Induction of interleukin-1alpha production in murine Sertoli cells by interleukin-1

In the present study we examined the involvement of interleukin (IL)-1alpha, -1beta, FSH, and lipopolysaccharide (LPS) in the regulation of IL-1alpha and -1beta production by Sertoli cells under in vitro conditions. Sertoli cell cultures from immature mice produced constitutively basal levels of intracellular IL-1alpha. Stimulation of Sertoli cell cultures with LPS (5 microgram/ml) resulted in a maximal production of intracellular IL-1alpha 2 h after the stimulation. Thereafter, these levels decreased but remained significantly higher within 24 h after stimulation than those in control cultures. The effect of LPS on IL-1alpha production was dose dependent. FSH did not show any effect on intracellular IL-1alpha production by Sertoli cells. IL-1alpha could not be detected in supernatants of unstimulated or stimulated Sertoli cell cultures. Sertoli cell cultures stimulated with recombinant IL-1alpha induced optimal intracellular levels of IL-1alpha within 2 h of stimulation. These levels remained high 24 h after stimulation. However, stimulation of Sertoli cell cultures with IL-1beta induced a peak of IL-1alpha production 8 h after stimulation. These levels decreased 24 h after the stimulation but were still found to be significantly higher than those in control cultures. The addition of IL-1 receptor antagonist (IL-1ra) to Sertoli cell cultures did not significantly alter their capacity to produce IL-1alpha. However, the stimulatory effects of recombinant IL-1alpha on IL-1alpha production by Sertoli cell cultures were reversed by the concomitant addition of recombinant IL-1ra. No immunoreactive IL-1beta could be detected in lysates or conditioned media of immature murine Sertoli cells under any of the stimulatory conditions outlined. Our results may suggest the involvement of physiological (IL-1) and pathophysiological factors (LPS) in the regulation of spermatogenesis and spermiogenesis processes and male fertility.     

Recombinant human interleukin-8, but not human interleukin-1beta, induces bovine neutrophil migration in an in vitro co-culture system

A co-culture system was established by culturing a bovine mammary epithelial cell line (MAC-T) and a bovine aortic endothelial cell line on calf tail collagen pre-coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration was investigated using this system. rHIL-8 stimulated bovine neutrophil migration in a dose-dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100 ng/ml of rHIL-8 was used. On the other hand, rHIL-1beta at concentrations up to 100 ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre-incubation with 5 ng/ml of rHIL-1beta in the co-culture system for 4 or 24 h failed to have any effect. These results suggest that IL-8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.     

Gingival and dermal fibroblasts produce interleukin-1 beta converting enzyme and interleukin-1 beta but not interleukin-18 even after stimulation with lipopolysaccharide

Epithelial cells play a critical role in periodontal disease through the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18). However, the role played by fibroblasts is still unclear. The rationale of this study was to throw light on the role of gingival fibroblasts in periodontal disease. We thus investigated the expression of IL-1 beta, IL-18, and ICE mRNA and the secretion of the corresponding proteins by human normal gingival fibroblasts before and after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli. IL-1 beta, IL-18, and ICE mRNA expression was evaluated by RT-PCR. Proteins were analyzed by Western blot and ELISA. We demonstrated that gingival fibroblasts expressed ICE mRNA. Basal expression of ICE was modulated following cell stimulation with lipopolysaccharide (5 mug/ml). However, gingival fibroblasts expressed low levels of IL-1 beta mRNA. The expression was potentiated by LPS. The expression of IL-1 beta mRNA was followed by the secretion of IL-1 beta but not IL-18 protein. Our study suggests that fibroblasts may be involved in the defense against infections via an IL-1 beta-mediated but not an IL-18-mediated mechanism.     

Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation.

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic "cobblestone" morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in beta-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) alpha s- and beta-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins.

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.     

Human endometrial stromal interleukin-1 beta: autocrine secretion and inhibition by interleukin-1 receptor antagonist

Decidualization of human endometrial stromal cells is suppressed by endometrial IL-1 in an autocrine or paracrine manner, indicating that constant suppression of stromal decidualization by IL-1 requires a neutralizing mechanism for IL-1 action to accept embryo implantation. Since production of IL-1ra in human endometrium is reported to be 10- to 30-fold higher than that of IL-1 alpha/beta, we investigated whether endogenous IL-1 beta secreted from human endometrial stromal cells can be inhibited by IL-1ra by using an in vitro decidualization culture. Human stromal cells were cultured with 8-Br-cAMP to induce decidualization, and concentrations of IL-1 beta, IL-8, and prolactin in the culture supernatants were assayed before and after decidualization. There was no significant difference in mean IL-1 beta concentrations measured before and after decidualization. Addition of IL-1ra to endometrial stromal cell cultures strongly inhibited endogenous IL-8 secretion from the cells. Although IL-1 beta showed a biphasic effect on cell proliferation and a suppressive effect on decidualization of stromal cells, these effects were completely inhibited by IL-1ra. The results imply that a high in vivo concentration of IL-1ra in human endometrial tissues may regulate IL-1 effects on decidualization and cell proliferation of human endometrial stromal cells.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.     

Induction of interleukin-3 by interleukin-1 in the absence of other exogenous stimuli

We have previously reported that lipopolysaccharide (LPS) could induce the production of interleukin-3 (IL-3) by mouse spleen cells. In the present study, we show that recombinant human interleukin-1, in the absence of other stimuli, is able to induce the production of IL-3. IL-3 was detected in the supernatants of adult, although neither in young nor in nude mouse splenocytes and was assessed by its capacity to support the growth of the IL-3-dependent FDC-P2 cell line. The presence of IL-3 was antigenically confirmed with a monoclonal anti-IL-3 antibody. Both recombinant IL-1 alpha and IL-1 beta had similar potential for inducing IL-3 production. IL-3 activity was detected in the supernatants of cells cultured in the presence of 100 pg/ml IL-1; maximal IL-3 levels were obtained with 10-30 ng/ml IL-1. Kinetic studies of IL-1-induced IL-3 production indicated that 4-6 days of culture were required for optimal production, whereas 1-2 days were sufficient in cultures stimulated with concanavalin A. Recombinant IL-6 failed to induce significant amounts of IL-3, and TNF alpha induced only weak IL-3 production. GM-CSF but not M-CSF could lead to the appearance of IL-3 in spleen cell culture supernatants. Removal of macrophages decreased the production of IL-3 induced by LPS and GMF-CSF though did not affect the IL-3 production induced by IL-1. This observation suggests that IL-1 production might be an intermediate event in IL-3 production induced by LPS and GM-CSF through the activation of macrophages. IL-3 was detected in culture supernatants of B-cell-depleted splenocytes indicating that T-cells were the source of IL-3. Surprisingly T-cell-depleted populations could also produce IL-3 upon IL-1 stimulation. Preliminary experiments with an autoreactive CD4- CD8- V beta 8+ clone suggested that these cells might also be involved in the described IL-3 production.     

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24h elicited a marked increase in mRNA expression for IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway, although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8

Neovascularization by sprouting angiogenesis is critical for inflammation-mediated tissue remodeling and wound healing. We report here that human polymorphonuclear neutrophils (PMN) stimulated for 1 h with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) released a proangiogenic entity that induced sprouting of capillary-like structures in an in vitro angiogenesis assay. The effect was comparable to the response obtained on stimulation with 100 ng/ml basic FGF. The PMN-mediated response was inhibited by neutralizing antibodies against VEGF or IL-8. As measured by ELISA technique, we found that fMLP-activated PMN (5 x 10(6)/ml) released 78 pg/ml IL-8 and 39 pg/ml VEGF within 1 h after stimulation. IL-8 release was blocked by actinomycin D or cycloheximide, but the inhibitors had no effect on VEGF release, suggesting that IL-8 secretion required de novo synthesis whereas VEGF was secreted from preformed stores. Accordingly, RT-PCR analysis revealed that IL-8 mRNA was upregulated on PMN stimulation, whereas the expression of VEGF mRNA was not affected. Moreover, supernatant derived from activated PMN induced upregulation of endothelial IL-8 mRNA expression, suggesting that release of VEGF and IL-8 from activated PMN may activate a paracrine feedforward mechanism involving endothelial IL-8. Moreover, VEGF-induced upregulation of endothelial IL-8 expression as well as sprouting of capillary-like structures was inhibited by a neutralizing anti-IL-8 antibody. These findings suggest that bacteria-derived tripeptides stimulate human PMN to release VEGF and IL-8, which activate endothelial cells and induce angiogenesis by a paracrine feedforward mechanism involving endothelial IL-8 upregulation.     

Establishment of bovine mammary epithelial cells (MAC-T): An in vitro model for bovine lactation

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in β-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) α_s- and β-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells

Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.