Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication

Abstract Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.

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Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication

Blood ◽

10.1182/blood.v77.3.461.bloodjournal773461 ◽

1991 ◽

Vol 77 (3) ◽

pp. 461-471 ◽

Cited By ~ 1

Author(s):

S Navarro ◽

N Debili ◽

JP Le Couedic ◽

B Klein ◽

J Breton-Gorius ◽

...

Keyword(s):

In Situ Hybridization ◽

Interleukin 6 ◽

Dot Blot ◽

Autocrine Loop ◽

Megakaryocytic Differentiation ◽

Normal Human ◽

In Vitro Effects ◽

A Minor

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.

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Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes

Blood ◽

10.1182/blood.v81.10.2744.bloodjournal81102744 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2744-2749 ◽

Cited By ~ 1

Author(s):

C Melani ◽

GF Mattia ◽

A Silvani ◽

A Care ◽

L Rivoltini ◽

...

Keyword(s):

In Situ Hybridization ◽

Interleukin 6 ◽

Peripheral Blood ◽

B Lymphocyte ◽

Human Peripheral Blood ◽

Constitutive Expression ◽

Neutrophil Granulocytes ◽

Gm Csf

Human peripheral blood granulocytes were analyzed for expression of interleukin-6 (IL-6) using reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Neutrophil granulocytes from healthy donors were shown to express variable levels of IL-6. This expression was rapidly down-regulated after the removal of the cells from the circulating blood. In vitro culture of neutrophils abolished IL-6 expression, which could be reactivated by addition of GM-CSF to the culture medium. Constitutive expression of IL-6 was instead demonstrated in eosinophil granulocytes purified from normal donors and from a hypereosinophilic patient. In situ hybridization of unstimulated granulocytes confirmed that IL-6 expression occurs both in eosinophils and in neutrophils from peripheral blood. These findings show that granulocytes can actively contribute to cytokine expression in the peripheral blood and suggest their role in the afferent limb of the immune response, since by IL-6 production they might modulate T- and B- lymphocyte functions, granulocyte self-priming, and endothelial interaction.

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In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization

Journal of Virological Methods ◽

10.1016/0166-0934(86)90022-4 ◽

1986 ◽

Vol 14 (3-4) ◽

pp. 195-211 ◽

Cited By ~ 7

Author(s):

M. Oglesbee ◽

D. Jackwood ◽

K. Perrine ◽

M. Axthelm ◽

S. Krakowka ◽

...

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Canine Distemper Virus ◽

Cdna Probe ◽

Canine Distemper ◽

Dot Blot

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Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes

Blood ◽

10.1182/blood.v81.10.2744.2744 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2744-2749 ◽

Cited By ~ 55

Author(s):

C Melani ◽

GF Mattia ◽

A Silvani ◽

A Care ◽

L Rivoltini ◽

...

Keyword(s):

In Situ Hybridization ◽

Interleukin 6 ◽

Peripheral Blood ◽

B Lymphocyte ◽

Human Peripheral Blood ◽

Constitutive Expression ◽

Neutrophil Granulocytes ◽

Gm Csf

Abstract Human peripheral blood granulocytes were analyzed for expression of interleukin-6 (IL-6) using reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Neutrophil granulocytes from healthy donors were shown to express variable levels of IL-6. This expression was rapidly down-regulated after the removal of the cells from the circulating blood. In vitro culture of neutrophils abolished IL-6 expression, which could be reactivated by addition of GM-CSF to the culture medium. Constitutive expression of IL-6 was instead demonstrated in eosinophil granulocytes purified from normal donors and from a hypereosinophilic patient. In situ hybridization of unstimulated granulocytes confirmed that IL-6 expression occurs both in eosinophils and in neutrophils from peripheral blood. These findings show that granulocytes can actively contribute to cytokine expression in the peripheral blood and suggest their role in the afferent limb of the immune response, since by IL-6 production they might modulate T- and B- lymphocyte functions, granulocyte self-priming, and endothelial interaction.

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Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

Protein and Peptide Letters ◽

10.2174/0929866526666191025102902 ◽

2020 ◽

Vol 27 (5) ◽

pp. 432-446

Author(s):

Akiko Yamamoto ◽

Ken-ichiro Matsunaga ◽

Toyoaki Anai ◽

Hitoshi Kawano ◽

Toshihisa Ueda ◽

...

Keyword(s):

In Situ Hybridization ◽

Immunohistochemical Analysis ◽

Protein Characterization ◽

Intermediate Filament Protein ◽

Tail Domain ◽

Animal Cells ◽

Dugesia Japonica ◽

Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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Human neutrophilic and eosinophilic granulocytes display different levels of c-fos proto-oncogene expression: an in situ hybridization study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/35.8.3298425 ◽

1987 ◽

Vol 35 (8) ◽

pp. 837-842 ◽

Cited By ~ 9

Author(s):

H Kreipe ◽

H J Radzun ◽

K Heidorn ◽

C Mäder ◽

M R Parwaresch

Keyword(s):

In Situ Hybridization ◽

Neutrophilic Granulocytes ◽

Blood Monocytes ◽

In Vitro Differentiation ◽

Monocytic Differentiation ◽

Eosinophilic Granulocytes ◽

Fos Expression ◽

High Level

The cellular homologue of the retroviral oncogene v-fos has been shown to be involved in cell differentiation of hematopoietic cells. By use of the human promyelocyte cell line HL-60, several in vitro differentiation studies suggested a selective activation of c-fos during monocytic differentiation of myeloid precursor cells. In contrast to these observations, we found high levels of c-fos mRNA in purified normal human granulocytes, whereas c-fos was only faintly expressed in blood monocytes. In situ hybridization revealed that the high level of c-fos expression is restricted to neutrophilic granulocytes, whereas c-fos transcription is not detectable in eosinophilic granulocytes. These results indicate that in vitro differentiation systems can be misleading and may not reflect the in vivo situation. The high level of c-fos expression in neutrophilic granulocytes may be caused by superinduction due to the reduced capacity for protein synthesis in these cells.

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Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos

Chromosome Research ◽

10.1007/s10577-007-1125-2 ◽

2007 ◽

Vol 15 (3) ◽

pp. 399-408 ◽

Cited By ~ 11

Author(s):

Gianfranco Coppola ◽

Basil Alexander ◽

Dino Di Berardino ◽

Elizabeth St John ◽

Parvathi K. Basrur ◽

...

Keyword(s):

In Situ Hybridization ◽

Chromosome Abnormalities

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Human Immunodeficiency Virus Infection of Early Passage Cervical Epithelial Cultures

International Journal of STD & AIDS ◽

10.1177/095646249300400608 ◽

1993 ◽

Vol 4 (6) ◽

pp. 342-345 ◽

Cited By ~ 5

Author(s):

S L Patrick ◽

T C Wright ◽

H E Fox ◽

H S Ginsberg

Keyword(s):

In Situ Hybridization ◽

Epithelial Cells ◽

Female Genital Tract ◽

Virus Production ◽

Heterosexual Transmission ◽

Early Passage ◽

Epithelial Cultures ◽

Cervical Epithelial Cells

Women are infected with HIV in increasing numbers; the predominant mode of spread is through heterosexual transmission. Little is known regarding the mechanism of HIV transit through the female genital tract. We investigated whether early passaage cervical epithelial cells could be directly infected with HIV-1LAI*. Virus production was measured using the reverse transcriptase (RT) assay and direct assay for syncytia-forming units. In-situ hybridization was performed on infected cervical cell cultures. Immunostaining was carried out using a monoclonal antibody to leukocyte common antigen (LCA). Virus was recovered in the supernatants of all infected cervical cultures. Localization of HIV infection using in-situ hybridization identified rare cells in the population which gave a strong signal. These infected cells had a lymphoid morphology and were also detected using immunostaining for LAC. Cervical epithelial cells were uninfected in this in vitro model; cells in this population which supported viral replication were most likely of the macrophage/monocyte lineage.

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Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development

Development ◽

10.1242/dev.122.3.839 ◽

1996 ◽

Vol 122 (3) ◽

pp. 839-847 ◽

Cited By ~ 1

Author(s):

G.I. Adam ◽

H. Cui ◽

S.J. Miller ◽

F. Flam ◽

R. Ohlsson

Keyword(s):

In Situ Hybridization ◽

First Trimester ◽

Paternal Allele ◽

Monoallelic Expression ◽

Hybridization Technique ◽

Placental Development ◽

Foetal Development ◽

Biallelic Expression ◽

Normal Human

Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra-embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo.

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