Goblet cells are specialized secretory cells endowed with a variety of receptors
coupled to intracellular signalling pathways that regulate the exocytotic machinery. Several models have
been used to identify and characterize
1) neuro-immuno-endocrine agents that regulate the rate of mucus exocytosis,
2) their receptors at the cell surface,
3) their intracellular signalling pathways,
4) their distal effectors, i.e. the molecular mechanisms underlying the exocytotic machinery.
These models fall into three main categories, i.e. explant cultures of the intestinal mucosa,
isolated intestinal cells, and stable goblet cell lines. As this latter model system has contributed a
great deal to our current concepts of mechanisms of mucin secretion by goblet cells, this overview will
focus mainly on it.
MUCOSAL EXPLANT CULTURES
As a first in vitro approach to identify agents that could potentially alter the mucin secretory response from goblet cells, Neutra et al. (1984) have used rabbit mucosal biopsies in explant culture in combination with a morphological/ autoradiographic assay. They found that acetylcholine and cholinomimetic drugs are potent goblet cell secretagogues in normal intestinal tissue. The ultrastructural analysis of the goblet cells stimulated by acetylcholine showed that the rapid secretion of the stored granules was accomplished by sequential fusion of secretory granule membranes with the plasma membrane and with each other, a process referred to as "compound exocytosis".
However the interpretation of the mechanisms involved in the secretory response of goblet cells to secretagogues was hampered by the cellular complexity and heterogeneity of the mucosal explants. To further complicate interpretation of organ culture studies, some transmitters may be present in the mucosa, closely associated with the final target, the mucous cell.
Other studies (Smith & Podolsky 1987) have identified six mucin species resolved by DEAE-cellulose chromatography in cultured mucosal explants of human colon. Interestingly, this model allowed comparison of the mucin profile of normal mucosa with that of mucosal explants from patients with inflammatory bowel disease.
Finally maintaining the integrity of goblet cells in explant cultures remains difficult, as time-course studies of the morphological changes associated with explant culture of adult human colonic mucosa have shown that the loss of mucins from crypt cells was an early modification of the epithelial cells (Senior et al. , 1982).
ISOLATED INTESTINAL CELLS
The rationale for using isolated cells and epithelial sheets for studying the
secretion of mucins is that certain fundamental problems, particularly the elucidation
of the site of action of secretagogues, have been difficult to solve in studies
based on organ culture techniques.
Several methods have been devised for isolating either epithelial sheets or
monodispersed populations of rat (Brasitus 1982, Ahnen et al. 1988) or rabbit
(Kaunitz 1988) colonocytes. There is considerable similarity in the steps involved
in these procedures. Ahnen et al. (1988) emphasized the importance of combining
EDTA chelation and mechanical dissociation of everted colon segments. Functional
activity of the dispersed cells as assessed by linear rates of incorporation
of (3H)-leucine and (3H)-fucose was
found to be maintained for 2.5-3h (Ahnen et al. 1988).
However colonic mucosa represents such a complex mixture of cell types that enrichment of various subpopulations becomes essential for certain studies. Although the isolation of populations of mature and immature rat colonocytes has been achieved by using a sequential dissociation technique (Ahnen et al. 1988), a method for separating highly enriched populations of goblet cells is still needed.
CULTURED CELLS
The ideal model for studying the secretion of mucins from goblet cells would
be a monolayer culture of goblet cells that have retained in vitro the regulatory
mechanisms operative in the same cells "in vivo". Such a model would have several
advantages :
(1) the mucins would be produced in aseptic conditions,
(2) they
would be less contaminated than those produced "in vivo" or in organ culture,
(3) a continuous monolayer culture would provide mucins with stable characteristics
allowing reproducible experiments.
Thus much effort has been devoted in recent
years to the culture of epithelial cells from the human colonic mucosa. Attempts
at culturing disaggregated tissue have generally been unsuccessful.
Due to the difficulty of culturing normal intestinal cells, some human colonic cancer cell lines have
attracted a great deal of research interest (Laboisse 1989). As early as 1984, we reported the isolation
and the characterization of stably differentiated clones (Augeron & Laboisse 1984) from the human colonic
adenocarcinoma cell line HT-29 (Fogh et al. 1975). The development of these clonal cell lines was made
possible by the discovery in our laboratory that the treatment of HT-29 cells with sodium butyrate was
able to cause the emergence of stably differentiated sub-populations from the undifferentiated parental
cells in culture (Augeron & Laboisse 1984).
This observation and the ability to grow these subpopulations as independent clonal cell lines have
made possible the development of a goblet cell line (Cl.16E) which is a very powerful model to study the
regulation of mucus secretion.
Cl.16E cells can be maintained as homogeneous monolayers of goblet cells on microporous membranes in
chambers. In this system the apical culture medium is separated from the basolateral culture medium.
Using this culture system, it is possible to check the polarized distribution of receptors. It is also
possible to evaluate the paracellular permeability of the monolayers by monitoring the electrical
resistance.
In addition, morphological studies are facilitated as filter-grown cells can be routinely processed
in exactly the same way as biopsy samples.
The exocytotic response of goblet cells in vitro can be measured by using a very sensitive and specific
assay, referred to by us as the "electrophoretic assay" (Augeron et al. 1992). In this assay the
secretory glycoproteins are metabolically labelled with 3H-Glucosamine.
Then 3H-mucins are measured as 3H-labeled macromolecules
trapped at the stacker-gel/interface of 3% polyacrylamide gels.
In efforts to identify agents that could potentially interact with goblet cell basolateral membranes to
evoke a mucin exocytotic response, we have considered and tested neurotransmitters and peptides as
candidate secretagogues. Acetylcholine, VIP, neurotensin and its analog neuromedin were agents that
consistently stimulated an exocytotic response from Cl.16E goblet cells.
Combined measurements of intracellular messengers (Ca2+, cAMP) and mucus
exocytosis allowed us to study in detail the signalling pathways coupled to the activation of receptors
in goblet cells (Bou-Hanna et al. 1994).
Inflammatory conditions lead to the local production of high amounts of inflammatory and immune mediators
(cytokines). Among these, extracellular ATP and interleukin-1 (IL-1) have been shown to function as goblet
cell secretagogues. Extracellular ATP elicits mucus secretion from the respiratory mucosa.
The effects of extracellular ATP and ATP analogs on Cl.16E goblet cells grown in chamber cultures are
tested. When applied to the apical medium, ATP elicited a strong and rapid exocytotic response.
This response was mediated by the activation of P2 receptors located at the apical surface of the goblet
cells (Merlin et al. 1994). In vivo, the source of luminal ATP is unclear at the moment, but could be
inflammatory cells that migrate into the intestinal lumen or a spill-over from the tissue because of
leaky "tight junctions". In situ, the consequences of stimulation of the P2 receptors would be massive
secretion of both fluid and mucin from the epithelial surface. These secretions should have a major
cleansing effect, by removing noxious agents, including bacteria, from the surface and therefore may be
part of the defense mechanisms of epithelia.
IL-1 is a proinflammatory cytokine present in the gastrointestinal mucosa, where it is released by lamina propria activated immune cells during inflammation. IL- 1 was recently found to induce mucin release from explant cultures of mouse duodenum (Cohan et al. 1991). The recent identification of specific receptors for IL-1 on several human colonic cell lines including Cl.16E (Jarry et al. 1996) provided the rationale for testing the effect of this cytokine on mucus secretion. Our results showed that IL-1 can directly stimulate mucin exocytosis from a cultured goblet cell line.
CONCLUSION
A great advance has recently been made in our understanding of the biology of mucus secreting cells through the exploitation of differentiated cell lines. These cultured cells however are transformed permanent cell lines. The recent development in our laboratory of a new culture medium allowing maintenance of normal human colonocytes in culture for up to several days without loss of their specialized functions (paper in preparation) now makes it possible to identify and characterize on primary normal intestinal epithelial cells the receptors and signalling systems regulating the mucus exocytotic response.