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1.
J Investig Allergol Clin Immunol ; 32(5): 383-392, 2022 Oct 11.
Article in English | MEDLINE | ID: mdl-34489229

ABSTRACT

BACKGROUND: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. METHODS: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. RESULTS: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. CONCLUSIONS: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins.


Subject(s)
Dander , Dermatitis, Atopic , Allergens , Animals , Cats , Cystatin A , DNA, Complementary , Dogs , Humans , Immunoglobulin E , Interleukin-4 , Mammals/genetics , T-Lymphocytes
2.
J. investig. allergol. clin. immunol ; 32(5): 383-392, 2022. ilus, graf
Article in English | IBECS | ID: ibc-212734

ABSTRACT

Background: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. Methods: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. Results: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from non–cystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. Conclusion: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins. (AU)


Antecedentes: Las reacciones de hipersensibilidad de tipo inmediato y retardado a los alérgenos que están en las mascotas son comunes en las enfermedades atópicas. En este estudio, en pacientes con dermatitis atopica (DA), se analiza la reactividad cruzada con las autoproteínas y su contribución a la enfermedad. Tanto la cistatina A humana como el alérgeno felino Fel d 3 pertenecen a la familia de las cistatinas, una familia de proteínas conservadas evolutivamente. El objetivo del presente estudio fue evaluar la reactividad cruzada entre las cistatinas de mamíferos y analizar la respuestas de las células T a la cistatina en pacientes con DA sensibilizados a la caspa de las mascotas. Métodos: El ADNc que codifica la cistatina de perro se clonó a partir de piel de perro. Se analizaron sueros de 245 pacientes con sensibilización por IgE a la caspa de gato y perro para determinar la unión de IgE a cistatina felina, canina y humana expresada de forma recombinante, respectivamente. De estos 245 pacientes, 141 fueron diagnosticados de DA. Resultados: Se detectó IgE específica frente a cistatina en el 14,7% (36) de los pacientes, de los cuales 19 padecían DA. Dentro de los pacientes con DA, 9 tenían IgE medible contra las tres cistatinas. Los pacientes con DA sensibilizados frente a cistatina no difirieron de los pacientes no sensibilizados con cistatina en términos de gravedad de la enfermedad, edad o niveles totales de IgE. El análisis de citocinas de células T reveló niveles elevados de IL-4 después de la estimulación con cistatina felina y humana. Conclusión: La respuesta humoral sugiere que, además de Fel d 3, la proteína homóloga de perro también podría desempeñar un papel en la alergia. Además, la cistatina humana parece ser capaz de promover una respuesta inmune de tipo 2 en pacientes con DA sensibilizados y, por lo tanto, puede considerarse un autoalérgeno, como se ha propuesto para otras proteínas conservadas evolutivamente. (AU)


Subject(s)
Humans , Animals , Cats , Dogs , Dermatitis, Atopic/etiology , Pets , Cross-Priming , Cystatins/immunology , T-Lymphocytes/immunology , Dermatitis, Atopic/immunology , Enzyme-Linked Immunosorbent Assay , Electrophoresis, Polyacrylamide Gel
4.
Sci Rep ; 9(1): 11210, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31371767

ABSTRACT

Human thioredoxin (hTrx), which can be secreted from cells upon stress, functions in allergic skin inflammation as a T cell antigen due to homology and cross-reactivity with the fungal allergen Mala s13 of the skin-colonizing yeast Malassezia sympodialis. Recent studies have shown that cell wall polysaccharides of Malassezia are detected by the immune system via the C-type lectin receptors Dectin-1 and Dectin-2, which are expressed on myeloid cells. Therefore, this study aimed to investigate a putative interaction between Dectin-1, Dectin-2 and the allergens Mala s13 and hTrx. Stimulation of human monocyte-derived dendritic cells or macrophages with Mala s13 or hTrx resulted in remarkable secretion of IL-1ß and IL-23. Blocking experiments suggest that hTrx induces IL-23 by Dectin-1 binding and IL-1ß by binding to either Dectin-1 or Dectin-2. Regarding Mala s13, Dectin-1 appears to be involved in IL-1ß signaling. Interference of Syk kinase function was performed to investigate downstream signaling, which led to diminished hTrx responses. In our experiments, we observed rapid internalization of Mala s13 and hTrx upon cell contact and we were able to confirm direct interaction with Dectin-1 as well as Dectin-2 applying a fusion protein screening platform. We hypothesize that this cytokine response may result in a Th2/Th17-polarizing milieu, which may play a key role during the allergic sensitization in the skin, where allergen presentation to T cells is accompanied by microbial colonization and skin inflammation.


Subject(s)
Alarmins/immunology , Allergens/immunology , Dermatitis, Atopic/immunology , Fungal Polysaccharides/immunology , Lectins, C-Type/metabolism , Thioredoxins/immunology , Antigen Presentation/immunology , Autoantigens/immunology , Blood Buffy Coat/cytology , Cross Reactions/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Interleukin-1beta/metabolism , Interleukin-23/metabolism , Macrophages/immunology , Macrophages/metabolism , Malassezia/immunology , Monocytes/immunology , Monocytes/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Primary Cell Culture , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Signal Transduction/immunology , Skin/immunology , Skin/microbiology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism
5.
Dev Biol ; 271(1): 119-29, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15196955

ABSTRACT

Retinoic acid (RA) has been identified as a key signal involved in the posteriorization of vertebrate neural ectoderm. The main biosynthetic enzyme responsible for RA signaling in the hindbrain and spinal cord is Raldh2. However, neckless/raldh2-mutant (nls) zebrafish exhibit only mild degrees of anteriorization in the neural ectoderm, compared to full vitamin A deficiency in amniotes and the Raldh2-/- mouse. Here we investigated the role of RA during neuronal development in the zebrafish hindbrain and anterior spinal cord using DEAB, an inhibitor of retinaldehyde dehydrogenases. We show that the nls hindbrain and spinal cord are not fully devoid of RA, since blocking Raldh-mediated RA signaling leads to a more severe hindbrain phenotype than in nls. The anteroposterior distribution of branchiomotor neurons in the facial and more posterior nuclei depends on full RA signaling throughout early and late gastrula stages. In contrast, inhibition of RA synthesis after gastrulation reduces the number of branchiomotor neurons in the vagal nucleus, but has no effect on anteroposterior cell fates. In addition, blockage of RA-mediated signaling not only interferes with the differentiation of branchiomotor neurons and their axons in the hindbrain, but also affects the development of the posterior lateral line nerve.


Subject(s)
Aldehyde Oxidoreductases/physiology , Motor Neurons/physiology , Phenotype , Rhombencephalon/embryology , Signal Transduction/physiology , Zebrafish/embryology , p-Aminoazobenzene/analogs & derivatives , Aldehyde Oxidoreductases/genetics , Animals , Immunohistochemistry , In Situ Hybridization , Morphogenesis , Retinal Dehydrogenase , Tretinoin/physiology
6.
Bioessays ; 23(11): 981-6, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11746213

ABSTRACT

Retinoids play a critical role in patterning, segmentation, and neurogenesis of the posterior hindbrain and it has been proposed that they act as a posteriorising signal during hindbrain development. Until now, direct evidence that endogenous retinoid signalling acts through a gradient to specify cell fates along the anteroposterior axis has been missing. Two recent studies tested the requirement for retinoid signalling in the developing hindbrain through systematic application of a pan-retinoic acid receptor antagonist. They demonstrate a stage-dependent requirement for increasing retinoid signalling activity along the hindbrain that proceeds from anterior to posterior. Together these findings challenge the concept of a stable gradient of retinoic acid across the hindbrain and warrant a re-interpretation of the phenotypes obtained by genetic and nutritional disruption of retinoid signalling in the amniote embryo.


Subject(s)
Body Patterning/physiology , Rhombencephalon/embryology , Signal Transduction/physiology , Tretinoin/metabolism , Animals , Dose-Response Relationship, Drug , Somites/metabolism , Time Factors
7.
Development ; 128(16): 3081-94, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11688558

ABSTRACT

We describe a new zebrafish mutation, neckless, and present evidence that it inactivates retinaldehyde dehydrogenase type 2, an enzyme involved in retinoic acid biosynthesis. neckless embryos are characterised by a truncation of the anteroposterior axis anterior to the somites, defects in midline mesendodermal tissues and absence of pectoral fins. At a similar anteroposterior level within the nervous system, expression of the retinoic acid receptor a and hoxb4 genes is delayed and significantly reduced. Consistent with a primary defect in retinoic acid signalling, some of these defects in neckless mutants can be rescued by application of exogenous retinoic acid. We use mosaic analysis to show that the reduction in hoxb4 expression in the nervous system is a non-cell autonomous effect, reflecting a requirement for retinoic acid signalling from adjacent paraxial mesoderm. Together, our results demonstrate a conserved role for retinaldehyde dehydrogenase type 2 in patterning the posterior cranial mesoderm of the vertebrate embryo and provide definitive evidence for an involvement of endogenous retinoic acid in signalling between the paraxial mesoderm and neural tube.


Subject(s)
Aldehyde Oxidoreductases/genetics , Mesoderm/metabolism , Mutation , Rhombencephalon/metabolism , Aldehyde Oxidoreductases/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Ectoderm/metabolism , Genetic Linkage , Homeodomain Proteins/metabolism , In Situ Hybridization , In Situ Nick-End Labeling , Models, Biological , Models, Genetic , Molecular Sequence Data , Neural Crest/embryology , Notochord/embryology , Phenotype , RNA, Messenger/metabolism , Receptors, Retinoic Acid/metabolism , Retinal Dehydrogenase , Retinoic Acid Receptor alpha , Rhombencephalon/embryology , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/metabolism , Tretinoin/metabolism , Tretinoin/pharmacology , Zebrafish , Zebrafish Proteins/agonists
8.
Immunology ; 103(2): 210-7, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11412308

ABSTRACT

Dendritic cells (DC) are recruited to sites of inflammation for the initiation of immune responses. As the anaphylatoxins C5a and C3a are important mediators of inflammation, we investigated the expression of their receptors (C3aR and C5aR) on human DC. DC were isolated from human skin or generated from purified blood monocytes and were identified by their expression of CD1a or CD83. Freshly isolated or cultured dermal CD1a+ and CD83+ DC bound anti-C5aR and anti-C3aR monoclonal antibodies (mAbs), as detected by flow cytometry. C5a induced calcium fluxes in dermal CD1a+ and CD83+ DC, which could be inhibited by C17/5, an anti-C5a mAb. C3a did not induce calcium fluxes in these cells. Anaphylatoxin receptor expression was down-regulated on dermal DC by adding tumour necrosis factor-alpha (TNF-alpha) to the culture medium. On CD1a+ CD83- cells generated from isolated blood monocytes by culture with 6.25 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 125 U/ml of interleukin-4 (IL-4), expression of both C5aR and C3aR was observed. In these cells, both C5a and C3a induced calcium fluxes. After addition of TNF-alpha to the culture medium, the majority of the CD1a+ cells expressed CD83+. These cells - expressing a phenotype of 'mature DC' - down-regulated the expression of the anaphylatoxin receptors and lost their reactivity to the respective ligands. Our results demonstrate the expression of the anaphylatoxin receptors C5aR and C3aR on human skin-derived DC and blood-derived cells expressing the DC-associated membrane molecule, CD1a. Furthermore, the expression of anaphylatoxin receptors on CD83+ dermal DC is indicative of an intermediate stage of maturation of these cells, which was not observed on in vitro-differentiated CD83+ cells.


Subject(s)
Antigens, CD/metabolism , Dendritic Cells/immunology , Immunoglobulins/analysis , Membrane Glycoproteins/analysis , Membrane Proteins , Receptors, Complement/metabolism , Skin/immunology , Calcium/metabolism , Cell Culture Techniques , Cell Differentiation/immunology , Complement C3a/metabolism , Complement C5a/metabolism , Dendritic Cells/metabolism , Down-Regulation/immunology , Female , Flow Cytometry , Humans , Monocytes/immunology , Receptor, Anaphylatoxin C5a , Tumor Necrosis Factor-alpha/immunology , CD83 Antigen
9.
J Immunol ; 165(11): 6599-605, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11086104

ABSTRACT

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4(+) and CD8(+) blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4(+) or CD8(+) TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR(+) T cells were found at the sites of injection in IFN-beta-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.


Subject(s)
Complement C3a/metabolism , Lymphocyte Activation/immunology , Membrane Proteins , Receptors, Complement/biosynthesis , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Antibodies, Monoclonal/metabolism , Cell Separation , Clone Cells , Dermatitis, Atopic/immunology , Dermatitis, Atopic/metabolism , Dermatitis, Atopic/pathology , Humans , Immunity, Cellular , Immunity, Innate , Interferon Type I/pharmacology , Receptors, Complement/immunology , Receptors, Complement/physiology , Skin/immunology , Skin/metabolism , Skin/pathology
10.
Mech Dev ; 90(2): 299-304, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10640716

ABSTRACT

T-box (tbx) genes constitute a large family of transcriptional regulators involved in developmental patterning processes. In tetrapods, tbx5 has been implicated in specifying forelimb type identity. Here, we report the cloning of the zebrafish tbx5.1 gene and characterise its expression during zebrafish embryogenesis and early larval development of wild type and mutant embryos that affect pectoral fin patterning. tbx5.1 is expressed during development of the heart, the pectoral fins and the eye. Notably, its expression in the lateral plate mesoderm defines a single and continuous region of heart and pectoral fin precursor cells, and constitutes the earliest specific marker for pectoral fin development in the zebrafish.


Subject(s)
Gene Expression Regulation, Developmental , T-Box Domain Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Humans , Mice , Molecular Sequence Data , Sequence Homology, Amino Acid , Tissue Distribution , Zebrafish
11.
Blood ; 94(5): 1717-26, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10477697

ABSTRACT

Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-gamma- or GM-CSF-primed human monocytes can be completely suppressed by preincubation with LPS (from Escherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-gamma-induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.


Subject(s)
Interleukin-12/biosynthesis , Lipopolysaccharides/pharmacology , Monocytes/metabolism , Antiviral Agents/pharmacology , Cells, Cultured , Down-Regulation/drug effects , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interferon-gamma/pharmacology , Monocytes/immunology , RNA, Messenger/biosynthesis
12.
Immunology ; 97(1): 166-72, 1999 May.
Article in English | MEDLINE | ID: mdl-10447728

ABSTRACT

Varying results have been published in the past regarding the reactivity of different leucocyte subpopulations, including neutrophils, monocytes and B lymphocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To better characterize the cellular distribution of C3a receptor (C3aR) expression, monoclonal antibodies against two different epitopes on the third extracellular domain of the human C3aR were generated. Quantification of C3aR as compared with C5aR densities was performed on peripheral blood leucocytes by quantitative indirect immunofluorescence. Eosinophils and basophils expressed similar numbers of C3aR and C5aR molecules/cell. On eosinophils 10 700+/-4500 (mean+/-SD) C3aR and 14 700+/-4100 C5aR were found, whereas basophils carried 8100+/-2100 C3aR and 13 500+/-3800 C5aR. Monocytes expressed approximately six times more C5aR than C3aR molecules on their surface (6000+/-2500 C3aR versus 34 100+/-9300 C5aR molecules) whereas on neutrophils, the expression of C5aR was more than 20 times higher than the expression of C3aR (3100+/-1000 C3aR versus 63 500+/-12 200 C5aR). No C3aR expression was detectable on peripheral blood-derived B lymphocytes and on tonsillar B cells before and after stimulation with interleukin-2/Staphylococcus aureus Cowan strain I. Our findings correspond well with the paucity of data on C3a-induced functional activities in monocytes and neutrophils and suggest that eosinophilic and basophilic granulocytes represent the primary effector cells in the peripheral blood which can be stimulated by C3a.


Subject(s)
Antibodies, Monoclonal/immunology , Leukocytes/immunology , Membrane Proteins , Receptors, Complement/metabolism , Animals , Antigens, CD/metabolism , B-Lymphocytes/immunology , Basophils/immunology , Eosinophils/immunology , Fluorescent Antibody Technique, Indirect , Humans , Monocytes/immunology , Neutrophils/immunology , Rats , Receptor, Anaphylatoxin C5a , Tumor Cells, Cultured
13.
J Immunol ; 162(11): 6763-9, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10352296

ABSTRACT

IL-12 is a key mediator of the immune response, skewing T lymphocytes toward a type 1 cytokine pattern. Priming with IFN-gamma or GM-CSF is required for expression of IL-12p70 by cells in which IL-12 is inducible by bacterial products such as LPS. We here show for the first time that the production of bioactive IL-12 by human monocytes can be significantly suppressed by C5a if applied to IFN-gamma-primed monocytes before LPS stimulation. There was a dose-dependent suppression by IL-12 (p70) on the levels of intracellular cytokine production and cytokine secretion. mRNA studies consistently showed a reduction of IL-12p40 and IL-12p35 expression by stimulation in the presence of C5a. The results of several different experimental approaches suggest that IL-12 down-regulation was not due to endogenous IL-10, IL-4, or PGE2 production induced by C5a. Moreover, stimulation of IFN-gamma-primed monocytes with C5a did not lead to a down-regulation of the CD14 Ag, which is an LPS receptor. These findings show that the anaphylatoxin C5a has the capacity to directly interact with the complex regulation of IL-12.


Subject(s)
Complement C5a/pharmacology , Immunosuppressive Agents/pharmacology , Interferon-gamma/pharmacology , Interleukin-12/antagonists & inhibitors , Interleukin-12/biosynthesis , Lipopolysaccharides/pharmacology , Monocytes/metabolism , Adjuvants, Immunologic/biosynthesis , Cells, Cultured , Dinoprostone/biosynthesis , Dose-Response Relationship, Immunologic , Humans , Interleukin-10/biosynthesis , Interleukin-4/biosynthesis , Monocytes/immunology
14.
Dev Biol ; 195(2): 131-43, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9520330

ABSTRACT

We report the embryonic phenotype of muscleblind (mbl), a recently described Drosophila gene involved in terminal differentiation of adult ommatidia. mbl is a nuclear protein expressed late in the embryo in pharyngeal, visceral, and somatic muscles, the ventral nerve cord, and the larval photoreceptor system. All three mbl alleles studied exhibit a lethal phenotype and die as stage 17 embryos or first instar larvae. These larvae are partially paralyzed, show a characteristically contracted abdomen, and lack striation of muscles. Our analysis of the somatic musculature shows that the pattern of muscles is established correctly, and they form morphologically normal synapses. Ultrastructural analysis, however, reveals two defects in the terminal differentiation of the muscles: inability to differentiate Z-bands in the sarcomeric apparatus and reduction of extracellular tendon matrix at attachment sites to the epidermis. Failure to differentiate both structures could explain the partial paralysis and contracted abdomen phenotype. Analysis of mbl expression in embryos that are either mutant for Dmef2 or ectopically express Dmef2 places mbl downstream of Dmef2 function in the myogenic differentiation program. mbl, therefore, may act as a critical element in the execution of two Dmef2-dependent processes in the terminal differentiation of muscles.


Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila/embryology , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Cell Differentiation , Cell Nucleus/chemistry , Central Nervous System/chemistry , Connectin , DNA-Binding Proteins/genetics , Drosophila/genetics , Ectoderm/chemistry , Gene Expression Regulation, Developmental , Genes, Insect/physiology , Insect Proteins/analysis , MEF2 Transcription Factors , Mesoderm/chemistry , Muscle Fibers, Skeletal/cytology , Muscle Proteins/analysis , Muscle, Skeletal/chemistry , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Mutation , Myogenic Regulatory Factors , Neuromuscular Junction/embryology , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Photoreceptor Cells, Invertebrate/chemistry , RNA, Messenger/analysis , Tendons/embryology , Transcription Factors/genetics
15.
Scand J Immunol ; 47(1): 19-24, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9467653

ABSTRACT

Controversial results have been published in the past regarding the functional reactivity of different cell types to the anaphylatoxin C3a and its degradation product C3a(desArg). To understand better the effects of C3a and C3a(desArg) on human mast cells, the authors performed binding experiments and calcium mobilization studies on the human mast cell line HMC-1 which has been shown previously to express C3a binding sites. For this purpose, functionally active, recombinant C3a (rC3a) was constructed with an 11 amino acid peptide attached to the N-terminus of the molecule. Using a monoclonal antibody (MoAb) against this tag, binding of rC3a to HMC-1 cells could be demonstrated by flow cytometry. Its binding was specific as it could be blocked with serum-derived C3a. In contrast, no binding of rC3a(desArg) to HMC-1 cells was detectable. Recombinant C3a led to a transient mobilization of intracellular calcium [Ca2+]i in HMC-1 which was inhibitable by the C3a-specific MoAb K13/16. No increase of [Ca2+]i was detected when the cells were treated with C3a(desArg). The authors found C3a receptor (C3aR)-specific mRNA in HMC-1 cells indicating that this receptor represents the binding site for C3a on these cells. These results demonstrate a specific binding for C3a but not for C3a(desArg) on cells of the human mast cell line HMC-1. As a consequence, functional activity was restricted to C3a with C3a(desArg) being completely inactive. Therefore, the data strongly suggest that the recently cloned high affinity C3aR which is assumed to represent the binding site for the anaphylatoxin on HMC-1 cells is unresponsive to C3a(desArg).


Subject(s)
Complement C3a/analogs & derivatives , Complement C3a/metabolism , Macrophage-1 Antigen/metabolism , Mast Cells/metabolism , Animals , Calcium/metabolism , Cell Line , Complement C3a/biosynthesis , Complement C3a/pharmacology , Fluorescent Antibody Technique , Humans , Mast Cells/drug effects , Mast Cells/ultrastructure , Mice , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transcription, Genetic
16.
Eur J Immunol ; 27(9): 2317-22, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9341775

ABSTRACT

Controversial results have been published in the past regarding the functional reactivity of monocytes (Mo) and macrophages (M phi) to the anaphylatoxin C3a and its degradation product C3a(desArg). In this study we performed binding and calcium mobilization experiments with recombinant human C3a (rC3a) and rC3a(desArg). Blood Mo displayed non-inhibitable binding of FITC-labeled rC3a (rC3aFITC) but responded to rC3a with a transient release of the intracellular calcium concentration ([Ca2+]i), whereas rC3a(desArg) was completely inactive. In contrast, binding of rC3aFITC to eosinophilic granulocytes and the mast cell line HMC-1 which have been shown previously to express C3a binding sites could be blocked by a monoclonal anti-C3a antibody. The rC3a-induced [Ca2+]i release in blood Mo was pertussis toxin (PTX)-sensitive suggesting the involvement of G-proteins in the signal transduction pathway. Skin-derived Mo/M phi reacted similarly to blood Mo as no specific binding of rC3aFITC to these cells could be demonstrated, whereas an intracellular release of calcium ions in response to the anaphylatoxin was observed. Homologous desensitization to rC3a but not heterologous desensitization to rC5a was detected in further experiments. The functional effect of C3a, but not the unspecific binding of rC3aFITC to blood Mo and skin-derived Mo/M phi could be blocked by the monoclonal anti-C3a antibody. These results suggest the expression of the recently cloned G-protein-coupled receptor for C3a on human blood Mo and skin-derived Mo/M phi. However, the total number of specific C3a binding sites on these cells is distinctly lower as compared to eosinophilic granulocytes and cells of the mast cell line HMC-1. The small number of C3a receptors on Mo/M phi may be masked by a pronounced non-inhibitable binding of rC3aFITC. This binding, however, may contribute to the recently described biological effects of C3a(desArg) on Mo.


Subject(s)
Calcium/physiology , Complement C3a/physiology , Eosinophils/physiology , Membrane Proteins , Monocytes/physiology , Receptors, Complement/physiology , Arginine , Cells, Cultured , Cytosol/metabolism , GTP-Binding Proteins/physiology , Humans , Mast Cells/metabolism , Pertussis Toxin , Recombinant Proteins , Signal Transduction/drug effects , Skin/cytology , Structure-Activity Relationship , Virulence Factors, Bordetella/pharmacology
17.
Development ; 124(21): 4321-31, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9334280

ABSTRACT

We have isolated the embryonic lethal gene muscleblind (mbl) as a suppressor of the sev-svp2 eye phenotype. Analysis of clones mutant for mbl during eye development shows that it is autonomously required for photoreceptor differentiation. Mutant cells are recruited into developing ommatidia and initiate neural differentiation, but they fail to properly differentiate as photoreceptors. Molecular analysis reveals that the mbl locus is large and complex, giving rise to multiple different proteins with common 5' sequences but different carboxy termini. Mbl proteins are nuclear and share a Cys3His zinc-finger motif which is also found in the TIS11/NUP475/TTP family of proteins and is highly conserved in vertebrates and invertebrates. Functional analysis of mbl, the observation that it also dominantly suppresses the sE-Jun(Asp) gain-of-function phenotype and the phenotypic similarity to mutants in the photoreceptor-specific glass gene suggest that mbl is a general factor required for photoreceptor differentiation.


Subject(s)
Drosophila Proteins , Drosophila/genetics , Insect Proteins/genetics , Nuclear Proteins/genetics , Photoreceptor Cells, Invertebrate/embryology , Receptor Protein-Tyrosine Kinases , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Cell Differentiation/genetics , DNA Transposable Elements , DNA-Binding Proteins/genetics , Drosophila/embryology , Drosophila/growth & development , Eye Proteins/genetics , Genes, Lethal , Genes, Suppressor , Membrane Glycoproteins/genetics , Molecular Sequence Data , Mutation , Nuclear Proteins/chemistry , Proto-Oncogene Proteins c-jun/genetics , Receptors, Steroid/genetics , Sequence Homology, Amino Acid , Transcription, Genetic , Vertebrates/genetics
18.
Arch Dermatol Res ; 289(2): 83-6, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9049041

ABSTRACT

The expression of the receptor for the anaphylatoxin C5a on mast cells was studied with three monoclonal antibodies directed to the N-terminal domain of the C5a receptor. Human skin was investigated by immunohistology applied to sequential 2 micron sections of acrylate-embedded tissues. All anti-C5a receptor antibodies stained c-kit+ or tryptase+ cells which were metachromatic after toluidine blue staining in normal human skin. The binding of anti-C5a receptor antibodies was inhibitable by a peptide representing the first 31 amino acids of the C5a receptor. A similar expression of C5a receptors was found on mast cells in chronic psoriatic plaques. However, C5a receptors were not detectable on mast cells in weal and flare reactions or in lesional skin of uticaria pigmentosa. These findings suggest that (1) anti-C5a receptor antibodies directed to the N-terminal domain of the receptor are suitable tools for the identification of mast cells in acrylate-embedded sections of human skin, (2) mast cell activation in weal and flare reactions results in C5a receptor downregulation or receptor blockade and (3) mast cells in urticaria pigmentosa lack a typical marker of normal human skin mast cells.


Subject(s)
Antigens, CD/analysis , Complement C5a , Mast Cells/chemistry , Psoriasis/metabolism , Receptors, Complement/analysis , Skin/metabolism , Urticaria Pigmentosa/metabolism , Antibodies, Monoclonal , Antigen-Antibody Reactions , Case-Control Studies , Drug Hypersensitivity , Humans , Hypersensitivity, Immediate/chemically induced , Immunohistochemistry , Psoriasis/pathology , Receptor, Anaphylatoxin C5a , Reference Values , Tissue Embedding , Urticaria Pigmentosa/pathology
19.
J Immunol ; 157(4): 1729-35, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8759762

ABSTRACT

The expression of the C5aR (CD88) on human epidermal and dermal cells was studied with five anti-C5aR mAb directed to the N-terminal domain of the receptor. All mAb bound to suspended dermal CD117+ mast cells and to dermal CD14+ cells. The binding to CD14+ and CD117+ cells could be blocked by rC5a and by peptide EX-1 representing amino acid residues 1-31 of the C5aR. In acetone-fixed frozen or in paraformaldehyde-fixed, paraffin-embedded tissue, we detected a binding of the Abs to dermal perivascular cells and, additionally, to keratinocytes and dermal epithelial cells that could be blocked by EX-1. Immunoelectromicroscopy revealed a binding of anti-C5aR mAb to desmosomal regions in human epidermis. However, the following results indicate that CD88 mAb cross-react with epithelium in a specific way: 1) the binding to suspended epidermal cells and to the epidermal cell line HaCat could be blocked by EX-1 but not by rC5a; 2) FITC-labeled C5a bound to CD117+ and to CD14+ cells but not to epidermal cells; 3) C5a led to transient calcium fluxes in CD14+ and CD117+ dermal but not in epidermal cells; 4) C5aR mRNA was detectable by reverse transcription PCR in granulocytes but not in keratinocytes or in HaCat. Our results show that CD88 mAb are good tools for the investigation of the C5aR on hemopoietic cells. Results with epithelial cells should be considered with caution, as the binding of CD88 mAb that were raised to a synthetic peptide sequence may be due to a cross-reactivity.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, CD/immunology , Desmosomes/immunology , Lipopolysaccharide Receptors/analysis , Proto-Oncogene Proteins c-kit/analysis , Receptors, Complement/immunology , Skin/immunology , Antigens, CD/biosynthesis , Antigens, CD/genetics , Base Sequence , Epidermal Cells , Epidermis/immunology , Humans , Keratinocytes/immunology , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Receptor, Anaphylatoxin C5a , Receptors, Complement/biosynthesis , Receptors, Complement/genetics , Skin/cytology
20.
Scand J Immunol ; 44(1): 30-6, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8693289

ABSTRACT

The expression of the receptor for the anaphylatoxin C5a (C5aR, CD88) on the human mast cell line HMC-1 was studied with four anti-C5aR monoclonal antibodies directed to the N-terminal domain of the receptor. All antibodies bound to the human mast cell line HMC-1. The binding could be blocked by recombinant C5a and by peptide EX-1 representing amino residues 1-31 on the N-terminal domain of the C5aR. In addition, FITC-labelled C5a bound to HMC-1, and this binding could be blocked by unlabelled C5a or C5aR antibodies. C5aR-specific mRNA was detected in HMC-1 cells by RT-PCR which confirmed the expression of the C5aR gene made by these cells. Lymphocyte-conditioned medium, interferon-gamma or phorbol esters which have been shown to induce a down-regulation of C5aR on myeloid cells did not influence the expression of C5aR on HMC-1. C5a led to a transient mobilization of intracellular calcium in HMC-1 which could be inhibited by pre incubation of C5a with a C5a-specific antibody. In contrast to findings with granulocytes, HMC-1 did not respond to C5a(desArg), confirming previous findings with human skin mast cells. The findings show that (i) although HMC-1 differ from granulocytes in their responsiveness to C5a(desArg), they express similar C5aR and (ii) HMC-1 cells resemble skin mast cells in the expression and function of C5aR and may therefore serve as a model in future studies addressing the biology of this anaphylatoxin receptor on skin mast cells.


Subject(s)
Antigens, CD/biosynthesis , Complement C5a, des-Arginine/physiology , Complement C5a/physiology , Mast Cells/metabolism , Receptors, Complement/biosynthesis , Antibodies, Monoclonal/chemistry , Antigens, CD/genetics , Antigens, CD/immunology , Binding Sites, Antibody , Complement C5a/immunology , Complement C5a/metabolism , Humans , Leukemia, Mast-Cell/genetics , Leukemia, Mast-Cell/immunology , Leukemia, Mast-Cell/metabolism , Mast Cells/drug effects , RNA, Messenger/analysis , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Receptors, Complement/immunology , Tumor Cells, Cultured
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