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Results for “Phenotypic modulation”

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PubMed2006

Macrophage activation and human immunodeficiency virus infection: HIV replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production.

Macrophages are pivotal for the regulation of immune and inflammatory responses, but whether their role in HIV infection is protective or deleterious remains unclear. In this study, we investigated the effect of pro- and anti-inflammatory stimuli on macrophage sensitivity to two different aspects of HIV infection: their susceptibility to infection stricto sensu, which we measured by endpoint titration method, and their ability to support virus spread, which we measured by using an RT activity assay in infection kinetics. We show a partially protective role for pro-inflammatory agents as well as for IL-4. We also illustrate that various different stimuli display differential effects on macrophage susceptibility to HIV and on virus replication that occurs thereafter. On the other hand, HIV replication strongly repressed CD206 and CD163 expression, thus clearly orientating macrophages towards a pro-inflammatory phenotype, but independently of TNF. Taken together, our results emphasize that HIV infection of macrophages sets up inflammation at the cell level but through unexpected mechanisms. This may limit target susceptibility and participate in virus clearance but may also result in tissue damage.

Antigens, CD↗
PubMed1992

Modulation of the antigenic phenotype of human melanoma cells by differentiation-inducing and growth-suppressing agents.

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-beta), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-beta results in an induction and/or increased expression of ICAM-1, HLA Class I antigens and HLA Class II antigens. IFN-beta and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-gamma), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-beta plus IFN-gamma which synergistically but reversibly suppresses HO-1 growth, to induce melanin synthesis or terminal differentiation in HO-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons.

Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN beta) and immune (IFN gamma) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN beta than to IFN gamma and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN beta plus IFN gamma was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN beta and IFN gamma differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN beta plus IFN gamma can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN beta or IFN gamma, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.

Animals↗
PubMedDate not supplied

DRD3 and DAT1 genes in schizophrenia: an association study.

OBJECTIVE: To investigate the role of the dopamine receptor 3 (DRD3) and transporter 1 (DAT1) genes in schizophrenia or in modulating its phenotype. METHODS: a Ser9Gly polymorphism in codon 9 of the DRD3 and a VNTR polymorphism in the DAT1genes were examined in two groups of schizophrenic patients, one of excellent neuroleptic responders (N=42) and one of nonresponders (N=64). A group of healthy volunteers screened for major psychiatric disorders was also included (N=89). In addition, age at onset of psychotic symptoms, attention performance and family loading for schizophrenia spectrum disorders were compared between patients with different genotypes in the DRD3 and DAT1 genes. RESULTS: No significant differences in the allelic distribution of the DRD3 and DAT1 polymorphisms were detected between schizophrenic patients and controls. A trend toward an excess of DRD3 genotype Gly/Gly was observed in neuroleptic nonresponder schizophrenic patients compared to controls (chi(2)=3. 30, df=1, p=0.07). No significant differences in age at onset of psychotic symptoms, attention task performance or family loading for schizophrenia spectrum disorders were observed between groups with different DRD3 and DAT1 genotypes. CONCLUSION: These results do not support the role of either of these genes in increasing susceptibility to schizophrenia or in modulating its phenotype in the studied population.

Adult↗
PubMed1993

In vitro modulation of proliferation and phenotype of resting and mitogen-stimulated bovine mononuclear leukocytes by 1,25-dihydroxyvitamin D3.

Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the proliferation and phenotype of normal bovine peripheral blood mononuclear leukocytes (MNL) were studied in vitro. Resting and pokeweed mitogen (PWM)-stimulated MNL cultures were supplemented with 1.0 nM of 1,25(OH)2D3 at the beginning of the culture period. Leukocytes were removed from 2-, 4-, 6-, 8-, 10-, 12-, and 14-day unsupplemented and 1.25(OH)2D3-supplemented cultures, and were counted and phenotyped using monoclonal antibodies to bovine leukocyte surface antigens. Cell numbers in resting MNL cultures decreased with time and were unaffected by 1,25(OH)2D3 supplementation. The progressive increase in cell numbers in PWM-stimulated MNL cultures was suppressed, but not abolished by 1,25(OH)2D3. Suppression was greatest in PWM-stimulated 6- to 12-day cultures. Cellular composition of resting MNL cultures was unaffected by 1,25(OH)2D3. Pokeweed mitogen-stimulated cultures supplemented with 1,25(OH)2D3 had fewer total T-cells and CD4+ T-cells at 6-14 days. In contrast, numbers of CD8+ T-cells were significantly higher in 1,25(OH)2D3-supplemented cultures at 6, 10, and 14 days. Effects of 1,25(OH)2D3 on CD4+ and CD8+ T-cell populations were manifested by a significant reduction in the CD4+:CD8+ T-cell ratios in 6- to 14-day cultures. Proliferation of IL-2 receptor+ and MHC class II antigen+ cells was also reduced in supplemented 6- to 14-day cultures, indicating events associated with PWM-induced MNL activation were suppressed by 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 modulates the proliferation and differentiation of bovine MNL in vitro. Our results also suggest that changes in plasma or tissue 1,25(OH)2D3 concentrations that occur during the peripartum period and in clinical cases of milk fever may regulate the bovine immune system in vivo.

Animals↗
PubMed1990

Collagen synthesis by cultured rabbit aortic smooth-muscle cells. Alteration with phenotype.

Enzymically isolated rabbit aortic smooth-muscle cells (SMC) in the first few days of primary culture express a 'contractile phenotype', but with time these cells modulate to a 'synthetic phenotype'. Synthetic-state SMC are able to proliferate, and, provided that they undergo fewer than 5 cumulative population doublings, return to the contractile phenotype after reaching confluency [Campbell, Kocher, Skalli, Gabbiani & Campbell (1989) Arteriosclerosis 9, 633-643]. The present study has determined the synthesis of collagen, at the protein and mRNA levels, by cultured SMC as they undergo a change in phenotypic state. The results show that, upon modulating to the synthetic phenotype, SMC synthesized 25-30 times more collagen than did contractile cells. At the same time, non-collagen-protein synthesis increased only 5-6-fold, indicating a specific stimulation of collagen synthesis. Steady-state mRNA levels are also elevated, with alpha 2(I) and alpha 1(III) mRNA levels 30 times and 20 times higher respectively, probably reflecting increased transcriptional activity. Phenotypic modulation was also associated with an alteration in the relative proportions of type I and III collagens synthesized, contractile SMC synthesizing 78.1 +/- 3.6% (mean +/- S.D.) type I collagen and 17.5 +/- 4.7% type III collagen, and synthetic cells synthesizing 90.3 +/- 2.0% type I collagen and 5.8% +/- 1.8% type III collagen. Enrichment of type I collagen was similarly noted at the mRNA level. On return to the contractile state, at confluency, collagen production and the percentage of type I collagen decreased. This further illustrates the close association between the phenotypic state of SMC and their collagen-biosynthetic phenotype.

Animals↗
PubMed1997

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro.

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.

Animals↗
PubMed2006

Induction of parathyroid hormone-related peptide following peripheral nerve injury: role as a modulator of Schwann cell phenotype.

Parathyroid hormone-related peptide (PTHrP) is widely distributed in the rat nervous system, including the peripheral nervous system, where its function is unknown. PTHrP mRNA expression has recently been shown to be significantly elevated following axotomy of sympathetic ganglia, although the role of PTHrP was not investigated. The role of PTHrP in peripheral nerve injury was investigated in this study using the sciatic nerve injury model and dorsal root ganglion (DRG) explant model of nerve regeneration. We find that PTHrP is a constitutively secreted peptide of proliferating Schwann cells and that the PTHrP receptor (PTH1R) mRNA is expressed in isolated DRG and in sciatic nerve. Using the sciatic nerve injury model, we show that PTHrP is significantly upregulated in DRG and in sciatic nerve. In addition, in situ hybridization revealed significant localization of PTHrP mRNA to Schwann cells in the injured sciatic nerve. We also find that PTHrP causes a dramatic increase in the number of Schwann cells that align with and bundle regrowing axons in explants, characteristic of immature, dedifferentiated Schwann cells. In addition to stimulating migration of Schwann cells along the axonal membrane, PTHrP also stimulates migration on a type 1 collagen matrix. Furthermore, treatment of purified Schwann cell cultures with PTHrP results in the rapid phosphorylation of the cAMP response element protein, CREB. We propose that PTHrP acts by promoting the dedifferentiation of Schwann cells, a critical requirement for successful nerve regeneration and an effect consistent with known PTHrP functions in other cellular differentiation programs.

Animals↗
PubMed1993

Modulation of Schwann cell phenotype by TGF-beta 1: inhibition of P0 mRNA expression and downregulation of the low affinity NGF receptor.

The phenotype of a fully differentiated, mature Schwann cell is apparently largely determined by Schwann cell-axon interactions. In vitro, elevation of intracellular cAMP levels in Schwann cells induces a phenotype which resembles that of a mature, i.e., axon-related, Schwann cell. Therefore, an important role for cAMP as a second messenger of axon-Schwann cell interactions in vivo is assumed. However, the effects of cAMP on Schwann cells are not restricted to induction of features of a mature phenotype. For example, elevation of intracellular cAMP levels results also in a markedly increased synthesis of nerve growth factor (NGF) mRNA, which is barely detectable in intact sciatic nerves of adult animals. Furthermore, since cAMP induces myelin gene expression in cultured Schwann cells, additional regulatory mechanisms have to be postulated for the induction and maintenance of a mature non-myelinating Schwann cell phenotype. Here we show that a soluble protein "growth factor" can partially induce a non-myelinating nature Schwann cell phenotype in vitro. Treatment with transforming growth factor beta 1 (TGF-beta 1) results in a marked and rapid downregulation of the low affinity NGF receptor (NGFR) on cultured Schwann cells without induction of P0 gene expression. In contrast, in agreement with previous studies, an increase in P0 mRNA levels and a reduction in NGFR mRNA levels are observed after cAMP elevation. Downregulation of NGFR mRNA after cAMP elevation is much slower when compared with the effect of TGF-beta 1, suggesting the involvement of different intracellular mechanisms. Consistent with this hypothesis, we did not observe an induction of mRNA coding for TGF-beta isoforms after cAMP elevation in cultured Schwann cells which constitutively synthesize TGF-beta 1 mRNA.

Animals↗
PubMed1990

Differential modulation of growth and phenotypic expression of chondrocytes in sparse and confluent cultures by growth factors in cartilage.

The growth-promoting actions of cartilage extracts (CE) on rabbit cultured chondrocytes were studied to assess the role of local acting growth factors in the generation and expansion of highly differentiated cells. In the present study, DNA synthesis and proteoglycan synthesis in the cultured chondrocytes were monitored by flow cytofluorometry and double-isotope autoradiography by using [3H]thymidine and [35S]sulfate. We report here that actions of the same set of growth factors extracted from cartilage evokes differential cellular responses depending upon cell density. Growth factors in the optimal dose of CE (2 micrograms/ml) or epidermal growth factor (EGF, 40 ng/ml) did not reveal such a cell density-dependent effect on cellular proliferation. However, growth factors in CE induced proteoglycan synthesis selectively in nonproliferating and expressing cells in confluent culture.

Animals↗
PubMed1993

Modulation of the enkephalinergic phenotype of rat sympathetic neurons by hormonal and transsynaptic mechanisms.

Most sympathetic neurons contain one or more neuropeptides in addition to catecholamines. Although the regulation of catecholamines has been studied extensively, comparatively little is known about the regulation of neuropeptides. Since glucocorticoids and preganglionic innervation regulate catecholaminergic properties in chromaffin cells, we examined the effects of these factors on a co-localized neuropeptide, leucine enkephalin (L-Enk), in adult rat sympathetic neurons in vivo. Lowered serum glucocorticoid levels as a consequence of bilateral adrenalectomy resulted in a reduction of ganglionic L-Enk content that was reversed by exposure of adrenalectomized animals to the synthetic glucocorticoid, dexamethasone. Surgical denervation of the SCG eliminated L-Enk-IR preganglionic fibers and caused a dramatic increase in the number of L-Enk-IR neurons. Inhibition of the enkephalinergic component of the preganglionic innervation by chronic exposure to the opiate receptor antagonist naloxone increases the number of L-Enk-IR cell bodies and total ganglionic L-Enk content. None of the experimental manipulations noticeably altered the number or distribution of NPY-IR neurons, suggesting that the effects of glucocorticoids and the innervation on ganglionic L-Enk levels were specific and not simply an alteration of the biosynthetic state of the cells. These results demonstrate that circulating glucocorticoids and the preganglionic innervation regulate L-Enk levels in sympathetic neurons. Since both glucocorticoid levels and preganglionic activity are known to be altered by stressful stimuli, acute regulation of sympathetic L-Enk levels may constitute an important component of the autonomic response to stress.

Adrenalectomy↗
PubMed1998

Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.

Hepatocyte growth factor (HGF) is a potent renotropic factor that has been shown to play important roles in kidney development and recovery from acute renal injury. To examine the effects of HGF on renal tubular epithelium, we generated HGF-producing renal epithelial cells by stably transfecting mIMCD-3 and OK cells with an expression plasmid containing human HGF cDNA. Expression of HGF in the transfected cells was confirmed by detection of HGF mRNA by Northern blot analysis and detection of HGF secretion into the conditioned medium by ELISA. HGF-transfected cells exhibited fibroblast-like scattered morphology and increased cell motility. They formed branching tubules when grown in 3-D collagen gel. In addition, HGF-producing cells grew faster than their parental cells, but failed to form colonies in soft agar. These phenotypic changes were inhibited by a specific, neutralizing anti-HGF antibody. Interestingly, both c-met transcript and c-met protein were increased in HGF-transfected cells, suggesting that HGF amplifies its own action via stimulation of c-met expression. Autocrine expression of HGF and c-met in renal epithelial cells also stimulated fibronectin gene expression, which was totally blocked by incubation with a neutralizing anti-HGF but not a pan-specific anti-TGF-beta antibody, suggesting that it is independent of TGF-beta production. Our data demonstrate that HGF as a single factor stimulates renal epithelial cell proliferation, migration, differentiation, and extracellular matrix remodeling, making it uniquely suited to promote renal tubulogenesis during development, as well as to reconstitute tubular integrity following acute injury.

Animals↗
PubMed1998

Neutralizing TNF-alpha activity modulates T-cell phenotype and function in experimental autoimmune uveoretinitis.

Inhibiting TNF-alpha activity prevents tissue destruction without inhibiting retinal T cell infiltration in experimental autoimmune uveoretinitis (EAU) in Lewis rats. To further determine the role of TNF-alpha in autoimmune uveitis we characterized T cells isolated from retinae after treatment with a TNF-alpha antagonist. TNF-alpha activity was neutralized in vivo with a p55 TNF-alpha receptor-Ig fusion protein (sTNFr-Ig), administered 8 and 10 days after induction of EAU with heterologous retinal antigens. Retinal T-cell phenotype expression was examined by flow cytometry with respect to OX22 status (CD45RBlow or CD45RBhigh), activation (OX40 and CD25 expression) and rate of T-cell apoptosis (Annexin V+PI- expression). Lymphocyte reactivity was assessed by proliferation responses and cytokine production to retinal antigens. Despite greater than 40% of CD4+ T cells being activated at the height of disease, the proportion of OX22low expression was reduced and T cells exhibited reduced IFN-gamma and elevated IL-4 production. Retinal T cells maintained antigen-specific proliferation and demonstrated a low apoptotic rate. Although in both animal groups, comparable numbers of T cells were isolated, neutralizing TNF activity suppressed Th1 effector mechanisms protecting against target organ damage.

Animals↗
PubMedDate not supplied

In vitro modulation of the metastatic phenotype. I. Analysis of differentiation forms of the B16 melanoma expressing Met-72 determinants and metastatic activity.

In vitro cultures of a highly metastatic B16 melanoma clone (BL6-10) were found to undergo dramatic changes in morphology and differentiation upon transfer to another culture medium. Specifically, BL6-10 melanoma cells which had been originally selected and adapted for growth in Eagles' Hanks' amino acid supplemented media with 10 per cent newborn calf serum were amelanotic and epitheliod in shape. When these cells were shifted into Dulbecco's modified Eagles medium with 10 per cent fetal calf serum, they became highly melanotic and of spindle/dendritic morphology within 4 days of culture. These morphological changes as well as other parameters were all characteristic of established criteria of melanoma differentiation. Alterations in the differentiation state of our highly metastatic variant, BL6-10, did not result in any change in tumorigenicity but did have profound effects on metastatic potential. All of the morphological and functional characteristics of the differentiated melanoma were found to be reversible by re-plating the cells in their original growth medium and 4 days of in vitro growth. These studies have allowed us to follow and more firmly establish Met-72 antigen expression as a surface marker for metastatic cells of the B16 melanoma, and have provided direct experimental evidence that the less differentiated, Met-72 positive melanoma form is the dominant cell type capable of metastatic potential.

Animals↗
PubMed1990

Modulation of endothelial cell phenotype by cyclic stretch: inhibition of collagen production.

Endothelial cells (EC) are constantly subjected to pulsatile stretch in vivo, but most studies of EC are performed under stationary tissue culture conditions. The aim of this study was to examine the effect of repetitive mechanical stretching on EC collagen production. Bovine EC were seeded in 35-mm flexible-bottom culture wells, allowed to attach for 24 hr, and then subjected to up to 24% elongation cyclic deformation, 3 cycles/min, 10 sec of elongation alternating with 10 sec of relaxation, for 5 days. Twenty-four hours prior to harvesting, serum-free media containing 50 microCi [3H]proline (PRO), an amino acid hydroxylated (OH) in collagen, and 50 micrograms/ml ascorbate were added per well. On Days 1, 3, and 5, the media and cells were collected, precipitated with TCA, sedimented, lyophilized, and analyzed by HPLC for OH-PRO and PRO. The results of this study indicate that EC production of collagen is inhibited with repetitive deformation. Since previous studies have shown that EC proliferation is increased after 3 cycles/min stretching, this supports the theory that collagen gene expression varies inversely with the proliferative state.

Animals↗
PubMed1992

Modulation of the cellular phenotype by integrated adeno-associated virus.

The adeno-associated virus (AAV) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990, Microbiol. Rev. 54, 316-329). To investigate the expression of the AAV rep gene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neo hybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV and neo DNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418R cell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAV rep DNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplification in vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418R by AAV/neo DNA constructs with a deleted rep gene, or by a neo DNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAV rep gene interferes with cellular processes connected with DNA repair and gene amplification.

Animals↗
PubMed1991

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗