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Morphologic and biochemical evidence for a contractile cell network within the rat intestinal mucosa
GASTROENTEROLOGY
1987;92:68-81
Morphologic and Biochemical Evidence for a Contractile Cell Network Within the Rat Intestinal Mucosa NANCY C. JOYCE, MARCY F. HAIRE, and GEORGE Department
of Cell Biology,
Yale University
School
Subepithelial and pericryptal fibroblastlike cells form a two-dimensional network immediately subjacent to the epithelial basal lamina in the small intestine and colon in several mammalian species. Stellate-shaped cells with similar, but not identical characteristics, form a three-dimensional network deep within the villar lamina propria. Electron microscopic studies indicate that these cells (a) contain a putative contractile apparatus, (b) are attached to each other and to apparently organized elements of the extracelluIar matrix by typical adhesive devices, and (c) form gap junctions with each other. Comparative in situ immunoperoxidase localization studies document the presence in these cells offour contraction-associated proteins (smooth muscle isotropomyosin, cyclic guanosine monophosphate-dependent protein kinase, both nonmuscle and smooth muscle isomyosin, and actin) in amounts generally greater than those found in connective tissue fibroblasts, but less than in smooth muscle cells. Taken together, these results strongly suggest a smooth muscle-like, contractile function for these cells and indicate that this cellular network may provide a supportive tonus for the epithelium, as well as provide the force needed for active movement of the villus, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and lacteals of the intestinal villus. A network of fusiform cells, with the general appearance of fibroblasts, subtends the epithelial basal lamina in both the small intestine (l-7) and colon Received February 10, 1986. Accepted June 23, 1986. Address requests for reprints to: Nancy C. Joyce, Ph.D., Department of Cell Biology. Yale University School of Medicine, P.O. Box 3333, 333 Cedar Street, New Haven, Connecticut 06510. This work was supported by National Research Service Award S-T32-GM07223-09 to N.C.J. and National Institutes of Health grant HL17080 to G.E.P. 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50
of Medicine,
E. PALADE
New Haven,
Connecticut
(1,8-11) in several species, including rat (2,3,5,7,10), mouse (1,4), rabbit (6,7,9,11), and humans (8,9). The cells comprising this network have elongated cell bodies and long, attenuated cytoplasmic processes, which closely appose the overlying basal lamina. The number of cells in the network appears to be greatest at the base of the crypts of Lieberkiihn and to decrease toward the surface of the villus, although this decrease is more pronounced in the small intesIn addition, steltine than in the colon (1,4,6,8,11). late-shaped cells with large, oval nuclei, scant perinuclear cytoplasm, and long cytoplasmic processes have been reported (3) to form a cellular network deep within the villar lamina propria of the rat small intestine. The foot processes of these cells closely appose the cells forming the subepithelial network and the smooth muscle cells that parallel the lacteal, as well as the basal lamina of capillaries. Several functions have been proposed for these cell types. Hirosawa and Yamada (12) have suggested that they may function as vitamin A-storing cells, because lipid droplets are frequently found within their cytoplasm. They may also synthesize proteins, including collagens, for the extracellular matrix, as they possess an abundant, often dilated, rough endoplasmic reticulum and a well-developed Golgi complex (2-4,6,7,9,11). Of particular importance for the overall function of the intestine is the suggestion (2,3,7) that these cells may form a contractile network within the intestinal mucosa capable of resisting mechanical stress and of affecting intestinal absorption. Evidence in support of the latter potential function comes mainly from ultrastructural studies that suggest that the subepithelial cells and stellate cells within the villar lamina propria contain a putative contractile apparatus. Well-defined microfilaments, 5-8 nm in diameter, are present within the periphAbbreviations used in this paper: PBS, phosphate-buffered saline.
DAB, 3,3’-diaminobenzidine;
INTESTINAL CONTRACTILE CELL NETWORK
January 1987
era1 cytoplasm of the perikaryon and become concentrated within the foot processes of both cell types (2,3,7). Myosinlike thick filaments, 15-18 nm in diameter, have been observed in glycerinated tissue (3), and structures resembling the dense bodies (2) and attachment plaques (3) of smooth muscle cells have also been described. Force may be transmitted during contraction not only to the plasmalemma of these cells, but also to components of the extracellular matrix and to neighboring cells. Filament bundles occasionally terminate at densities associated with the cytoplasmic aspect of the plasma membrane in regions where a corresponding condensation of the extracellular matrix can be observed. These structures have been described by Pitha (7) as “hemidesmosomes” and were thought to anchor the cell within the tissue matrix. In addition, the foot processes of subepithelial cells come into close contact with those of adjacent cells of the same type and with those of stellate cells, and, in these regions of close apposition, the intercellular gap is reduced to -20 nm (7). Within the lamina propria, close contacts have also been observed between stellate cell processes and the smooth muscle cells that parallel the lacteal (3). A close association has also been observed between nerve endings and the plasma membranes of both the subepithelial (2) and stellate cells (3), suggesting that both cell types could be induced to contract by neuronal stimulation. In addition, the presence of gap junctions between the processes of individual subepithelial cells (2) suggests that they may be able to communicate with each other and thereby propagate a contraction wave throughout the cellular network. The present studies confirm and extend previous electron microscopic findings suggesting that both the subepithelial and stellate cells possess a putative contractile apparatus; moreover, they establish by in situ immunoperoxidase localization procedures that these cells contain contraction-associated proteins in concentrations high enough to suggest a differentiated contractile function. The combined morphologic and biochemical evidence permits the conclusion that both the subepithelial cells and stellate cells are resident myofibroblasts that form a contractile network within the rat intestinal mucosa. Materials Electron
and Methods Microscopic
Studies
Tissue preparation. The vasculature of anesthetized Sprague-Dawley rats (male retired breeder) (Charles River Breeding Laboratories Inc., Wilmington, Mass.) was perfused at room temperature first for 10 min with 14 mM
69
glucose in Dulbecco’s phosphate-buffered saline [PBS) (Gibco, Grand Island, N.Y.) and then for 20 min with ice-cold 3% glutaraldehyde, 2% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M cacodylate buffer, pH 7.4. During the perfusion, a segment of the small intestine was isolated between two ligatures and fixative was injected into the lumen and also dripped onto the serosa to facilitate fixation of the intestinal wall from both sides. After the vascular perfusion was completed, the ligated segment was excised, cut into small pieces, and immersed in ice-cold fixative for a total of 90 min. Tissue pieces were washed first in 0.1 M cacodylate buffer, pH 7.4, and then in veronal-acetate buffer, pH 7.4, before postfixation in 2% osmium tetroxide in the same (veronal-acetate) buffer. After washes in the last buffer and in distilled water, the tissue was stained en bloc with 2% many1 acetate; then dehydrated in graded ethanols and propylene oxide: and finally flat-embedded in PolylBed 812 Embedding Medium (Polysciences, Inc., Warrington, Pa.). Thin sections for electron microscopy, cut on a Porter-Blum MT2-B ultramicrotome, were stained with Reynold’s lead, and micrographed on a Philips 301 electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.).
Immunoperoxidase
Localization
Studies
Antibodies. Five antibodies (all raised in rabbits) were used in these studies. Affinity-purified immunoglobulin G (IgG) raised against pigeon gizzard tropomyosin and IgG raised against chicken gizzard actin were kind gifts of Dr. Jan DeMey (Beerse, Belgium). The antitropomyosin IgG specifically recognizes smooth muscle tropomyosin in immunoblots and in immunoperoxidase localization studies (13). The specificity of the antiactin IgG for its homol(14). Immunoglobulin ogous antigen has been documented G raised against the heavy chain of human platelet myosin was obtained from Biomedical Technologies, Inc., Cambridge, Mass. Immunoglobulin G directed against the heavy chain of rat intestinal smooth muscle myosin was produced in our laboratory. The specificity of each IgG for the cognate myosin isoform was established by immunoblots and immunoperoxidase localization studies (15). An antiserum raised against bovine lung cyclic guanosine monophosphate (GMP)-dependent protein kinase was a kind gift of Dr. Ulrich Walter (Wurzburg, Federal Republic of Germany). The specificity of this serum for cyclic GMP-dependent protein kinase has been determined by a number of methods (1618). Nonimmune serum was collected from four normal rabbits and nonimmune IgG was prepared from this pool by precipitation with ammonium sulfate and subsequent solubilization and dialysis against PBS. Both nonimmune IgG and serum were stored frozen in the presence of O.OE~~ sodium azide. Fixation. Two fixatives were tested for their ability to retain antigenicity and preserve tissue structure for immunoperoxidase localization. Using the protocol described for morphologic studies, the vasculature was perfused at room temperature for 10 min with 14 mM glucose in PBS and then with PBS containing 4% formaldehyde
70 JOYCE ET AL.
(freshly prepared from paraformaldehyde); the ligated intestinal segment was excised, placed in fresh 4% formaldehyde, cut into small pieces, and further fixed by immersion in the same solution at room temperature for -20 min. Alternatively, the same protocol was followed using a “periodate-lysine-paraformaldehyde” fixative, prepared according to the method of McLean and Nakane (19). In this case, the small pieces cut from the intestinal segment were immersed in the same fixative at 0°C for 3 h. After preparation of the tissue for immunocytochemistry [described below), results of the two fixation methods were compared. Fixation with 4% formaldehyde permitted low concentrations of antigen to be detected, although tissue structure was poorly preserved. Intestinal epithelial cells were occasionally separated from one another and the membranes surrounding mucous droplets in the apical portion of goblet cells were somewhat disrupted. The McLean-Nakane fixative preserved structural integrity to a greater degree; however, antigenicity was reduced and low concentrations of antigen, which had been consistently demonstrable after 4% formaldehyde fixation, were no longer detected. To test for the presence of possibly low concentrations of some contraction-associated proteins, fixation was carried out according to the first protocol in all subsequent experiments. Tissue preparation. The method used for preparation and reaction of tissue sections for immunocytochemistry has been described previously (13). Briefly, tissue pieces, treated for cryoprotection with 10% dimethylsulfoxide in PBS, were frozen in liquid nitrogen-cooled isopentane. Cryostat sections (-32 pm) were cut and residual aldehydes were quenched for 30 min at room temperature in 0.15 M glycine in half-strength PBS. All steps subsequent to quenching were performed at ~4% to minimize structural artifacts. Generally, tissue sections were permeabilized with 0.3% Triton X-100 in PBS supplemented with 0.015 M glycine and 0.1% bovine serum albumin. This same solution was also used as an antibody diluent and washing medium. For the immunolocalization of cyclic GMP-dependent protein kinase, additional permeabilization beyond the original freeze-thaw was unnecessary (20), and therefore Triton X-100 was omitted from the incubation solution. After permeabilization, sections were incubated for 18-24 h in -10 pg/ml of either immune or nonimmune IgG or a 1:50 dilution of immune or nonimmune serum, washed for 18-24 h, and then treated for 12-24 h with 12.5 pg/ml of horseradish peroxidaseconjugated sheep antirabbit IgG Fab (Pasteur Institute, Paris, France). They were subsequently reacted with 3,3’diaminobenzidine (DAB) and 0.01% hydrogen peroxide for lo-60 min, washed in PBS, and then plastic-embedded in PolyiBed 812. Thick sections (-1 pm) of the reacted tissue were examined and micrographed with a Zeiss Photomicroscope II (Carl Zeiss, Inc., Thornwood, N.J.) using Kodak Technical Pan film (Eastman Kodak Co., Rochester, N.Y.). Nomenclature. To facilitate the description of the cells under discussion, the following nomenclature will be used. The elongated cells subtending the villar epithelium will be called subepithelial myofibroblasts. Similar cells subtending the epithelium of the crypts of Lieberkuhn will
GASTROENTEROLOGY Vol. 92. No. 1
be termed pericryptal myofibroblasts, and the stellateshaped cells deep within the lamina propria will be referred to as lamina propria myofibroblasts. Data analysis. The immunoperoxidase localization technique provides a semiquantitative measure of antigen concentration. The color intensity developed in tissue incubated with an antibody and reacted with DAB and hydrogen peroxide reflects the relative concentration of antigen within individual cell types. Several staining intensities can be easily discerned and graded under standardized conditions for the peroxidase reaction. Cells strongly reactive for an antigen stain dark brown to black; moderately reactive cells stain medium brown; weakly reactive cells stain tan to light brown, whereas cells that are not obviously stained above background levels are considered unreactive. By observing the color intensity developed in individual cell types, their relative reactivity for a specific antigen, and therefore the relative concentration of antigen, can be compared. This comparison is valid for the same antigen detected by the same antibody in different cell types within a given tissue specimen. It does not apply for different antigens in the same or different cell types. Several cell types were often positively stained in tissues incubated for each of the proteins tested; however, only the relative reactivities of smooth muscle cells (as an example of a differentiated contractile cell], connective tissue fibroblasts (as an example of a nonmuscle cell), and subepithelial, pericryptal, and lamina propria myofibroblasts will be reported.
Results Morphologic
Studies
For electron subepithelial examined.
studies,
pericryptal
A survey
the presence reticulum
microscopic
and
of their
cell
of a well-developed
and Golgi
complex
(Figure
mitochondria,
icles,
occasional
coated
formed
a plate
including
thelial the
aspect
foot
completely were
filled
frequently
the microfilament bling the dense
(Figure the
lb)
confirmed
endoplasmic la),
as well
vesicles. within
and
Bundles
and
extended they
Thick
appeared
bundles. Fibrillar bodies of smooth
as
ves-
the adepi-
where
cytoplasm.
observed
the were
and cytoplasmic
of the perikaryon
processes
bodies rough
free polysomes, of microfilaments
only
myofibroblasts
into almost
filaments to parallel
densities resemmuscle cells ap-
peared to be associated with microfilaments within both the perikaryon and the cytoplasmic processes. Fibrillar plaques,
thickenings, were present
suggestive of attachment on both the adepithelial and
the abepithelial aspects of the processes. In these regions, a corresponding lamellar condensation of the extracellular matrix was occasionally seen. These focal concentrations seemed to be the only regions of organized matrix associated with these cells, which generally did not appear to elaborate a well-defined basal lamina.
January
Figure
1987
INTESTINAL
CONTRACTILE
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I. cr. A representative electron micrograph of a portion of the cell body of a subepithelial myofibroblast from the rat intestinal villus illustrates some characteristic features of this cell type. The cell lies adjacent to the basal lamina (BL) of the epithelial cells (EC] and, on its abepithelial aspect, may be closely approached by other cell types that populate the villar lamina propria. A well-developed rough endoplasmic reticulum (RER) and Golgi complex (G) are located within the perinuclear cytoplasm. A plate of microfilaments (MF) lies within the adepithelial aspect of the perikaryon and the proximal portion of the cytoplasmic process (P). Several dense bodies (D) are associated with the microfilament bundles in this region of the cell. CH, chylomicra. Magnification, X31,006. b. Electron micrograph of the more distal portions of several subepithelial myofibroblast foot processes (large arrows) illustrates the high fibrillar content of the cytoplasm in this region of the cell and the multiple layering of the individual processes. Microfilaments and thick filaments [arrowheads) almost completely fill the cytoplasm. Fibrillar densities (D) are associated with microfilament bundles both within the cytoplasm and on the cytoplasmic aspect of the abepithelial plasma membrane. The extracellular matrix forms an organized plate in the region directly opposite the plasmalemma-associated density (curved arrow) forming a hemidesmosomelike structure. Note the coated pit (small arrow) on the adepithelial aspect of a foot process. Magnification, X26.500.
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JOYCE ET AL.
Figure
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Vol. 92, No. 1
2. The electron micrograph in (a) illustrates three junctions formed by separate cytoplasmic processes with a single subepithelial myofibroblast process. Gap junctions (GJ) are formed between the larger process and two processes probably belonging to two other subepithelial myofibroblasts. (The left junction is enlarged in b; the right junction is enlarged in d.) An adhering junction, shown at the arrowhead in (a) and at the m-row in (c), is characterized by symmetric fibrillar thickenings within the cytoplasmic aspect of the plasma membranes of the two opposing processes. The enlargement of this area in (c) also illustrates more clearly a density (arrowhead) similar to that found in hemidesmosomes, which is present on the abepithelial aspect of the upper process. Note the adepithelial location of microfilaments (mfl in the larger process in (a], as well as the thick filaments (small arrows) also located in this region of the cell. EC, intestinal epithelial cell; BL, basal lamina. Magnification, (a) ~51,000; (b) X148.000; (c) X78.000; (d) ~114,000.
INTESTINAL
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lanuary
1987
Figure
3. Immunoperoxidase staining for smooth muscle tropomyosin reveals several characteristics of the myofibroblast network within the intestinal mucosa. Darkly stained pericryptal myofibroblasts (arrows in a) form a layered network immediately subjacent to the cryptal epithelium. Around the crypts. the network is reasonably tight, with myofibroblast cell bodies or their processes in close apposition to a relatively large percentage of the epithelial cell basal surface. The network formed by subepithelial myofibroblasts (arrows) in the villus (b) is somewhat looser and thinner, generally consisting of only one to two cell layers. Lamina propria myofibroblasts (LPM) within the villus appear to form a three-dimensional network, with their processes in close apposition to capillaries, subepithelial myofibroblasts, and other cell types that populate this region. Note that the most intense staining is seen in the peripheral cytoplasm and foot processes of the cells. EC, epithelial cells; Ly, lymphatic vessel; SM, smooth muscle cells; La, lacteal; Eo, eosinophil; P, microvascular pericyte. Magnification, (n) x 1000: (b) x 1050.
Subepithelial and pericryptal myofibroblasts formed two types of close contacts with each other, as illustrated in Figure 2. One type of contact, which was frequently observed, resembled an adhering junction in which dense plaques of fibrillar material were present in phase on the cytoplasmic aspect of the two opposing plasma membranes. In three dimensions, however, such structures appeared to involve a small circumscribed area (a macula, rather than a zonule). Communicating (gap) junctions, characterized by their closely apposed, parallel membranes and central gap, were seen less often. In the micrograph in Figure Za, gap junctions are formed by two adjacent cells with a single cytoplasmic process. Immunoperoxidase staining (discussed below) revealed some characteristics of the cellular network within the intestinal mucosa. Pericryptal myofibroblasts formed a relatively tight network, generally consisting of two or three cell layers. Such a network is illustrated in Figure 3a. Subepithelial myofibroblasts within the villus (Figure 3b) were somewhat more loosely arranged and formed a network of one to two cell layers. The multiple layering in both the
crypts and the villi seemed to be local, not general, so that the network appeared somewhat uneven in randomly sectioned specimens. Lamina propria myofibroblasts were relatively numerous and appeared to form a reticular network, coming in close contact with many of the cells populating this area, as well as with subepithelial myofibroblasts. Immunoperoxidase
Localization
Studies
Control experiments. Tissue sections incubated in nonimmune IgG followed by secondary antibody (Figures 4a and 4c) showed no evidence of nonspecific staining beyond that due to endogenous peroxidase activity or nonspecific adsorption of DAB. Intense staining due to endogenous peroxidase activity was visible in leukocytes, especially eosinophils, within the lamina propria and in erythrocytes occasionally seen within capillary lumina (see Figure 8a). In addition, a low intensity staining, probably due to endogenous peroxidase activity, was observable in the granules of Paneth cells located at the base of the crypts of Lieberkiihn (Figure ad). Light staining was also observed in chylomicra oc-
74
Figure
IOYCE ET AL.
GASTROENTEROLOGY
Vol. 92. No. 1
4. Micrographs of sections taken from intestinal villi (a and b) and crypts of Lieberkiihn (c and d] incubated in either nonimmune IgG (a and c) or nonimmune serum (b and d) illustrate typical staining patterns obtained under control conditions. Endogenous peroxidase activity is observable in leukocytes, especially eosinophils (Eo) within the lamina propria and in Paneth cell granules (arrowheads) at the base of the crypts. Note that the eosinophil at the right in (a] has migrated between epithelial cells. No staining is visible in subepithelial (a and b) or pericryptal myofibroblasts (c and d) shown at the arrows. In tissue incubated with nonimmune serum, certain structures (possibly granule membranes) within the apical region of goblet cells (GC) are nonspecifically stained. C, capillary; La, lacteal; Ly, lymphatic vessel: L, intestinal lumen; MM, muscularis mucosae; VSM, vascular smooth muscle cells. Magnification, (a) x1150; (b) x1250; (c) x950; (d] x1200.
casionally found in the intercellular spaces at the base of the intestinal epithelial cells within the villus (Figure 5a). This type of staining may be caused by nonspecific adsorption of DAB, as similar nonspecific adsorption has been observed in lipid droplets in striated muscle cells and in adipose cells incubated in DAB alone (13,151. Control specimens
incubated only with secondary antibody followed by DAB reaction showed no additional nonspecific staining (micrographs not shown). Controls incubated in nonimmune serum followed by horseradish peroxidase-conjugated secondary antibody were very similar to those described above. The only observable difference was the nonspecific
January
Figure
INTESTINAL
1987
CONTRACTILE
CELL NETWORK
75
5. Longitudinal section through part of an intestinal villus (a] and transverse section of a crypt (b) taken from specimens incubated with anti-smooth muscle tropomyosin IgG show positive staining in subepithelial, pericryptal, and lamina propria myofibroblasts. Pericryptal myofibroblasts (arrows in b) appear to stain more intensely than subepithelial myofibroblasts (arrows in a). Lamina propria myofibroblasts (LPM). although weakly reactive, are clearly stained above background levels. Smooth muscle cells are strongly reactive, whereas connective tissue fibroblasts (arrowhead in b) are unreactive for this tropomyosin isoform. Note the light staining, probably due to nonspecific adsorption of DAB, in chylomicra [arrowheads in a) at the base of the epithelial cells. SM, smooth muscle cells; Eo, eosinophil; VSM, vascular smooth muscle cells; MM, muscularis mucosae; L, intestinal lumen. Magnification, (a) X1000; (b) X800.
staining of certain structures (possibly mucous granule membranes) located in the apical region of goblet cells incubated with nonimmune serum (Figures 4b and ad). Immunoperoxidase Localization Muscle Tropomyosin
of Smooth
Positive staining for smooth muscle tropomyosin was consistently observed in subepithelial, pericryptal, and lamina propria myofibroblasts, and in smooth muscle cells, as illustrated in Figures 5a and 5b. The smooth muscle cells disposed parallel to the lacteal (Figure 5a) and the cells comprising the muscularis mucosae (Figure Sb], as well as vascular smooth muscle cells, were strongly stained. Pericryptal myofibroblasts were less intensely stained than smooth muscle cells, but were more reactive than subepithelial or lamina propria myofibroblasts. Connective tissue fibroblasts were consistently unreactive for this antigen. In all positive cells, DAB reaction product was diffusely distributed within the cell cytoplasm, whereas the nucleus was unstained. Staining in all three types of myofibroblasts was generally most intense within the foot processes and at the periphery of the cell body. In some sections,
the adepithelial aspect of the perikaryon in both the subepithelial and the pericryptal myofibroblasts appeared more darkly stained than their abepithelial aspect. Immunoperoxidase Localization of Cyclic Guanosine Monophosphate-Dependent Protein Kinase Both subepithelial and pericryptal myofibroblasts reacted positively for cyclic GMP-dependent protein kinase [Figures 6a and 6b). The intensity of reaction in these cells was less than the strongly positive staining seen in smooth muscle cells, however. A comparison of relative staining intensities suggests that subepithelial myofibroblasts were somewhat more reactive than pericryptal myofibroblasts. Both myofibroblasts within the lamina propria and connective tissue fibroblasts appeared unstained. The staining pattern for cyclic GMP-dependent protein kinase was different from that detected for smooth muscle tropomyosin. For the kinase, DAB reaction product was distributed mainly within the cell body and proximal portions of the foot processes of the subepithelial and pericryptal myofibroblasts;
76
JOYCE ET AL.
Figure
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6. These sections, taken from an intestinal villus (a) and a crypt of Lieberkiihn (b), show the comparative reactivity of cells for cyclic GMP-dependent protein kinase. Smooth muscle cells (SM) within the villus and the cells comprising the muscularis mucosae (MM) are strongly positive for cyclic GMP-dependent protein kinase. Subepithelial (a) and pericryptal myofibroblasts (b), shown at the arrows, are less intensely stained, although the myofibroblasts in the villus appear more reactive than those surrounding the crypts. Lamina propria myofibroblasts (LPM) and connective tissue fibroblasts (arrowhead in b) are unstained. *, nucleus of a leukocyte that has migrated between epithelial cells; La. lacteal; L, intestinal lumen. Magnification, (a] x9.50; (b) XIOOO.
in contrast, the more distal segments of their foot processes were either lightly stained or unstained, suggesting that these regions of the cell did not contain significant amounts of antigen. For smooth muscle tropomyosin, the foot processes appeared to be stained over considerably longer distances (compare Figure 5 with Figure 6).
Immunoperoxidase
Localization
of Actin
Subepithelial, pericryptal, and lamina propria myofibroblasts stained positively for actin to about the same extent (Figures 7a and 7b), but the intensity of their reaction was less than that of smooth muscle cells. The actin staining patterns of the three cell types were similar to those generated by staining for smooth muscle tropomyosin: in both cases, diffuse reaction product was visible in the perinuclear cytoplasm, as well as in the foot processes. Staining of the peripheral cytoplasm within a lamina propria myofibroblast is particularly well-illustrated in Figure 7a. The brush border and basolateral regions of the epithelial cells were intensely stained for actin, whereas connective tissue fibroblasts were only weakly reactive.
lmmunoperoxidase Isomyosins
Localization
of
Nonmuscle myosin. In tissue sections stained for nonmuscle myosin, subepithelial, pericryptal, and lamina propria myofibroblasts, as well as connective tissue fibroblasts, all reacted positively (Figures 8a and 8b), whereas smooth muscle cells were consistently unreactive. Pericryptal myofibroblasts appeared to be somewhat more intensely stained for nonmuscle myosin than either subepithelial or lamina propria myofibroblasts; however, all these cells were more strongly positive than connective tissue fibroblasts, which appeared only weakly reactive. The staining patterns for all these cells were comparable to those obtained for actin and smooth muscle tropomyosin. Smooth muscle myosin. As shown in Figures 9a and 9b, subepithelial and pericryptal myofibroblasts stained weakly for smooth muscle isomyosin. Although the reactivity was above background level, the staining intensity was much less than that observed for smooth muscle cells. Pericryptal myofibroblasts stained somewhat more intensely than subepithelial myofibroblasts and both cell types were more reactive than myofibroblasts within the lamina propria. Connective tissue fibroblasts ap-
January 1987
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77
Figure
7. Part of a longitudinal section through an intestinal villus (a] and an oblique (grazing] section of two crypts of Lieberkiihn (b) illustrate the positive reaction of subepithelial (arrows in a), pericryptal (arrows in b), and lamina propria myofibroblasts (LPM) for actin. All three cell types contain DAB reaction product, particularly toward the periphery of the cell body, and within the foot processes. This staining pattern is clearly evident in the lamina propria myofibroblast in (a). The staining intensity for actin in these cells is less than that in the smooth muscle cells of the muscularis mucosae (M), seen in (b). Connective tissue fibroblasts (arrowhead in b) are weakly reactive. Staining for actin is intense in the brush border and periphery of epithelial cells and in migrating leukocytes (*). L. intestinal lumen. Magnification, (a) x 1300; (b) X 1000.
Figure
8. Sections of the lamina propria of a villus (a) and a crypt of Lieberkiihn (b) illustrate the positive reactivity of subepithelial (arrows in a), pericryptal (arrows in b), and lamina propria myofibroblasts (LPM) for nonmuscle isomyosin. Diffuse DAB reaction product is present within the perikaryon and foot processes of these cells. Connective tissue fibroblasts (arrowhead in b) also react positively, but are weakly stained. Epithelial cells in both the villus and crypt, as well as the lacteal endothelium (a) stain diffusely, whereas smooth muscle cells (SM) are consistently unreactive for nonmuscle isomyosin. Arrowhead in (a) indicates dark staining of erythrocytes due to endogenous peroxidase activity; *, nucleus of a leukocyte that has migrated between two epithelial cells; Eo, eosinophil; La, lacteal; L, intestinal lumen. Magnification, (a) x950; (b) ~1450.
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JOYCE ET AL.
Figure
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9. These longitudinal sections of the lamina propria within a villus (a) and a crypt of Lieberktihn (b) illustrate relatively weak reactivity of subepithelial and pericryptal myofibroblasts (arrows) for smooth muscle isomyosin. Pericryptal myofibroblasts appear somewhat more reactive than subepithelial myofibroblasts, whereas lamina propria myofibroblasts (LPM) and connective tissue fibroblasts (arrowheads in b) appear unreactive. Smooth muscle cells (SM) are strongly reactive for this myosin isoform. Eo, eosinophil; La, lacteal; C, capillary; L, intestinal lumen. Magnification, (a] x1159; (b) x959.
peared unreactive. As with nonmuscle isomyosin, diffuse reaction product was visible in both the perinuclear cytoplasm and foot processes of the cells. The signal-to-background ratio did not increase in these cells with longer periods of reaction with DAB and hydrogen peroxide, and attempts to intensify the staining by published techniques (21-23) were unsuccessful.
Discussion Electron
Microscopic
Observations
The results obtained from our electron microscopic study of intestinal subepithelial and pericryptal myofibroblasts confirmed many of the observations made by Pitha (3, Desaki et al. (2), and Giildner et al. (3). Specifically, this applies to the presence of both thick and thin filaments within the adepithelial cytoplasm of the perikaryon and foot processes. In addition, we extended the observations by identifying structures within the cytoplasm that have the morphology of dense bodies. Their presence within both subepithelial and pericryptal myofibroblasts, as well as the presence of densities similar to adhesion plaques located on both the adluminal and abluminal aspects of their plasmalemma, suggests that these cells contain a putative contractile apparatus capable of generating and transmitting force. In our studies, we further characterized the “close
contacts” formed by subepithelial and pericryptal myofibroblasts. We confirmed the fact that gap junctions are present between these cells and assume that such junctions permit communication among several adjacent cells within the network as suggested by the finding (Figure 2a) that a single foot process can form gap junctions with at least two neighboring cells. In addition, adhering junctions are frequently observed connecting the cells within the network. The morphologic evidence thus obtained confirms and extends previous findings and supports the hypotheses advanced by both Giildner et al. (3) and Desaki et al. (2) according to which these cells form a cellular network, and are capable of communication with one another and of transmitting the force generated during their contraction both to neighboring cells and to elements of the extracellular matrix. lmmunoperoxidase Localization Contraction-Associated Proteins
of
The results of the immunoperoxidase localization studies provide direct evidence for the presence of at least four contraction-associated proteins in subepithelial, pericryptal, and lamina propria myofibroblasts. Both actin and myosin are considered essential for contractility, and tropomyosin is thought to be required for efficient regulation of
INTESTINAL CONTRACTILE CELL NETWORK
January 1987
Table
1. Relative
Reactivities
of Intestinal
for Four Contraction-Associated
Cell type Smooth muscle cells Pericryptal myofibroblasts Subepithelial myofibroblasts Lamina propria myofibroblasts Connective tissue fibroblasts ” cGK, cyclic GMP-dependent
Myofibroblasts, Connective Tissue Fibroblasts, and Smooth Muscle Cells Proteins
Smooth muscle tropomyosin +++ +++to++ ++ + protein kinase.
79
cGK” +++ ++to+ ++ -
+ + + , Strongly reactive;
acto-myosin interaction in smooth muscle (24,25) and nonmuscle cells (26,27), as well as in skeletal muscle. The role of cyclic GMP-dependent protein kinase in cellular contraction is not yet fully understood; however, it has been suggested (28,29) that this enzyme is involved in smooth muscle cell relaxation. of these Table 1 shows the relative reactivities cells for each of the proteins studied. In general, the intensity of the reaction in subepithelial, pericryptal, and lamina propria myofibroblasts was less than that found for either visceral or vascular smooth muscle cells, but was greater than the reactivity of cells differentiated for functions other than contraction, such as connective tissue fibroblasts. The results also indicate that the relative concentration of each of these proteins differs in each of the three cell types under study. Pericryptal myofibroblasts were somewhat more reactive for contraction-associated proteins (except for cyclic GMP-dependent protein kinase) than subepithelial myofibroblasts, and both of these cell types appeared more reactive than myofibroblasts within the lamina propria, indicating a gradation in the amount of contraction-associated proteins, and thus possibly a modulation in contractile activity, within these three cell types. The presence of cyclic GMP-dependent protein kinase in these cells, as well as the smooth muscle isoforms of tropomyosin and myosin suggest that subepithelial, pericryptal, and lamina propria myofibroblasts are rather closely related to smooth muscle cells in terms of the contraction-associated proteins and isoforms they contain. The fact that they stain positively for both nonmuscle and smooth muscle isomyosin suggests, however, that their contractile protein equipment is not identical with that of smooth muscle cells. Therefore, their particular type of contraction, and possibly its mode of regulation, may differ from that of the latter. The results reported in this study are generally similar to those obtained for the in situ immunoperoxidase localization of the same contraction-associated proteins in microvascular pericytes (13,15,
Actin
Nonmuscle isomyosin
+++ ++ ++ ++ +
++ ++ ++ +
+ + , moderately
reactive;
+ , weakly reactive; -,
Smooth muscle isomyosin +++ ++to+ + unreactive.
20). Overall comparison of the findings indicates that microvascular pericytes are generally more intensely stained for each of the proteins tested than are the subepithelial or pericryptal myofibroblasts. The immunocytochemical results presented in this study establish that subepithelial, pericryptal, and lamina propria myofibroblasts contain essential contraction-associated proteins in concentrations approaching those found in microvascular pericytes and smooth muscle cells and distinctly higher than those detected in cells differentiated for other, noncontractile functions. The morphologic findings suggest that these cells contain a putative contractile apparatus, as well as a means for intercellular communication and force-generation. Taken together, these results provide strong evidence that the three types of fibroblastlike cells found within the rat intestinal mucosa are differentiated for a contractile function. There is still insufficient information to allow a specific determination as to the direction of force that may be generated by these cells as well as to the effects of their contraction. However, the structural arrangement indicated by both electron and light microscopy suggests that they form an essentially two-dimensional cellular network subjacent to the intestinal epithelium and a three-dimensional network within the lamina propria of the intestinal villi. Tension generated by the contraction of such cellular networks may provide a supportive tonus to the epithelium, enabling it to withstand forces that may deform or dislocate it. It may also provide the force needed for active movement of the villi, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and the lacteals of the intestinal villi. The evidence presented in this study, although highly suggestive, does not prove that intestinal mucosal myofibroblasts contract in vivo. There is a similar lack of direct evidence for in vivo contraction in studies of the intestinal epithelial cell brush border. Biochemical (30-34), morphologic (35,36), and immunocytochemical localization studies (31,
80
IOYCE ET AL.
37-39) together have established the presence of essential contractile proteins within the terminal web region in a spatial arrangement suggestive of a contractile function; however, brush border contraction has only been observed in disrupted systems (30,32,33). New approaches must be applied to the study of both these cell types in order to understand more fully their contractile function in vivo.
References 1. Deane HW. Some electron microscopic observations on the lamina propria of the gut, with comments on the close association of macrophages, plasma cells and eosinophils. Anat Ret 1964;149:453-74. 2. Desaki J, Fujiwara T, Komuro T. A cellular reticulum of fibroblast-like cells in the rat intestine: scanning and transmission electron microscopy. Arch Histol Jpn 1984;47: 179-86. 3. Giildner FH, Wolff JR, Graf Keyserlingk D. Fibroblasts as part of the contractile system in duodenal villi of rat. Z Zellforsch 1972;135:349-60. 4. Marsh MN, Trier JS. Morphology and cell proliferation of subepithelial fibroblasts in adult mouse jejunum. I. Structural features. Gastroenterology 1974;67:622-35. 5. Palay SL, Karlin LJ. An electron microscope study of the intestinal villus. I. The fasting animal. Biophys Biochem Cytol 1959;5:363-95. 6. Parker FG, Baries EN, Kaye GI. The pericryptal fibroblast sheath. IV. Replication, migration, and differentiation of the subepithelial fibroblasts of the crypt and villus of the rabbit jejunum. Gastroenterology 1974;67:607-21. 7. Pitha J. The fine structure of clear fibroblast-like cells in the lamina propria of the small intestine. J Ultrastruct Res 1968; 22:231-g. WL. The structure of the colonic mucosa. The 8. Donnellan epithelium and subepithelial reticula-histiocytic complex. 1965;49:496-514. Gastroenterology 9. Kaye GI, Lane N, Pascal RR. Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchymal cell system in adult tissue. II. Fine structural aspects of normal rabbit and human colon. Gastroenterology 1968;54:852-65. FP, Dujardin-Loits R-M, 10. Maskens AP, Rahier JR, Meersseman Haot JG. Cell proliferation of pericryptal fibroblasts in the rat colon mucosa. Gut 1979;20:775-9. pericryptal fibroblast 11. Pascal RR, Kaye GI, Lane N. Colonic sheath: replication, migration, and cytodifferentiation of a mesenchymal cell system in adult tissue. 1. Autoradiographic studies of normal rabbit colon. Gastroenterology 1968;54: 835-51. K, Yamada E. Localization of vitamin A in the small 12. Hirosawa intestine of mouse: an electron microscope radioautographic study. Cell Tissue Res 1977;177:57-65. GE. Contractile proteins in 13. Joyce NC, Haire MF, Palade pericytes. 1. Immunoperoxidase localization of tropomyosin. J Cell Biol 1985;100:1379-86, G, DeMey J, Moeremans M, Daneels G, DeBraban14. Langanger der M, Small JV. Ultrastructural localization of cy-actinin and filamin in cultured cells with the immunogold staining (IGS) method. J Cell Biol 1984;99:1324-34. GE. Contractile proteins in 15. Joyce NC, Haire MF, Palade pericytes. II. Immunocytochemical evidence for the presence of two isomyosins in graded concentrations. J Cell Biol 1985; 100:1387-95.
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16. Lohmann SM, Walter U, Greengard P, DeCamilli P. Immunohistochemical localization of cyclic GMP-dependent protein kinase in mammalian brain. Proc Nat1 Acad Sci USA 1981; 78:653-7. 17. Walter U. Distribution of cyclic GMP-dependent protein kinase in various rat tissues and cell lines determined by a sensitive and specific radioimmunoassay. Eur J Biochem 1981:118:339-46, 18. Walter U, Miller P, Wilson F, Menkes D, Greengard P. Immunological distinction between guanosine 3’:5’-monophosphate-dependent and adenosine 3’:5’-monophosphatedependent protein kinases. J Biol Chem 1980;255:3757-62. 19. McLean IW, Nakane PK. Periodate-lysine-formaldehyde fixative. A new fixative for immunoelectron microscopy. J Histothem Cytochem 1974;22:1077-83. 20. Joyce NC, DeCamilli P, Boyles J. Pericytes, like vascular smooth muscle cells, are immunocytochemically positive for cyclic GMP-dependent protein kinase. Microvasc Res 1984; 28:206-19. 21. Adams JC. Technical considerations on the use of horseradish peroxidase as a neuronal marker. Neuroscience 1977;2:141-5. 22. Gallyas F. Gores T, Merchenthaler I. High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J Histochem Cytochem 1982;30: 183-4. 23. Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18:315-33. 24. Chacko S. Effects of phosphorylation. calcium ion, and tropomyosin on actin-activated adenosine 5’-triphosphatase activity of mammalian smooth muscle myosin. Biochemistry 1981;20:702-7, A, Small JV. Regulation of the actin-myosin inter25. Sobieszek action in vertebrate smooth muscle: activation via a myosin light-chain kinase and the effect of tropomyosin. J Mol Biol 1977;112:559-76. N. Tropomyosin enhances actomyosin 26. Onji T, Shibata ATPase activity in platelets. Biophys Biochem Res Commun 1982;109:697-703, kinetic studies on the actin activa27. Sobieszek A. Steady-state tion of skeletal muscle heavy meromyosin subfragments. J Mol Biol 1982;157:275-86. and biological role 28. Lincoln TM, Corbin JD. Characterization of the cGMP-dependent protein kinase. Adv Cyclic Nucleotide Res 1983;15:139-92. TM, Johnson RM. Possible role of cyclic GMP29. Lincoln dependent protein kinase in vascular smooth muscle function. Adv Cyclic Nucleotide Res 1984;17:285-96. R, Newman SB, Karnovsky MJ. Contraction of 30. Rodewald isolated brush borders from the intestinal epithelium. J Cell Biol 1976;70:541-54. MS, Pollard TD, Fujiwara K. Characterization and 31. Mooseker localization of myosin in the brush border of intestinal epithelial cells. J Cell Biol 1978;79:444-53. TCS, Mooseker MS. Ca++-calmodulin-dependent 32. Keller phosphorylation of myosin, and its role in brush border contraction in vitro. J Cell Biol 1982;95:943-59. con33. Broschat KO, Stidwell RP, Burgess DR. Phosphorylation trols brush border motility by regulating myosin structure and association with the cytoskeleton. Cell 1983:35:561-71. KA. Chasan R, Mooseker MS. The role 34. Keller TCS, Conzelman of myosin in terminal web contraction in isolated intestinal epithelial brush borders. J Cell Biol 1985;100:1647-55. N, Keller TCS, Chasan R, Mooseker MS. Mecha35. Hirokawa
January 1987
nism of brush border contractility studied by the quickfreeze-deep-etch method. J Cell Biol 1983;96:1325-36. 36. Hirokawa N, Tilney L, Fujiwara K, Heuser JE. The organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells. J Cell Biol 1982;94: 425-43. 37. Bretscher A, Weber K. Localization of actin and microfilament associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J Cell Biol 1978;79:839-45.
INTESTINAL CONTRACTILE CELL NETWORK
81
38. Drenckhahn D, Groschel-Stewart U. Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels. J Cell Biol 1980;86:475-82. 39. Geiger B, Dutton A, Tokuyasu T, Singer S. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol 1981;91:614-28.
1987;92:68-81
Morphologic and Biochemical Evidence for a Contractile Cell Network Within the Rat Intestinal Mucosa NANCY C. JOYCE, MARCY F. HAIRE, and GEORGE Department
of Cell Biology,
Yale University
School
Subepithelial and pericryptal fibroblastlike cells form a two-dimensional network immediately subjacent to the epithelial basal lamina in the small intestine and colon in several mammalian species. Stellate-shaped cells with similar, but not identical characteristics, form a three-dimensional network deep within the villar lamina propria. Electron microscopic studies indicate that these cells (a) contain a putative contractile apparatus, (b) are attached to each other and to apparently organized elements of the extracelluIar matrix by typical adhesive devices, and (c) form gap junctions with each other. Comparative in situ immunoperoxidase localization studies document the presence in these cells offour contraction-associated proteins (smooth muscle isotropomyosin, cyclic guanosine monophosphate-dependent protein kinase, both nonmuscle and smooth muscle isomyosin, and actin) in amounts generally greater than those found in connective tissue fibroblasts, but less than in smooth muscle cells. Taken together, these results strongly suggest a smooth muscle-like, contractile function for these cells and indicate that this cellular network may provide a supportive tonus for the epithelium, as well as provide the force needed for active movement of the villus, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and lacteals of the intestinal villus. A network of fusiform cells, with the general appearance of fibroblasts, subtends the epithelial basal lamina in both the small intestine (l-7) and colon Received February 10, 1986. Accepted June 23, 1986. Address requests for reprints to: Nancy C. Joyce, Ph.D., Department of Cell Biology. Yale University School of Medicine, P.O. Box 3333, 333 Cedar Street, New Haven, Connecticut 06510. This work was supported by National Research Service Award S-T32-GM07223-09 to N.C.J. and National Institutes of Health grant HL17080 to G.E.P. 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50
of Medicine,
E. PALADE
New Haven,
Connecticut
(1,8-11) in several species, including rat (2,3,5,7,10), mouse (1,4), rabbit (6,7,9,11), and humans (8,9). The cells comprising this network have elongated cell bodies and long, attenuated cytoplasmic processes, which closely appose the overlying basal lamina. The number of cells in the network appears to be greatest at the base of the crypts of Lieberkiihn and to decrease toward the surface of the villus, although this decrease is more pronounced in the small intesIn addition, steltine than in the colon (1,4,6,8,11). late-shaped cells with large, oval nuclei, scant perinuclear cytoplasm, and long cytoplasmic processes have been reported (3) to form a cellular network deep within the villar lamina propria of the rat small intestine. The foot processes of these cells closely appose the cells forming the subepithelial network and the smooth muscle cells that parallel the lacteal, as well as the basal lamina of capillaries. Several functions have been proposed for these cell types. Hirosawa and Yamada (12) have suggested that they may function as vitamin A-storing cells, because lipid droplets are frequently found within their cytoplasm. They may also synthesize proteins, including collagens, for the extracellular matrix, as they possess an abundant, often dilated, rough endoplasmic reticulum and a well-developed Golgi complex (2-4,6,7,9,11). Of particular importance for the overall function of the intestine is the suggestion (2,3,7) that these cells may form a contractile network within the intestinal mucosa capable of resisting mechanical stress and of affecting intestinal absorption. Evidence in support of the latter potential function comes mainly from ultrastructural studies that suggest that the subepithelial cells and stellate cells within the villar lamina propria contain a putative contractile apparatus. Well-defined microfilaments, 5-8 nm in diameter, are present within the periphAbbreviations used in this paper: PBS, phosphate-buffered saline.
DAB, 3,3’-diaminobenzidine;
INTESTINAL CONTRACTILE CELL NETWORK
January 1987
era1 cytoplasm of the perikaryon and become concentrated within the foot processes of both cell types (2,3,7). Myosinlike thick filaments, 15-18 nm in diameter, have been observed in glycerinated tissue (3), and structures resembling the dense bodies (2) and attachment plaques (3) of smooth muscle cells have also been described. Force may be transmitted during contraction not only to the plasmalemma of these cells, but also to components of the extracellular matrix and to neighboring cells. Filament bundles occasionally terminate at densities associated with the cytoplasmic aspect of the plasma membrane in regions where a corresponding condensation of the extracellular matrix can be observed. These structures have been described by Pitha (7) as “hemidesmosomes” and were thought to anchor the cell within the tissue matrix. In addition, the foot processes of subepithelial cells come into close contact with those of adjacent cells of the same type and with those of stellate cells, and, in these regions of close apposition, the intercellular gap is reduced to -20 nm (7). Within the lamina propria, close contacts have also been observed between stellate cell processes and the smooth muscle cells that parallel the lacteal (3). A close association has also been observed between nerve endings and the plasma membranes of both the subepithelial (2) and stellate cells (3), suggesting that both cell types could be induced to contract by neuronal stimulation. In addition, the presence of gap junctions between the processes of individual subepithelial cells (2) suggests that they may be able to communicate with each other and thereby propagate a contraction wave throughout the cellular network. The present studies confirm and extend previous electron microscopic findings suggesting that both the subepithelial and stellate cells possess a putative contractile apparatus; moreover, they establish by in situ immunoperoxidase localization procedures that these cells contain contraction-associated proteins in concentrations high enough to suggest a differentiated contractile function. The combined morphologic and biochemical evidence permits the conclusion that both the subepithelial cells and stellate cells are resident myofibroblasts that form a contractile network within the rat intestinal mucosa. Materials Electron
and Methods Microscopic
Studies
Tissue preparation. The vasculature of anesthetized Sprague-Dawley rats (male retired breeder) (Charles River Breeding Laboratories Inc., Wilmington, Mass.) was perfused at room temperature first for 10 min with 14 mM
69
glucose in Dulbecco’s phosphate-buffered saline [PBS) (Gibco, Grand Island, N.Y.) and then for 20 min with ice-cold 3% glutaraldehyde, 2% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M cacodylate buffer, pH 7.4. During the perfusion, a segment of the small intestine was isolated between two ligatures and fixative was injected into the lumen and also dripped onto the serosa to facilitate fixation of the intestinal wall from both sides. After the vascular perfusion was completed, the ligated segment was excised, cut into small pieces, and immersed in ice-cold fixative for a total of 90 min. Tissue pieces were washed first in 0.1 M cacodylate buffer, pH 7.4, and then in veronal-acetate buffer, pH 7.4, before postfixation in 2% osmium tetroxide in the same (veronal-acetate) buffer. After washes in the last buffer and in distilled water, the tissue was stained en bloc with 2% many1 acetate; then dehydrated in graded ethanols and propylene oxide: and finally flat-embedded in PolylBed 812 Embedding Medium (Polysciences, Inc., Warrington, Pa.). Thin sections for electron microscopy, cut on a Porter-Blum MT2-B ultramicrotome, were stained with Reynold’s lead, and micrographed on a Philips 301 electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.).
Immunoperoxidase
Localization
Studies
Antibodies. Five antibodies (all raised in rabbits) were used in these studies. Affinity-purified immunoglobulin G (IgG) raised against pigeon gizzard tropomyosin and IgG raised against chicken gizzard actin were kind gifts of Dr. Jan DeMey (Beerse, Belgium). The antitropomyosin IgG specifically recognizes smooth muscle tropomyosin in immunoblots and in immunoperoxidase localization studies (13). The specificity of the antiactin IgG for its homol(14). Immunoglobulin ogous antigen has been documented G raised against the heavy chain of human platelet myosin was obtained from Biomedical Technologies, Inc., Cambridge, Mass. Immunoglobulin G directed against the heavy chain of rat intestinal smooth muscle myosin was produced in our laboratory. The specificity of each IgG for the cognate myosin isoform was established by immunoblots and immunoperoxidase localization studies (15). An antiserum raised against bovine lung cyclic guanosine monophosphate (GMP)-dependent protein kinase was a kind gift of Dr. Ulrich Walter (Wurzburg, Federal Republic of Germany). The specificity of this serum for cyclic GMP-dependent protein kinase has been determined by a number of methods (1618). Nonimmune serum was collected from four normal rabbits and nonimmune IgG was prepared from this pool by precipitation with ammonium sulfate and subsequent solubilization and dialysis against PBS. Both nonimmune IgG and serum were stored frozen in the presence of O.OE~~ sodium azide. Fixation. Two fixatives were tested for their ability to retain antigenicity and preserve tissue structure for immunoperoxidase localization. Using the protocol described for morphologic studies, the vasculature was perfused at room temperature for 10 min with 14 mM glucose in PBS and then with PBS containing 4% formaldehyde
70 JOYCE ET AL.
(freshly prepared from paraformaldehyde); the ligated intestinal segment was excised, placed in fresh 4% formaldehyde, cut into small pieces, and further fixed by immersion in the same solution at room temperature for -20 min. Alternatively, the same protocol was followed using a “periodate-lysine-paraformaldehyde” fixative, prepared according to the method of McLean and Nakane (19). In this case, the small pieces cut from the intestinal segment were immersed in the same fixative at 0°C for 3 h. After preparation of the tissue for immunocytochemistry [described below), results of the two fixation methods were compared. Fixation with 4% formaldehyde permitted low concentrations of antigen to be detected, although tissue structure was poorly preserved. Intestinal epithelial cells were occasionally separated from one another and the membranes surrounding mucous droplets in the apical portion of goblet cells were somewhat disrupted. The McLean-Nakane fixative preserved structural integrity to a greater degree; however, antigenicity was reduced and low concentrations of antigen, which had been consistently demonstrable after 4% formaldehyde fixation, were no longer detected. To test for the presence of possibly low concentrations of some contraction-associated proteins, fixation was carried out according to the first protocol in all subsequent experiments. Tissue preparation. The method used for preparation and reaction of tissue sections for immunocytochemistry has been described previously (13). Briefly, tissue pieces, treated for cryoprotection with 10% dimethylsulfoxide in PBS, were frozen in liquid nitrogen-cooled isopentane. Cryostat sections (-32 pm) were cut and residual aldehydes were quenched for 30 min at room temperature in 0.15 M glycine in half-strength PBS. All steps subsequent to quenching were performed at ~4% to minimize structural artifacts. Generally, tissue sections were permeabilized with 0.3% Triton X-100 in PBS supplemented with 0.015 M glycine and 0.1% bovine serum albumin. This same solution was also used as an antibody diluent and washing medium. For the immunolocalization of cyclic GMP-dependent protein kinase, additional permeabilization beyond the original freeze-thaw was unnecessary (20), and therefore Triton X-100 was omitted from the incubation solution. After permeabilization, sections were incubated for 18-24 h in -10 pg/ml of either immune or nonimmune IgG or a 1:50 dilution of immune or nonimmune serum, washed for 18-24 h, and then treated for 12-24 h with 12.5 pg/ml of horseradish peroxidaseconjugated sheep antirabbit IgG Fab (Pasteur Institute, Paris, France). They were subsequently reacted with 3,3’diaminobenzidine (DAB) and 0.01% hydrogen peroxide for lo-60 min, washed in PBS, and then plastic-embedded in PolyiBed 812. Thick sections (-1 pm) of the reacted tissue were examined and micrographed with a Zeiss Photomicroscope II (Carl Zeiss, Inc., Thornwood, N.J.) using Kodak Technical Pan film (Eastman Kodak Co., Rochester, N.Y.). Nomenclature. To facilitate the description of the cells under discussion, the following nomenclature will be used. The elongated cells subtending the villar epithelium will be called subepithelial myofibroblasts. Similar cells subtending the epithelium of the crypts of Lieberkuhn will
GASTROENTEROLOGY Vol. 92. No. 1
be termed pericryptal myofibroblasts, and the stellateshaped cells deep within the lamina propria will be referred to as lamina propria myofibroblasts. Data analysis. The immunoperoxidase localization technique provides a semiquantitative measure of antigen concentration. The color intensity developed in tissue incubated with an antibody and reacted with DAB and hydrogen peroxide reflects the relative concentration of antigen within individual cell types. Several staining intensities can be easily discerned and graded under standardized conditions for the peroxidase reaction. Cells strongly reactive for an antigen stain dark brown to black; moderately reactive cells stain medium brown; weakly reactive cells stain tan to light brown, whereas cells that are not obviously stained above background levels are considered unreactive. By observing the color intensity developed in individual cell types, their relative reactivity for a specific antigen, and therefore the relative concentration of antigen, can be compared. This comparison is valid for the same antigen detected by the same antibody in different cell types within a given tissue specimen. It does not apply for different antigens in the same or different cell types. Several cell types were often positively stained in tissues incubated for each of the proteins tested; however, only the relative reactivities of smooth muscle cells (as an example of a differentiated contractile cell], connective tissue fibroblasts (as an example of a nonmuscle cell), and subepithelial, pericryptal, and lamina propria myofibroblasts will be reported.
Results Morphologic
Studies
For electron subepithelial examined.
studies,
pericryptal
A survey
the presence reticulum
microscopic
and
of their
cell
of a well-developed
and Golgi
complex
(Figure
mitochondria,
icles,
occasional
coated
formed
a plate
including
thelial the
aspect
foot
completely were
filled
frequently
the microfilament bling the dense
(Figure the
lb)
confirmed
endoplasmic la),
as well
vesicles. within
and
Bundles
and
extended they
Thick
appeared
bundles. Fibrillar bodies of smooth
as
ves-
the adepi-
where
cytoplasm.
observed
the were
and cytoplasmic
of the perikaryon
processes
bodies rough
free polysomes, of microfilaments
only
myofibroblasts
into almost
filaments to parallel
densities resemmuscle cells ap-
peared to be associated with microfilaments within both the perikaryon and the cytoplasmic processes. Fibrillar plaques,
thickenings, were present
suggestive of attachment on both the adepithelial and
the abepithelial aspects of the processes. In these regions, a corresponding lamellar condensation of the extracellular matrix was occasionally seen. These focal concentrations seemed to be the only regions of organized matrix associated with these cells, which generally did not appear to elaborate a well-defined basal lamina.
January
Figure
1987
INTESTINAL
CONTRACTILE
CELL NETWORK
71
I. cr. A representative electron micrograph of a portion of the cell body of a subepithelial myofibroblast from the rat intestinal villus illustrates some characteristic features of this cell type. The cell lies adjacent to the basal lamina (BL) of the epithelial cells (EC] and, on its abepithelial aspect, may be closely approached by other cell types that populate the villar lamina propria. A well-developed rough endoplasmic reticulum (RER) and Golgi complex (G) are located within the perinuclear cytoplasm. A plate of microfilaments (MF) lies within the adepithelial aspect of the perikaryon and the proximal portion of the cytoplasmic process (P). Several dense bodies (D) are associated with the microfilament bundles in this region of the cell. CH, chylomicra. Magnification, X31,006. b. Electron micrograph of the more distal portions of several subepithelial myofibroblast foot processes (large arrows) illustrates the high fibrillar content of the cytoplasm in this region of the cell and the multiple layering of the individual processes. Microfilaments and thick filaments [arrowheads) almost completely fill the cytoplasm. Fibrillar densities (D) are associated with microfilament bundles both within the cytoplasm and on the cytoplasmic aspect of the abepithelial plasma membrane. The extracellular matrix forms an organized plate in the region directly opposite the plasmalemma-associated density (curved arrow) forming a hemidesmosomelike structure. Note the coated pit (small arrow) on the adepithelial aspect of a foot process. Magnification, X26.500.
72
JOYCE ET AL.
Figure
GASTROENTEROLOGY
Vol. 92, No. 1
2. The electron micrograph in (a) illustrates three junctions formed by separate cytoplasmic processes with a single subepithelial myofibroblast process. Gap junctions (GJ) are formed between the larger process and two processes probably belonging to two other subepithelial myofibroblasts. (The left junction is enlarged in b; the right junction is enlarged in d.) An adhering junction, shown at the arrowhead in (a) and at the m-row in (c), is characterized by symmetric fibrillar thickenings within the cytoplasmic aspect of the plasma membranes of the two opposing processes. The enlargement of this area in (c) also illustrates more clearly a density (arrowhead) similar to that found in hemidesmosomes, which is present on the abepithelial aspect of the upper process. Note the adepithelial location of microfilaments (mfl in the larger process in (a], as well as the thick filaments (small arrows) also located in this region of the cell. EC, intestinal epithelial cell; BL, basal lamina. Magnification, (a) ~51,000; (b) X148.000; (c) X78.000; (d) ~114,000.
INTESTINAL
CONTRACTILE
CELL NETWORK
73
lanuary
1987
Figure
3. Immunoperoxidase staining for smooth muscle tropomyosin reveals several characteristics of the myofibroblast network within the intestinal mucosa. Darkly stained pericryptal myofibroblasts (arrows in a) form a layered network immediately subjacent to the cryptal epithelium. Around the crypts. the network is reasonably tight, with myofibroblast cell bodies or their processes in close apposition to a relatively large percentage of the epithelial cell basal surface. The network formed by subepithelial myofibroblasts (arrows) in the villus (b) is somewhat looser and thinner, generally consisting of only one to two cell layers. Lamina propria myofibroblasts (LPM) within the villus appear to form a three-dimensional network, with their processes in close apposition to capillaries, subepithelial myofibroblasts, and other cell types that populate this region. Note that the most intense staining is seen in the peripheral cytoplasm and foot processes of the cells. EC, epithelial cells; Ly, lymphatic vessel; SM, smooth muscle cells; La, lacteal; Eo, eosinophil; P, microvascular pericyte. Magnification, (n) x 1000: (b) x 1050.
Subepithelial and pericryptal myofibroblasts formed two types of close contacts with each other, as illustrated in Figure 2. One type of contact, which was frequently observed, resembled an adhering junction in which dense plaques of fibrillar material were present in phase on the cytoplasmic aspect of the two opposing plasma membranes. In three dimensions, however, such structures appeared to involve a small circumscribed area (a macula, rather than a zonule). Communicating (gap) junctions, characterized by their closely apposed, parallel membranes and central gap, were seen less often. In the micrograph in Figure Za, gap junctions are formed by two adjacent cells with a single cytoplasmic process. Immunoperoxidase staining (discussed below) revealed some characteristics of the cellular network within the intestinal mucosa. Pericryptal myofibroblasts formed a relatively tight network, generally consisting of two or three cell layers. Such a network is illustrated in Figure 3a. Subepithelial myofibroblasts within the villus (Figure 3b) were somewhat more loosely arranged and formed a network of one to two cell layers. The multiple layering in both the
crypts and the villi seemed to be local, not general, so that the network appeared somewhat uneven in randomly sectioned specimens. Lamina propria myofibroblasts were relatively numerous and appeared to form a reticular network, coming in close contact with many of the cells populating this area, as well as with subepithelial myofibroblasts. Immunoperoxidase
Localization
Studies
Control experiments. Tissue sections incubated in nonimmune IgG followed by secondary antibody (Figures 4a and 4c) showed no evidence of nonspecific staining beyond that due to endogenous peroxidase activity or nonspecific adsorption of DAB. Intense staining due to endogenous peroxidase activity was visible in leukocytes, especially eosinophils, within the lamina propria and in erythrocytes occasionally seen within capillary lumina (see Figure 8a). In addition, a low intensity staining, probably due to endogenous peroxidase activity, was observable in the granules of Paneth cells located at the base of the crypts of Lieberkiihn (Figure ad). Light staining was also observed in chylomicra oc-
74
Figure
IOYCE ET AL.
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4. Micrographs of sections taken from intestinal villi (a and b) and crypts of Lieberkiihn (c and d] incubated in either nonimmune IgG (a and c) or nonimmune serum (b and d) illustrate typical staining patterns obtained under control conditions. Endogenous peroxidase activity is observable in leukocytes, especially eosinophils (Eo) within the lamina propria and in Paneth cell granules (arrowheads) at the base of the crypts. Note that the eosinophil at the right in (a] has migrated between epithelial cells. No staining is visible in subepithelial (a and b) or pericryptal myofibroblasts (c and d) shown at the arrows. In tissue incubated with nonimmune serum, certain structures (possibly granule membranes) within the apical region of goblet cells (GC) are nonspecifically stained. C, capillary; La, lacteal; Ly, lymphatic vessel: L, intestinal lumen; MM, muscularis mucosae; VSM, vascular smooth muscle cells. Magnification, (a) x1150; (b) x1250; (c) x950; (d] x1200.
casionally found in the intercellular spaces at the base of the intestinal epithelial cells within the villus (Figure 5a). This type of staining may be caused by nonspecific adsorption of DAB, as similar nonspecific adsorption has been observed in lipid droplets in striated muscle cells and in adipose cells incubated in DAB alone (13,151. Control specimens
incubated only with secondary antibody followed by DAB reaction showed no additional nonspecific staining (micrographs not shown). Controls incubated in nonimmune serum followed by horseradish peroxidase-conjugated secondary antibody were very similar to those described above. The only observable difference was the nonspecific
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Figure
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5. Longitudinal section through part of an intestinal villus (a] and transverse section of a crypt (b) taken from specimens incubated with anti-smooth muscle tropomyosin IgG show positive staining in subepithelial, pericryptal, and lamina propria myofibroblasts. Pericryptal myofibroblasts (arrows in b) appear to stain more intensely than subepithelial myofibroblasts (arrows in a). Lamina propria myofibroblasts (LPM). although weakly reactive, are clearly stained above background levels. Smooth muscle cells are strongly reactive, whereas connective tissue fibroblasts (arrowhead in b) are unreactive for this tropomyosin isoform. Note the light staining, probably due to nonspecific adsorption of DAB, in chylomicra [arrowheads in a) at the base of the epithelial cells. SM, smooth muscle cells; Eo, eosinophil; VSM, vascular smooth muscle cells; MM, muscularis mucosae; L, intestinal lumen. Magnification, (a) X1000; (b) X800.
staining of certain structures (possibly mucous granule membranes) located in the apical region of goblet cells incubated with nonimmune serum (Figures 4b and ad). Immunoperoxidase Localization Muscle Tropomyosin
of Smooth
Positive staining for smooth muscle tropomyosin was consistently observed in subepithelial, pericryptal, and lamina propria myofibroblasts, and in smooth muscle cells, as illustrated in Figures 5a and 5b. The smooth muscle cells disposed parallel to the lacteal (Figure 5a) and the cells comprising the muscularis mucosae (Figure Sb], as well as vascular smooth muscle cells, were strongly stained. Pericryptal myofibroblasts were less intensely stained than smooth muscle cells, but were more reactive than subepithelial or lamina propria myofibroblasts. Connective tissue fibroblasts were consistently unreactive for this antigen. In all positive cells, DAB reaction product was diffusely distributed within the cell cytoplasm, whereas the nucleus was unstained. Staining in all three types of myofibroblasts was generally most intense within the foot processes and at the periphery of the cell body. In some sections,
the adepithelial aspect of the perikaryon in both the subepithelial and the pericryptal myofibroblasts appeared more darkly stained than their abepithelial aspect. Immunoperoxidase Localization of Cyclic Guanosine Monophosphate-Dependent Protein Kinase Both subepithelial and pericryptal myofibroblasts reacted positively for cyclic GMP-dependent protein kinase [Figures 6a and 6b). The intensity of reaction in these cells was less than the strongly positive staining seen in smooth muscle cells, however. A comparison of relative staining intensities suggests that subepithelial myofibroblasts were somewhat more reactive than pericryptal myofibroblasts. Both myofibroblasts within the lamina propria and connective tissue fibroblasts appeared unstained. The staining pattern for cyclic GMP-dependent protein kinase was different from that detected for smooth muscle tropomyosin. For the kinase, DAB reaction product was distributed mainly within the cell body and proximal portions of the foot processes of the subepithelial and pericryptal myofibroblasts;
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Figure
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6. These sections, taken from an intestinal villus (a) and a crypt of Lieberkiihn (b), show the comparative reactivity of cells for cyclic GMP-dependent protein kinase. Smooth muscle cells (SM) within the villus and the cells comprising the muscularis mucosae (MM) are strongly positive for cyclic GMP-dependent protein kinase. Subepithelial (a) and pericryptal myofibroblasts (b), shown at the arrows, are less intensely stained, although the myofibroblasts in the villus appear more reactive than those surrounding the crypts. Lamina propria myofibroblasts (LPM) and connective tissue fibroblasts (arrowhead in b) are unstained. *, nucleus of a leukocyte that has migrated between epithelial cells; La. lacteal; L, intestinal lumen. Magnification, (a] x9.50; (b) XIOOO.
in contrast, the more distal segments of their foot processes were either lightly stained or unstained, suggesting that these regions of the cell did not contain significant amounts of antigen. For smooth muscle tropomyosin, the foot processes appeared to be stained over considerably longer distances (compare Figure 5 with Figure 6).
Immunoperoxidase
Localization
of Actin
Subepithelial, pericryptal, and lamina propria myofibroblasts stained positively for actin to about the same extent (Figures 7a and 7b), but the intensity of their reaction was less than that of smooth muscle cells. The actin staining patterns of the three cell types were similar to those generated by staining for smooth muscle tropomyosin: in both cases, diffuse reaction product was visible in the perinuclear cytoplasm, as well as in the foot processes. Staining of the peripheral cytoplasm within a lamina propria myofibroblast is particularly well-illustrated in Figure 7a. The brush border and basolateral regions of the epithelial cells were intensely stained for actin, whereas connective tissue fibroblasts were only weakly reactive.
lmmunoperoxidase Isomyosins
Localization
of
Nonmuscle myosin. In tissue sections stained for nonmuscle myosin, subepithelial, pericryptal, and lamina propria myofibroblasts, as well as connective tissue fibroblasts, all reacted positively (Figures 8a and 8b), whereas smooth muscle cells were consistently unreactive. Pericryptal myofibroblasts appeared to be somewhat more intensely stained for nonmuscle myosin than either subepithelial or lamina propria myofibroblasts; however, all these cells were more strongly positive than connective tissue fibroblasts, which appeared only weakly reactive. The staining patterns for all these cells were comparable to those obtained for actin and smooth muscle tropomyosin. Smooth muscle myosin. As shown in Figures 9a and 9b, subepithelial and pericryptal myofibroblasts stained weakly for smooth muscle isomyosin. Although the reactivity was above background level, the staining intensity was much less than that observed for smooth muscle cells. Pericryptal myofibroblasts stained somewhat more intensely than subepithelial myofibroblasts and both cell types were more reactive than myofibroblasts within the lamina propria. Connective tissue fibroblasts ap-
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Figure
7. Part of a longitudinal section through an intestinal villus (a] and an oblique (grazing] section of two crypts of Lieberkiihn (b) illustrate the positive reaction of subepithelial (arrows in a), pericryptal (arrows in b), and lamina propria myofibroblasts (LPM) for actin. All three cell types contain DAB reaction product, particularly toward the periphery of the cell body, and within the foot processes. This staining pattern is clearly evident in the lamina propria myofibroblast in (a). The staining intensity for actin in these cells is less than that in the smooth muscle cells of the muscularis mucosae (M), seen in (b). Connective tissue fibroblasts (arrowhead in b) are weakly reactive. Staining for actin is intense in the brush border and periphery of epithelial cells and in migrating leukocytes (*). L. intestinal lumen. Magnification, (a) x 1300; (b) X 1000.
Figure
8. Sections of the lamina propria of a villus (a) and a crypt of Lieberkiihn (b) illustrate the positive reactivity of subepithelial (arrows in a), pericryptal (arrows in b), and lamina propria myofibroblasts (LPM) for nonmuscle isomyosin. Diffuse DAB reaction product is present within the perikaryon and foot processes of these cells. Connective tissue fibroblasts (arrowhead in b) also react positively, but are weakly stained. Epithelial cells in both the villus and crypt, as well as the lacteal endothelium (a) stain diffusely, whereas smooth muscle cells (SM) are consistently unreactive for nonmuscle isomyosin. Arrowhead in (a) indicates dark staining of erythrocytes due to endogenous peroxidase activity; *, nucleus of a leukocyte that has migrated between two epithelial cells; Eo, eosinophil; La, lacteal; L, intestinal lumen. Magnification, (a) x950; (b) ~1450.
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Figure
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9. These longitudinal sections of the lamina propria within a villus (a) and a crypt of Lieberktihn (b) illustrate relatively weak reactivity of subepithelial and pericryptal myofibroblasts (arrows) for smooth muscle isomyosin. Pericryptal myofibroblasts appear somewhat more reactive than subepithelial myofibroblasts, whereas lamina propria myofibroblasts (LPM) and connective tissue fibroblasts (arrowheads in b) appear unreactive. Smooth muscle cells (SM) are strongly reactive for this myosin isoform. Eo, eosinophil; La, lacteal; C, capillary; L, intestinal lumen. Magnification, (a] x1159; (b) x959.
peared unreactive. As with nonmuscle isomyosin, diffuse reaction product was visible in both the perinuclear cytoplasm and foot processes of the cells. The signal-to-background ratio did not increase in these cells with longer periods of reaction with DAB and hydrogen peroxide, and attempts to intensify the staining by published techniques (21-23) were unsuccessful.
Discussion Electron
Microscopic
Observations
The results obtained from our electron microscopic study of intestinal subepithelial and pericryptal myofibroblasts confirmed many of the observations made by Pitha (3, Desaki et al. (2), and Giildner et al. (3). Specifically, this applies to the presence of both thick and thin filaments within the adepithelial cytoplasm of the perikaryon and foot processes. In addition, we extended the observations by identifying structures within the cytoplasm that have the morphology of dense bodies. Their presence within both subepithelial and pericryptal myofibroblasts, as well as the presence of densities similar to adhesion plaques located on both the adluminal and abluminal aspects of their plasmalemma, suggests that these cells contain a putative contractile apparatus capable of generating and transmitting force. In our studies, we further characterized the “close
contacts” formed by subepithelial and pericryptal myofibroblasts. We confirmed the fact that gap junctions are present between these cells and assume that such junctions permit communication among several adjacent cells within the network as suggested by the finding (Figure 2a) that a single foot process can form gap junctions with at least two neighboring cells. In addition, adhering junctions are frequently observed connecting the cells within the network. The morphologic evidence thus obtained confirms and extends previous findings and supports the hypotheses advanced by both Giildner et al. (3) and Desaki et al. (2) according to which these cells form a cellular network, and are capable of communication with one another and of transmitting the force generated during their contraction both to neighboring cells and to elements of the extracellular matrix. lmmunoperoxidase Localization Contraction-Associated Proteins
of
The results of the immunoperoxidase localization studies provide direct evidence for the presence of at least four contraction-associated proteins in subepithelial, pericryptal, and lamina propria myofibroblasts. Both actin and myosin are considered essential for contractility, and tropomyosin is thought to be required for efficient regulation of
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Table
1. Relative
Reactivities
of Intestinal
for Four Contraction-Associated
Cell type Smooth muscle cells Pericryptal myofibroblasts Subepithelial myofibroblasts Lamina propria myofibroblasts Connective tissue fibroblasts ” cGK, cyclic GMP-dependent
Myofibroblasts, Connective Tissue Fibroblasts, and Smooth Muscle Cells Proteins
Smooth muscle tropomyosin +++ +++to++ ++ + protein kinase.
79
cGK” +++ ++to+ ++ -
+ + + , Strongly reactive;
acto-myosin interaction in smooth muscle (24,25) and nonmuscle cells (26,27), as well as in skeletal muscle. The role of cyclic GMP-dependent protein kinase in cellular contraction is not yet fully understood; however, it has been suggested (28,29) that this enzyme is involved in smooth muscle cell relaxation. of these Table 1 shows the relative reactivities cells for each of the proteins studied. In general, the intensity of the reaction in subepithelial, pericryptal, and lamina propria myofibroblasts was less than that found for either visceral or vascular smooth muscle cells, but was greater than the reactivity of cells differentiated for functions other than contraction, such as connective tissue fibroblasts. The results also indicate that the relative concentration of each of these proteins differs in each of the three cell types under study. Pericryptal myofibroblasts were somewhat more reactive for contraction-associated proteins (except for cyclic GMP-dependent protein kinase) than subepithelial myofibroblasts, and both of these cell types appeared more reactive than myofibroblasts within the lamina propria, indicating a gradation in the amount of contraction-associated proteins, and thus possibly a modulation in contractile activity, within these three cell types. The presence of cyclic GMP-dependent protein kinase in these cells, as well as the smooth muscle isoforms of tropomyosin and myosin suggest that subepithelial, pericryptal, and lamina propria myofibroblasts are rather closely related to smooth muscle cells in terms of the contraction-associated proteins and isoforms they contain. The fact that they stain positively for both nonmuscle and smooth muscle isomyosin suggests, however, that their contractile protein equipment is not identical with that of smooth muscle cells. Therefore, their particular type of contraction, and possibly its mode of regulation, may differ from that of the latter. The results reported in this study are generally similar to those obtained for the in situ immunoperoxidase localization of the same contraction-associated proteins in microvascular pericytes (13,15,
Actin
Nonmuscle isomyosin
+++ ++ ++ ++ +
++ ++ ++ +
+ + , moderately
reactive;
+ , weakly reactive; -,
Smooth muscle isomyosin +++ ++to+ + unreactive.
20). Overall comparison of the findings indicates that microvascular pericytes are generally more intensely stained for each of the proteins tested than are the subepithelial or pericryptal myofibroblasts. The immunocytochemical results presented in this study establish that subepithelial, pericryptal, and lamina propria myofibroblasts contain essential contraction-associated proteins in concentrations approaching those found in microvascular pericytes and smooth muscle cells and distinctly higher than those detected in cells differentiated for other, noncontractile functions. The morphologic findings suggest that these cells contain a putative contractile apparatus, as well as a means for intercellular communication and force-generation. Taken together, these results provide strong evidence that the three types of fibroblastlike cells found within the rat intestinal mucosa are differentiated for a contractile function. There is still insufficient information to allow a specific determination as to the direction of force that may be generated by these cells as well as to the effects of their contraction. However, the structural arrangement indicated by both electron and light microscopy suggests that they form an essentially two-dimensional cellular network subjacent to the intestinal epithelium and a three-dimensional network within the lamina propria of the intestinal villi. Tension generated by the contraction of such cellular networks may provide a supportive tonus to the epithelium, enabling it to withstand forces that may deform or dislocate it. It may also provide the force needed for active movement of the villi, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and the lacteals of the intestinal villi. The evidence presented in this study, although highly suggestive, does not prove that intestinal mucosal myofibroblasts contract in vivo. There is a similar lack of direct evidence for in vivo contraction in studies of the intestinal epithelial cell brush border. Biochemical (30-34), morphologic (35,36), and immunocytochemical localization studies (31,
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37-39) together have established the presence of essential contractile proteins within the terminal web region in a spatial arrangement suggestive of a contractile function; however, brush border contraction has only been observed in disrupted systems (30,32,33). New approaches must be applied to the study of both these cell types in order to understand more fully their contractile function in vivo.
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16. Lohmann SM, Walter U, Greengard P, DeCamilli P. Immunohistochemical localization of cyclic GMP-dependent protein kinase in mammalian brain. Proc Nat1 Acad Sci USA 1981; 78:653-7. 17. Walter U. Distribution of cyclic GMP-dependent protein kinase in various rat tissues and cell lines determined by a sensitive and specific radioimmunoassay. Eur J Biochem 1981:118:339-46, 18. Walter U, Miller P, Wilson F, Menkes D, Greengard P. Immunological distinction between guanosine 3’:5’-monophosphate-dependent and adenosine 3’:5’-monophosphatedependent protein kinases. J Biol Chem 1980;255:3757-62. 19. McLean IW, Nakane PK. Periodate-lysine-formaldehyde fixative. A new fixative for immunoelectron microscopy. J Histothem Cytochem 1974;22:1077-83. 20. Joyce NC, DeCamilli P, Boyles J. Pericytes, like vascular smooth muscle cells, are immunocytochemically positive for cyclic GMP-dependent protein kinase. Microvasc Res 1984; 28:206-19. 21. Adams JC. Technical considerations on the use of horseradish peroxidase as a neuronal marker. Neuroscience 1977;2:141-5. 22. Gallyas F. Gores T, Merchenthaler I. High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J Histochem Cytochem 1982;30: 183-4. 23. Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18:315-33. 24. Chacko S. Effects of phosphorylation. calcium ion, and tropomyosin on actin-activated adenosine 5’-triphosphatase activity of mammalian smooth muscle myosin. Biochemistry 1981;20:702-7, A, Small JV. Regulation of the actin-myosin inter25. Sobieszek action in vertebrate smooth muscle: activation via a myosin light-chain kinase and the effect of tropomyosin. J Mol Biol 1977;112:559-76. N. Tropomyosin enhances actomyosin 26. Onji T, Shibata ATPase activity in platelets. Biophys Biochem Res Commun 1982;109:697-703, kinetic studies on the actin activa27. Sobieszek A. Steady-state tion of skeletal muscle heavy meromyosin subfragments. J Mol Biol 1982;157:275-86. and biological role 28. Lincoln TM, Corbin JD. Characterization of the cGMP-dependent protein kinase. Adv Cyclic Nucleotide Res 1983;15:139-92. TM, Johnson RM. Possible role of cyclic GMP29. Lincoln dependent protein kinase in vascular smooth muscle function. Adv Cyclic Nucleotide Res 1984;17:285-96. R, Newman SB, Karnovsky MJ. Contraction of 30. Rodewald isolated brush borders from the intestinal epithelium. J Cell Biol 1976;70:541-54. MS, Pollard TD, Fujiwara K. Characterization and 31. Mooseker localization of myosin in the brush border of intestinal epithelial cells. J Cell Biol 1978;79:444-53. TCS, Mooseker MS. Ca++-calmodulin-dependent 32. Keller phosphorylation of myosin, and its role in brush border contraction in vitro. J Cell Biol 1982;95:943-59. con33. Broschat KO, Stidwell RP, Burgess DR. Phosphorylation trols brush border motility by regulating myosin structure and association with the cytoskeleton. Cell 1983:35:561-71. KA. Chasan R, Mooseker MS. The role 34. Keller TCS, Conzelman of myosin in terminal web contraction in isolated intestinal epithelial brush borders. J Cell Biol 1985;100:1647-55. N, Keller TCS, Chasan R, Mooseker MS. Mecha35. Hirokawa
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nism of brush border contractility studied by the quickfreeze-deep-etch method. J Cell Biol 1983;96:1325-36. 36. Hirokawa N, Tilney L, Fujiwara K, Heuser JE. The organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells. J Cell Biol 1982;94: 425-43. 37. Bretscher A, Weber K. Localization of actin and microfilament associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J Cell Biol 1978;79:839-45.
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38. Drenckhahn D, Groschel-Stewart U. Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels. J Cell Biol 1980;86:475-82. 39. Geiger B, Dutton A, Tokuyasu T, Singer S. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol 1981;91:614-28.