previously described three-step procedure (15) and used at a mean purity of 99%. Purified basophils were incubated in round-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) at 37°C in humidified air containing 6% CO 2 at 10 6 basophils/ml in a final volume of 100 l of culture medium (Iscove's modified Dulbecco's medium supplemented with 100 units/ml penicillin G, 50 g/ml transferrin, 5 g/ml insulin, 100 g/ml streptomycin, 10% fetal calf serum).…”

Section: Purification Culture and Stimulation Of Basophilsmentioning

confidence: 99%

Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs

Schramm

Falcone

Gronow

et al. 2003

Journal of Biological Chemistry

175

192

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The eggs of the parasitic trematode Schistosoma mansoni are powerful inducers of a T helper type 2 (Th2) immune response and immunoglobulin E (IgE) production. S. mansoni egg extract (SmEA) stimulates human basophils to rapidly release large amounts of interleukin (IL)-4, the key promoter of a Th2 response. Here we show purification and sequence of the IL-4-inducing principle of S. mansoni eggs (IPSE). Stimulation studies with human basophils using SmEA fractions and natural and recombinant IPSE as well as neutralization and immunodepletion studies using antibodies to recombinant IPSE demonstrate that IPSE is the bioactive principle in SmEA leading to activation of basophils and to expression of IL-4 and IL-13. Regarding the mechanism of action, blot analysis showed that IPSE is an IgE-binding factor, suggesting that it becomes effective via crosslinking receptor-bound IgE on basophils. Immunohistology revealed that IPSE is enriched in and secreted from the subshell area of the schistosome egg. We conclude from these data that IPSE may be an important parasitederived component for skewing the immune response toward Th2.Infection with the parasitic trematode Schistosoma mansoni leads to a pronounced Th2 1 response and to elevated IgE production both in humans and in experimental animals. The definition of parasite-derived products capable of skewing the immune response toward Th2 would not only enhance our understanding of the defense mechanisms involved in helminth infections but may also lead to new insights into the pathogenesis of immediate-type hypersensitivity diseases such as asthma. However, in contrast to our increasing understanding of how pathogen-derived products can initiate Th1-type immune responses, there is so far little detailed knowledge about the nature of the parasite-derived molecule(s) and the underlying mechanisms that trigger and/or amplify a Th2-type reaction. In S. mansoni infection, a critical role in inducing a polarized Th2 response is played by the egg stage of the parasite (1), since a Th2 response and IgE production are only observed after egg deposition or following injection of schistosome eggs (2) or extracts thereof (3) into naive animals. By contrast, the initial larval (schistosomula) and adult worm stages rather induce a response skewed to Th1.It is now firmly established, both in vivo and in vitro, that the cytokine profile present during an immune reaction is an important element in directing the response to Th1 or Th2 and that IL-4 is the key cytokine responsible for biasing the immune reaction toward a Th2 phenotype (4 -7). In the human system, basophils are a prominent source of IL-4 and IL-13; these cells secrete large amounts of IL-4 and IL-13 in response to IgE-receptor cross-linking or activation by a combination of IL-3 and C5a (8, 9). Indeed, human basophils can be viewed as "innate Th2-type" effector cells, since IL-4 and IL-13 are expressed in a very restricted manner without production of any of the cytokines involved in Th1-type immune responses. We therefore ...

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Purification of morphologically and functionally intact human basophils to near homogeneity

Cited by 44 publications

References 17 publications

Differential Role for Mitogen-Activated Protein Kinases in IgE-Dependent Signaling in Human Peripheral Blood Basophils: In Contrast to p38 MAPK, c-Jun N-Terminal Kinase Is Poorly Expressed and Does Not Appear to Control Mediator Release

Differential Role for Mitogen-Activated Protein Kinases in IgE-Dependent Signaling in Human Peripheral Blood Basophils: In Contrast to p38 MAPK, c-Jun N-Terminal Kinase Is Poorly Expressed and Does Not Appear to Control Mediator Release

Interleukin-4-Inducing Principle from Schistosoma mansoni Eggs Contains a Functional C-Terminal Nuclear Localization Signal Necessary for Nuclear Translocation in Mammalian Cells but Not for Its Uptake

Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs

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