Carl Denef

Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers

BACKGROUND/AIMS Hepatic stellate cells (HSC) are commonly considered the precursor population of septal myofibroblasts (MF) in cirrhosis. We studied the distribution and expression profile of mesenchymal (myo)fibroblast-like populations in fibrotic and cirrhotic liver, in an attempt to elucidate their possible interrelationships. METHODS Fibrotic/cirrhotic livers (from 22 human explants and from two rat models: carbon tetrachloride intoxication, bile duct-ligation) were studied by means of immunohistochemistry (single and double immunostaining) with antibodies raised against desmin, alpha-smooth muscle actin (alpha SMA), glial fibrillary acidic protein (GFAP), neural-cell adhesion molecule (N-CAM), synaptophysin, neurotrophins, neurotrophin receptors and alpha B-crystallin (ABCRYS). RESULTS Septal MF showed the same expression profile as portal MF, in human and rat, being alpha SMA/ABCRYS/brain-derived nerve growth factor/GFAP-expression, with additional N-CAM- and desmin-expression in rat portal/septal MF. Perisinusoidally located HSC stained with all tested markers, MF at the septal/parenchymal interface showed an expression profile, intermediate between the profiles of HSC and portal/septal MF. CONCLUSIONS In advanced fibrosis and in cirrhosis, regardless of cause or species, three distinct mesenchymal (myo)fibroblast-like liver cell subpopulations can be discerned: portal/septal MF, interface MF and perisinusoidally located HSC. The fact that septal MF share more characteristics with portal MF than with HSC might suggest descent.

Production of Interleukin-6 by Folliculo-Stellate Cells of the Anterior Pituitary Gland in a Histiotypic Cell Aggregate Culture System

Reaggregate cell cultures of mouse or rat anterior pituitary were found to produce interleukin-6 (IL-6), a cytokine known for its multiple actions in the immune system. Studies on aggregates prepared from differentially enriched pituitary cell populations revealed the presence of folliculo-stellate (FS) cells to be essential for IL-6 production. Aggregates that contained only hormone-secreting, but no FS cells, failed to produce IL-6. Furthermore, the yield of IL-6 increased with increasing proportions of FS cells present in the aggregates. It is suggested that IL-6 participates in the local regulation of the secretory function of the hypophysis and may constitute a link between events in the immune system and those in the endocrine system.

New perspectives in the function of pituitary folliculo-stellate cells

Classical morphological studies of the folliculo-stellate (FS) cells of the anterior pituitary have suggested that these cells play roles as supporting cells, in metabolism and in macromolecular transport. Over the last 10 years the details of their activity in both trophic and catabolic processes has been clarified, and recent work has demonstrated several transport systems in these cells. Various novel peptides with growth factor or cytokine activity have been identified in FS cells and/or FS cell conditioned media. These recent functional experiments confirm and extend previous morphological and experimental studies, and in addition open new perspectives on the physiological roles of FS cells.

The analysis of heart rate variability in unrestrained rats. Validation of method and results

An experimental setting and software were developed to evaluate cardiac autonomic function in unrestrained rats. Subcutaneously implanted ECG electrodes and an indwelling venous catheter were tunneled to a tail cuff in five rats. The ECG was A/D converted at 1000 Hz. After peak detection, a time series of RR intervals was obtained. Programs for the analysis of heart rate variability (HRV) were implemented in LabVIEW. Statistical properties were determined in the time domain. After cubic spline function curve fitting, resampling at 0.1 s and test for stationarity, power spectral analysis was performed on sampled records of 30 min duration after applying a sliding Hanning window (Welch method: 256 points (duration 25.6 s), 50% overlap and 0.039 Hz resolution). Algorithms were tested with simulated signals consisting of isolated frequency components, which were retrieved at their exact locations. Physiological validation of the system was performed by, beta-adrenergic and cholinergic blockade and by forced breathing at a fixed rate. Measurements were performed on five unrestrained rats under basal conditions. Mean RR was 174.2 +/- 3.6 ms; S.D., 13.3 +/- 4.6 ms; rMSSD, 5.2 + /- 1.2 ms; pNN10, 3.5 +/- 1.9% and pNN5, 18.7 +/- 6.4%. Low (0.19-0.74 Hz) and high frequency (0.78-2.5 Hz) power were determined (and also percent of low to total and high to total): 18.42 +/- 10.74 ms2 (22.9 +/- 6.5%) and 15.66 +/- 5.56 ms2 (19.9 +/- 2.7%), and the ratio low/high: 1.16 +/- 0.39. In conclusion, HRV analysis programs were developed and thoroughly tested through simulations and in vivo, under basal conditions and after pharmacological blockades. Using this software, HRV data from unrestrained rats were obtained.

Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity.

We have previously shown that bioactive interleukin-6 (IL-6) is produced by rat and mouse (anterior) pituitary cells in vitro. Since the amount produced correlated with the presence of S-100-containing folliculostellate (FS) cells, these cells were suggested to be a source of IL-6 in the anterior pituitary (AP) lobe. In the present study we used immunocytochemical techniques to confirm this presumption. Freshly isolated mouse pituitary cells were subjected to immunocytochemical procedures whereby two different (neutralizing) monoclonal antibodies (MAb) against mouse IL-6 (6B4 and 20F3) and a polyclonal antiserum raised against bovine S-100 were used as primary antibodies. Single immunostaining revealed a small portion of mouse pituitary cells (about 6.5%) to be positive for IL-6 immunoreactivity with both antibodies. Importantly, the same proportion of cells was found to be IL-6 positive if only the AP was used as the cell source. About 7.5% of the pituitary cells stained for the presence of S-100 immunoreactivity. Positive staining for IL-6 was also found in pituitary cell samples from 2-day-old monolayer cultures and from redispersed 9-day-old histotypic aggregates, which both secreted bioassayable IL-6. In contrast, no IL-6 staining was found in AtT-20 cells, an established ACTH-secreting tumor cell line of the mouse pituitary which did not secrete bioactive IL-6. The specificity of the IL-6 immunostaining was demonstrated by a total loss of staining when MAb 6B4 was omitted or replaced by irrelevant rat IgG. Conclusively, pre-adsorption of the anti-IL-6 MAb (6B4) with recombinant mouse IL-6 totally abolished staining of pituitary cells. Double immunostaining for IL-6 and S-100 revealed that most if not all of the IL-6-containing pituitary cells were positive for S-100. Few of the S-100-containing cells did not stain for IL-6. These results confirm our previous hypothesis that FS cells, characterized by immunostaining of S-100 protein, contain bioactive and immunoreactive IL-6 and therefore are very likely producers of IL-6 in the AP. Furthermore, our results suggest that IL-6 is implicated in the local regulatory role ascribed to FS cells in the pituitary gland.

The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

Cell-to-cell communication in peptide target cells of anterior pituitary

Publisher Summary This chapter discusses the cell-to-cell communication in peptide target cells of anterior pituitary. It also describes intercellular communication in the anterior pituitary by preparing highly enriched populations of the various pituitary cell types and the influence of one of these cell types on the activity of others in three-dimensional cell cultures (also known as reaggregates, aggregates, or aggregate cell cultures). The existence of regulatory signals from one cell type to another was estimated from the change in secretory response induced by the co-aggregation of both cell types. Secretory responses were followed as a function of time in a perifusion system in that aggregates remained functional for periods up to several weeks. The three-dimensional configuration and tissue just as the organization of the cells in the aggregate most likely favor the expression of these communication systems. As has been shown for brain cells, aggregates express various functional and morphogenetic capabilities that are very similar to the in vivo capabilities of the tissue.

Paracrine Interactions in the Anterior Pituitary

The topographical affinity between certain cell types in rat anterior pituitary as well as the presence of biogenic amines, neuropeptides, growth and tissue factors in specific cell types suggest participation of paracrine control mechanisms in the regulation of anterior pituitary hormone secretion. Due to the recent advances in the separation of pituitary cell types and the development of three-dimensional cell cultures, direct experimental evidence for control by intercellular messengers has become available. The stimulation of PRL release from superfused pituitary cell aggregates by LHRH has been shown to be mediated by gonadotrophs. Gonadotrophs appear to secrete a factor with PRL-releasing activity. Gonadotrophs also modulate the stimulation of PRL release by angiotensin II. Interaction of somatotrophs with an unknown small-sized cell type strongly amplifies the GH response to adrenaline, GRF and VIP. The latter phenomenon requires the permissive action of glucocorticoids. Some of these in vitro observations can be correlated with recently reported in vivo actions of LHRH, PRL and angiotensin II and with pathophysiological changes in the pituitary.

Bioactive peptides in anterior pituitary cells

The anterior pituitary (AP) has been shown to contain a wide variety of bioactive peptides: brain-gut peptides, growth factors, hypothalamic releasing factors, posterior lobe peptides, opioids, and various other peptides. The localization of most of these peptides was first established by immunocytochemical methods and some of the peptides were localized in identified cell types. Although intracellular localization of a peptide may be the consequence of internalization from the plasma compartment, there is evidence for local synthesis of most of these peptides in the AP based on the identification of their messenger-RNA (mRNA). In several cases the release of the peptide from the AP cell has been shown and regulation of synthesis, storage and release have also been described. Because the amount of most of the AP peptides is very low (except for POMC peptides and galanin), endocrine functions are not expected. There is more evidence for paracrine, autocrine, or intracrine roles in growth, differentiation, and regeneration, or in the control of hormone release. To demonstrate such functions, in vitro AP experiments have been designed to avoid the interference of hypothalamic or peripheral hormones. The strategy is first to show a direct effect of the peptide after adding it to the in vitro system and, secondly, to explore if the endogenous AP peptide has a similar action by using blockers of peptide receptors or antisera immunoneutralizing the peptide.

Regulatory activity and topological distribution of folliculo-stellate cells in rat anterior pituitary cell aggregates

An enriched population of cells immunoreactive to antiserum against S-100 protein, a marker of folliculo-stellate (FS) cells in the rat pituitary, was obtained by separation of dispersed pituitary cells from adult female rats by gradient sedimentation at unit gravity. The effect of FS cells on the stimulation and inhibition of prolactin (PRL) and growth hormone (GH) release was studied by coaggregation experiments of the FS cell-enriched population with respectively a lactotroph-enriched and a somatotroph-enriched population from adult female rats. The FS cell population not only attenuated the stimulation of PRL and GH release, but also significantly attenuated the inhibition of PRL release by 10, 30 or 300 nM dopamine (DA), and the inhibition of GH release by 0.1 nM somatostatin (SRIF). The stimulatory action of angiotensin II (AII) on PRL secretion in the presence of DA was also attenuated by the FS cells. Light microscopic evaluation of immunostained semithin sections showed a meshwork of cytoplasmic extensions of FS cells as well as follicular structures in the aggregates. There was no preferential association of FS cells with certain cell types. The permeability of the aggregates to diffusing molecules was tested at the ultrastructural level by the lanthanum hydroxide tracing technique. Lanthanum traced the intercellular gaps over the entire aggregate irrespective of whether the proportional number of FS cells was high or low, indicating that FS cells do not seal off certain areas in the aggregate by the formation of tight junctions. It is suggested that FS cells attenuate the action not only of stimulatory but also inhibitory secretagogues on hormone-secreting pituitary cells. The possible physiological relevance of the present findings is supported by the topological distribution of the FS cells in the aggregates, which closely resembles that of the intact pituitary.