Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

Download Full-text

Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00054.2004 ◽

2004 ◽

Vol 287 (5) ◽

pp. L1056-L1065 ◽

Cited By ~ 10

Author(s):

D. L. H. Poelma ◽

L. J. Zimmermann ◽

W. A. van Cappellen ◽

J. J. Haitsma ◽

B. Lachmann ◽

...

Keyword(s):

Alveolar Macrophages ◽

Divalent Cations ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Cells ◽

Alveolar Type Ii Cells ◽

Lipid Ratio

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.

Download Full-text

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00478.2001 ◽

2002 ◽

Vol 283 (3) ◽

pp. L648-L654 ◽

Cited By ~ 27

Author(s):

D. L. H. Poelma ◽

L. J. I. Zimmermann ◽

H. H. Scholten ◽

B. Lachmann ◽

J. F. van Iwaarden

Keyword(s):

Alveolar Macrophages ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Cells ◽

Alveolar Type Ii Cells ◽

Small Subpopulation

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70–75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.

Download Full-text

Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.10.1527377 ◽

1992 ◽

Vol 40 (10) ◽

pp. 1589-1597 ◽

Cited By ~ 129

Author(s):

W F Voorhout ◽

T Veenendaal ◽

Y Kuroki ◽

Y Ogasawara ◽

L M van Golde ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Alveolar Macrophages ◽

Surfactant Protein ◽

Surfactant Protein D ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Clara Cells ◽

Double Labeling

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.

Download Full-text

Secretory proteins of the lung in rodents: immunocytochemistry.

Journal of Histochemistry & Cytochemistry ◽

10.1177/33.6.3889141 ◽

1985 ◽

Vol 33 (6) ◽

pp. 564-568 ◽

Cited By ~ 31

Author(s):

G Singh ◽

S L Katyal ◽

J M Ward ◽

S A Gottron ◽

M L Wong-Chong ◽

...

Keyword(s):

Serum Proteins ◽

Rabbit Antiserum ◽

Cell Types ◽

Lung Lavage ◽

Clara Cell ◽

Secretory Proteins ◽

Type Ii ◽

Clara Cells ◽

Type Ii Pneumocytes ◽

Surfactant Apoprotein

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.

Download Full-text

Francisella tularensis Replicates within Alveolar Type II Epithelial Cells In Vitro and In Vivo following Inhalation

Infection and Immunity ◽

10.1128/iai.01254-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 1034-1039 ◽

Cited By ~ 76

Author(s):

Joshua D. Hall ◽

Robin R. Craven ◽

James R. Fuller ◽

Raymond J. Pickles ◽

Thomas H. Kawula

Keyword(s):

Epithelial Cells ◽

Francisella Tularensis ◽

Alveolar Epithelial Cell ◽

Cell Types ◽

Alveolar Type ◽

Type Ii ◽

Alveolar Epithelial ◽

Epithelial Cell Lines

ABSTRACT Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells.

Download Full-text

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.9.2426341 ◽

1986 ◽

Vol 34 (9) ◽

pp. 1137-1148 ◽

Cited By ~ 149

Author(s):

S R Walker ◽

M C Williams ◽

B Benson

Keyword(s):

Alveolar Macrophages ◽

Lamellar Body ◽

Alveolar Type ◽

Multivesicular Bodies ◽

Type Ii Cells ◽

Type Ii ◽

Clara Cells ◽

Rat Lung ◽

Lamellar Bodies ◽

Thin Sections

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.

Download Full-text

Degradation of surfactant protein D by alveolar macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.1.l97 ◽

1998 ◽

Vol 274 (1) ◽

pp. L97-L105 ◽

Cited By ~ 20

Author(s):

Qun Dong ◽

Jo Rae Wright

Keyword(s):

Alveolar Macrophages ◽

Pulmonary Surfactant ◽

Degradation Products ◽

Surfactant Protein ◽

Surfactant Protein D ◽

Alveolar Type ◽

Clara Cells ◽

Temperature Dependent ◽

A Cell

Surfactant protein (SP) D is a pulmonary surfactant-associated protein that may function in lung host defense. SP-D is produced by alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells of the airway and is secreted into the air space. Here we investigated whether alveolar macrophages degraded SP-D in vitro. We also examined the effects of SP-A and lipids on SP-D metabolism. The results showed that alveolar macrophages bound and degraded SP-D in a time- and temperature-dependent fashion. After 100 min of incubation, the formation of trichloroacetic acid-soluble degradation products increased 4-fold in the medium and 30-fold in the cells. The degradation of SP-D was via a cell-associated process because SP-D was not degraded when incubated in medium previously conditioned by alveolar macrophages. Gel autoradiography of cell lysate samples after incubation with 125I-labeled SP-D demonstrated an increase in degradation products, further confirming the degradation of SP-D by alveolar macrophages. In addition, the degradation of SP-D was not affected by coincubation with SP-A or surfactant-like liposomes containing either phosphatidylglycerol or phosphatidylinositol. In conclusion, alveolar macrophages rapidly degrade SP-D and may play an important role in SP-D turnover and clearance.

Download Full-text

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180410125850 ◽

2018 ◽

Vol 18 (4) ◽

pp. 246-255 ◽

Cited By ~ 1

Author(s):

Lara Termini ◽

Enrique Boccardo

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Experimental Models ◽

Biological Research ◽

Specific Gene ◽

Specific Cell ◽

Organotypic Cultures ◽

Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

Download Full-text

Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽

10.3390/cells10010066 ◽

2021 ◽

Vol 10 (1) ◽

pp. 66

Author(s):

Rashmita Pradhan ◽

Phuong A. Ngo ◽

Luz d. C. Martínez-Sánchez ◽

Markus F. Neurath ◽

Rocío López-Posadas

Keyword(s):

Intestinal Mucosa ◽

Rho Gtpases ◽

Current Knowledge ◽

Cell Types ◽

Effector Proteins ◽

Special Focus ◽

Specific Cell ◽

Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

Download Full-text