Epithelial Cell-conditioned Medium Research Articles

Basic fibroblast growth factor: a potential mediator of stromal growth in the human prostate.

Studies were undertaken, using isolated prostatic epithelial and stromal cells, to evaluate the role of basic fibroblast growth factor (bFGF) in the regulation of benign prostatic growth. bFGF was detected in lysates, but not the conditioned media, of cultured prostatic epithelial and stromal cells by Western immunoblotting and immunoprecipitation of metabolically labeled proteins. Immunofluorescence analysis of benign human prostate localized the majority of bFGF to the prostatic stroma. In addition, bFGF was a potent stimulator of stromal cell proliferation in vitro, but was not mitogenic to cultured epithelial cells. Further studies demonstrated bFGF receptors (Kd = 258 pM; 61,400 receptors/cell) on stromal cells, but not epithelial cells. Epithelial cell-conditioned medium was mitogenic for stromal cells, suggesting the presence of paracrine interactions. However, bFGF does not appear to be the mediator of this interaction, since the mitogenic effect of epithelial cell-conditioned medium on stromal cells was not significantly reduced by the addition of anti-bFGF. Additional studies showed that concentrated stromal cell-conditioned medium was not mitogenic to cultured stromal cells under serum-free defined conditions, indicating the lack of an external autocine mechanism. These studies demonstrate that bFGF is actively synthesized by isolated prostatic epithelial and stromal cells, but is largely not secreted. Prostatic stroma, but not epithelia, are responsive to the mitogenic effect of bFGF in vitro. However, because of the limited secretion of bFGF by prostatic cells, the mechanism(s) of bFGF-mediated regulation of stromal growth remains unclear.

Human upper airway structural cell-derived cytokines support human peripheral blood monocyte survival: a potential mechanism for monocyte/macrophage accumulation in the tissue.

A central feature of chronic airway inflammation is accumulation of monocyte/macrophages in the tissue. It is well known that circulating monocytes are short-lived cells whereas tissue macrophages are longer-lived cells. One mechanism that may account for accumulation of inflammatory cells includes enhanced survival and/or differentiation of these cells. Recent studies imply that signals released by tissue structural cells may be crucial in these events. To investigate this notion, human blood monocytes were cultured with either culture medium alone as a control, human nasal epithelial cell-conditioned medium (EpCM), or fibroblast-conditioned medium (FCM) for more than 1 wk. Survival of monocytes in medium alone was 17% at day 7, whereas survival of those cultured with 50% of EpCM or FCM was 62% and 64%, respectively. The effect of EpCM and FCM was dose dependent. Preincubation of either conditioned medium (CM) with an antibody against granulocyte/macrophage colony-stimulating factor (GM-CSF) or an antibody against macrophage colony-stimulating factor (M-CSF) resulted in a partial abrogation of the survival-enhancing effect, to an average of 50% and 30%, respectively. Complete inhibition was obtained by preincubation of the CM with a combination of both antibodies. The effect of CM represented true survival because CM only induced a low profile of [3H]thymidine incorporation and, furthermore, less than 0.3% of the cells cultured with CM underwent DNA synthesis as assessed by autoradiography. In addition, ultrastructural observations demonstrated that most monocytes cultured with either CM but not with control culture medium assumed ultrastructural features of macrophages by day 8 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone‐related protein production by primary cultures of mammary epithelial cells

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult non-neoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/10(5) cells x 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)-beta, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.

A bronchial epithelial cell-derived factor in asthma that promotes eosinophil activation and survival as GM-CSF.

We have examined the in vitro interaction between bronchial epithelial cells and eosinophils derived from five asthmatics by determining the effect of epithelial cell-conditioned medium on the survival and activation of peripheral blood eosinophils. The supernatants of epithelial cells from six normal donors were used as control. The asthmatic epithelial cell-conditioned medium significantly increased the survival of eosinophils cultured for 3 (P less than 0.025) and 6 (P less than 0.05) days. The incubation of eosinophils with the supernatants of asthmatic epithelial cells for 1 h also increased the generation of superoxide anion and the release of leukotriene C4, triggered by phorbol myristate acetate and calcium ionophore, by more than twofold. The preincubation of asthmatic epithelial cell-conditioned media with saturating concentrations of a mono-specific antiserum against granulocyte-macrophage colony-stimulating factor (GM-CSF) completely abolished their activity, whereas the addition of recombinant human GM-CSF restored it. The supernatants of asthmatic epithelial cells contained 0.88 +/- 0.09 (SD) ng/5 X 10(5) cells immunoreactive GM-CSF, and this amount was significantly greater than that measured in the supernatants of normal epithelial cells (0.21 +/- 0.105, P less than 0.025). Bronchial epithelial cells from asthmatics also expressed increased levels of GM-CSF mRNA when compared with normal epithelial cells. Thus both the synthesis and release of GM-CSF by bronchial epithelial cells are upregulated in asthma, and this may contribute to the persistence of eosinophil infiltration and activation in asthmatic airways.

Bronchial Epithelial Cells Produce Chemotactic Activity for Bronchial Epithelial Cells: Possible Role for Fibronectin in Airway Repair

Bronchial mucosal injury initiates a complex series of repair mechanisms, one of which is reepithelialization of a denuded lumenal surface. This suggests the hypothesis that bronchial epithelial cells, the cells initially affected by bronchial injury, might be able to initiate repair of an injured area by producing a chemotactic activity for intact bronchial epithelial cells. To evaluate this, bronchial epithelial cells were prepared from bovine lung by protease digestion and cultured in medium 199 (M199) with 10% fetal calf serum (FCS) until confluence, after which the cells were rinsed with Hanks' balanced salt solution, and serum-free fresh M199 was added. This conditioned medium was then collected and used to test the chemotactic response of bronchial epithelial cells using a blindwell chamber technique. Target cells for this assay were isolated from airways by protease digestion, grown to confluence in M199 with 10% FCS, and then harvested with trypsin. Bronchial epithelial cell-conditioned medium harvested after 3 days attracted more cells (197.0 +/- 5.7 cells/10 high power fields) than did M199 without FCS alone (4.3 +/- 0.9) (p less than 0.01). Checkerboard analysis showed that the migration was chemotactic. The chemotactic activity was nondialyzable, pepsin-labile, acid-stable, heat-labile, and lipid-inextractable. The chemotactic activity accumulated in the culture medium with time. The addition of 25 micrograms/ml of cycloheximide inhibited this accumulation. Column chromatography with Sephadex G-150 revealed a single peak of chemotactic activity in the high molecular weight range. The chemotactic activity was bound to gelatin-Sepharose 4B and was eluted with 6 M urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Immature human thymocytes can be driven to differentiate into nonlymphoid lineages by cytokines from thymic epithelial cells.

The signals and cellular interactions required for hematopoietic stem-cell commitment to the T lineage are unknown, yet are central to understanding the early stages of normal T-cell development. To study the differentiative capacity of T-cell precursors, we isolated CD4-, CD8-, surface(s) CD3- thymocytes from postnatal human thymuses and determined their capacity to differentiate into lymphoid and nonlymphoid lineages in vitro. We found that CD4-, CD8-, sCD3- thymocytes, which differentiated in the presence of T-cell conditioned medium plus interleukin 2 into T cells expressing the gamma delta receptor for antigen, were capable of differentiating into myeloid or erythroid lineages in the presence of either 5637 bladder carcinoma cell line conditioned medium plus recombinant human erythropoietin or human thymic epithelial cell conditioned medium. Thymic epithelial cell conditioned medium was as effective as 5637 supernatant plus erythropoietin in inducing myeloerythroid differentiation in the CD4-, CD8-, sCD3- thymocytes. Sixty-eight +/- 14% of CD4-, CD8-, sCD3- thymocytes underwent nonlymphoid differentiation within 4 days in culture with 5637 supernatant plus erythropoietin. Twenty-six +/- 4% of freshly isolated CD4-, CD8-, sCD3- cells were CD34+, and clonal granulocyte/macrophage, granulocyte/erythrocyte/monocyte/megakaryocyte, and T-cell progenitors were found in both CD34+ and CD34- subsets of CD4-, CD8-, sCD3- thymocytes. Thus, cells within the human CD4-, CD8-, sCD3- thymocyte subset can give rise to gamma delta+ T cells as well as to cells of myeloerythroid lineages. Moreover, CD34+, CD4-, CD8-, sCD3- cells can give rise to clonal T-cell progenitors as well as to clonal myeloid progenitors.

Bronchial epithelial cells produce lung fibroblast chemotactic factor: fibronectin.

The interaction between the epithelial cells and the subjacent mesenchymal cells in the airway is thought to play a major role during tissue repair after airway injury and lung morphogenesis. To evaluate this interaction, we cultured human lung fibroblasts, and bovine and human bronchial epithelial cells, and determined that bronchial epithelial cell-conditioned medium has a chemotactic activity for lung fibroblasts. This activity had the characteristics of protein: it was nondialyzable, heat-labile, pepsin-labile, acid-stable, and lipid-inextractable. Molecular sieve chromatography on Sephadex G-150 and affinity chromatography on gelatin-Sepharose revealed that there was one peak of chemotactic activity in high molecular weight range, which bound to gelatin, thus suggesting that the chemotactic factor might be fibronectin. Production and secretion of fibronectin into the culture media were demonstrated by biosynthetic incorporation of radioactive amino acid into fibronectin followed by immunoprecipitation on SDS-PAGE and autoradiography. Release into the culture medium was confirmed by ELISA. The identity of fibronectin as the chemotactic activity was confirmed by the addition of antifibronectin antibody to the conditioned medium, which inhibited chemotaxis in dose-dependent manner. Thus, bronchial epithelial cells produce fibronectin which can function as a chemotactic factor for lung fibroblasts. This production of fibronectin by bronchial epithelial cells may play an important role in regulating interaction between the bronchial epithelial cells that line the lumenal surface of the bronchial epithelial wall and the mesenchymal fibroblasts that underlie the bronchial epithelial basement membrane.

The embryonic thymus produces chemotactic peptides involved in the homing of hemopoietic precursors

During ontogeny, T cell precursors must colonize the thymus to acquire immunocompetency. Using migration assays, a chemotactic activity was detected in conditioned media from avian embryonic thymic epithelial cells. The responding cells were shown to acquire T lymphocyte markers after homing into the thymus. Absorption experiments demonstrated surface receptors for the chemotactic substance on these hemopoietic precursors, which were not found on thymus-derived lymphocytes. Two peaks of chemotactic activity in the 1 kd–4 kd molecular weight range were detected after fractionation of thymic epithelial cell-conditioned medium. One of these activities was retained after heating to 95°C but was destroyed after proteolytic treatment. Thus chemotactic peptides may be responsible for the thymic recruitment of the first hemopoietic precursors and may also be involved in the renewal of these precursors throughout adult life.

Retinal pigment epithelial cells release a chemoattractant for astrocytes.

Astrocyte migration was studied using a modified Boyden chamber. Astrocytes migrated in response to a concentration gradient (chemotaxis) of retinal pigment epithelial cell-conditioned medium. In contrast, smooth muscle cell- and corneal fibroblast-conditioned media stimulated random migration (chemokinesis) of astrocytes but not chemotaxis. Furthermore, retinal pigment epithelial cell-conditioned medium stimulates only random migration of smooth muscle cells and corneal fibroblasts. The chemotactic factor produced by retinal pigment epithelial cells appears to have a molecular weight between 10,000 and 30,000 determined by ultrafiltration. Activity is lost after boiling but is stable, with a pH between 2 and 7. These results suggest that in proliferative vitreoretinopathy, retinal pigment epithelial cells on the retina and within the vitreous cavity may release a chemoattractant that stimulates astrocytes to migrate into the vitreous cavity.