close

Вход

Забыли?

вход по аккаунту

?

Intracellular variation of rat intestinal mucin granules localized by monoclonal antibodies.

код для вставкиСкачать
THE ANATOMICAL RECORD 230:513-518 (1991)
lntracellular Variation of Rat Intestinal Mucin
Granules Localized by Monoclonal Antibodies
MARY G. OLIVER AND ROBERT D. SPECIAN
Department of Cellular Biology and Anatomy, Louisiana State University Medical Center,
Shreveport, Louisiana 71130
ABSTRACT
Monoclonal antibodies produced against rat small intestinal mucins were utilized to study variability of stored mucin granules within rat ileal
goblet cells. Eleven antibody-secreting hybridoma cultures were produced; six of
these uniformly labeled stored mucin granules in virtually all goblet cells, suggesting that some antigenic features are common to all granules. The other five
stained goblet cells in the rat small intestinal epithelium nonuniformly. R803,
R805, and R807 localized within almost all goblet cells but revealed differential
labeling of centrally and peripherally located mucin granules. R804 uniformly
labeled the mucin granules of most villous goblet cells; some of the crypt goblet
cells were uniformly labeled, but the majority were only partially labeled, resulting in a mottled staining pattern. R808 stained only a small portion of crypt goblet
cells; there is, however, a n increase in both number of goblet cells labeled and in
uniformity of staining of the stored granule mass from the base of the crypt to the
surface, resulting in uniform labeling of virtually all goblet cells at the villus tip.
This study demonstrates for the first time that rat small intestinal mucin granules
are immunologically heterogeneous and nonuniformly distributed within the epithelium. Additionally, staining patterns within the stored granule mass suggest
that structurally distinct subpopulations of mucin granules may exist within a
single goblet cell.
Goblet cells are highly polarized exocrine cells distributed along the length of the intestinal tract. They
synthesize mucins, high molecular weight (-2 x lo6
daltons) glycoproteins that are packaged into mucin
granules that are contained in a tightly packed apical
granule mass. Preliminary evidence suggests that a
mannose-containing glycoprotein tightly associated
with mucins is also co-packaged into mucin granules
(Sibley and Specian, 1990). It has been suggested (Florey, 1962)that mucins have three functions in the gastrointestinal tract: 1)to protect the mucosa by forming
a physical barrier to chemical and physical irritants; 2)
to lubricate luminal contents and facilitate their movement; and 3) to entrap and agglutinate parasites in the
gut for elimination.
Histochemical studies in normal human colon (Filipe, 1969)and in normal rat colon (Thomopoulos et al.,
1983) have shown that more than one distinct mucin
type can be distinguished based upon carbohydrate
composition. These studies have also shown that the
population of goblet cells is heterogeneous with respect
to the type of stored mucin and that there are qualitative and quantitative differences in stored mucins between different regions of the gastrointestinal tract.
Biochemical analyses of rat colonic (Lamont and
Ventola, 1980), human colonic (Podolsky and Isselbacher, 19831, and human small intestinal mucins
(Wesley e t al., 1985) have demonstrated that more
than one chemically distinct species of mucin glycoprotein exists and that these mucin species vary in both
carbohydrate and amino acid composition. UnfortuQ 1991 WILEY-LISS, INC
nately, the differences between biochemically distinct
mucin species are not apparently dramatic enough to
account for differential staining properties, and no direct correlation has so far been made between histochemical and biochemical analyses. Biochemical analyses of mucins in ulcerative colitis (Podolsky and
Isselbacher, 1984) and in cystic fibrosis (Wesley et al.,
1983) have demonstrated that in these disease states
there are qualitative and quantitative alterations in
the mucin glycoproteins produced. These studies suggest that the individual mucin species may serve specific physiological functions in the intestinal tract. Biochemical analysis, however, leaves unclear whether
these glycoproteins are normally produced by all goblet
cells, or whether the production of individual mucin
species is relegated to distinct subpopulations of goblet
cells.
To attempt to localize distinct mucin glycoproteins
within the mucosa, current studies have now focused
upon detecting these mucin species by immunocytochemical methods. Podolsky and co-workers (1986),
using specific monoclonal antibodies to human colonic
mucins, have demonstrated distinct subpopulations of
goblet cells, containing specific species or combination
of species of mucin glycoproteins. The goals of the
present study are to demonstrate, by using monoclonal
Received June 14, 1990; accepted December 17, 1990
514
M.G. OLIVER AND R.D. SPECIAN
antibodies against rat small intestinal glycoproteins,
the existence of immunologically distinct subpopulations of goblet cells in rat small bowel. Furthermore,
mucin granules within a n individual goblet cell are
immunologically heterogeneous, and there are unique
storage patterns of structurally distinct mucin granules. The purpose of this report is to present the preliminary results of this study.
METHODS
Preparation of Rat Intestinal Mucin
All reagents are from Sigma Chemical Corporation,
St. Louis, MO, unless otherwise specified.
Male Wistar rats (150-300 g) were fasted overnight
and anesthetized by a n intraperitoneal injection of sodium pentobarbital. The intestinal epithelium was separated from the underlying tissue by vascular perfusion with 20 mM EDTA (ethylenediaminetetraacetic
acid) in 0.9% sodium chloride in phosphate buffer
(PBS) a t pH 7.3 for 5 minutes (modified after Bjerknes
and Cheng, 1981).The small intestine from the ligament of Trietz to the cecum was removed, bisected, and
then slit open. The mucosal epithelium was gently
scraped from the basement membrane with a microscope slide. This results in large quantities of intestinal
epithelium with virtually no contamination from underlying tissues.
Enrichment of rat intestinal mucin glycoproteins
was performed by modification of the method of Lamont and Ventola (1980). Isolated epithelium was homogenized in 0.1 M dibasic potassium phosphate buffer
with 5 mM EDTA at pH 7.3 for 6 minutes at 4°C. The
homogenate was centrifuged 2 x 60 minutes a t
lOO,OOOg, dialyzed against distilled water, resuspended
in 0.1 M phosphate buffer at a concentration of 10 mgl
ml, and centrifuged for one hour and 100,OOOg. The
high molecular weight glycoproteins were then enriched by gel filtration through a Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) column (2.4 x
40 cm). The void volume was determined independently with blue dextran. Void volume fractions were
pooled, dialyzed against distilled water, and lyophilized. Antibodies prepared against glycoproteins purified by this method localize exclusively within mucin
granules in goblet cells (Specian and Oliver, 1990).
Preparation of Antiserum Against Rat Small
Intestinal Mucins
Balblc mice were immunized with 200 pg of purified
mucins suspended in 0.2 ml of PBS emulsified with
Freund's complete adjuvant via three subcutaneous injections over 6 to 12 weeks. Mice were then bled by
cutting the distal region of the tail vein and collecting
blood in a microcentrifuge tube. Blood was allowed to
clot at room temperature for 30 min and was centrifuged in a clinical centrifuge for 10 min. Serum was
carefully decanted into another microcentrifuge tube
and centrifuged again for 10 min. Indirect immunofluorescence of the antiserum demonstrated localization of the antiserum to the apical granule mass of
intestinal goblet cells, results similar to antiserum produced in rabbit against this antigen (Specian and 01iver, 1990). No brush border or membrane localization
was evident.
Production of Hybridomas
Balblc mice were immunized with 200 yg of purified
mucins suspended in 0.2 ml of PBS emulsified with
Freund's complete adjuvant via three subcutaneous injections over 6 to 12 weeks and then were intravenously boosted with 200 pg of purified mucins suspended in 0.9% sodium chloride 4 days before fusion.
Four days after boosting, mice were sacrificed by cervical dislocation. The spleens were removed under sterile conditions and the cells were dispersed by passage
through a wire-mesh screen. Immunized mouse spleen
cells (4.5 x lo7) were fused with NS-1 myeloma cells
( 3 x 1 0 7 in RPMI 1640 (GIBCO, Grand Island, NY)
containing 40% polyethylene glycol by the method of
Gefter et al. (1977). Fusion products were dispensed
into 96-well culture plates a t a density of 7.5 x lo5 cells
per well. Microcultures were then maintained in RPMI
medium supplemented with 10% fetal calf serum and
containing 1x lo5 M hypoxanthine, 4 x lo7 M aminopterin, and 1.6 x lo5 M thymidine. Cultures producing
antibody were cloned by serial dilutions and expanded.
Screening for Antibody Production
At 14 days, microcultures were assayed for antibody
production by a n ELISA (modified from Pool et al.,
1983). Supernatants from each microculture were incubated overnight a t 4°C with a polystyrene ball coated
with antigen and blocked with bovine serum albumin
(BSA) and ovalbumin. A positive control (mouse antiserum) and a negative control (2% BSA in PBS) were
run with each set of supernatants. The balls were
rinsed with PBS/0.2% BSA/0.05% Tween and incubated with peroxidase conjugated to goat antimouse
IgG for 2 hours at 24°C. Balls were rinsed and incubated with substrate (0.04% o-phenylenediaminel
0.001% H202in citratelphosphate buffer, pH 5.0) for 20
min. The reaction was stopped by addition of 2.4 M
H2S04.Absorbance was read on a Hitachi spectrophotometer a t 492 nm. Supernatants with absorbance
readings at 60% of the positive control or higher were
judged to be producing antibody.
lmmunocytochemistry
Segments of ileum from male Wistar rats were immersion fixed with 2% paraformaldehyde in 0.1 M potassium phosphate buffer, rinsed, dehydrated to 95%
ethanol, and embedded in glycol methacrylate (JB-4,
Polysciences, Warrington, PA). This preparation results in preservation of intact mucin granules in both
villous and crypt goblet cells with little loss in antigenicity (Oliver et al., 1990). Immunocytochemical localizations were performed on 1.5 pm sections pretreated
with 0.3% H202in methanol to block endogenous peroxidase (Streefkrek, 1972) and ovalbumin and BSA to
block nonspecific staining. Sections were incubated
overnight a t 4°C with supernatant; a positive control
(mouse antiserum) and a negative control (2% BSA in
PBS) were run with each set of supernatants. Sections
were rinsed with PBSIBSAlTween and incubated with
peroxidase conjugated to goat antimouse IgG for 2
hours a t 24°C. Sections were rinsed and incubated with
substrate (0.05% diaminobenzidine with 0.001% H202
in 0.1 M Tris-HC1 buffer) for 20 min (Nakane and
Pierce, 1966). Sections were washed with distilled wa-
MONOCLONAL ANTIBODIES AGAINST RAT MUCINS
Figs. 1-8. All figures are immunoperoxidase localizations of either
mouse antiserum to rat small intestinal glycoproteins or a monoclonal
antibody to rat small intestinal glycoproteins. All slides have been
counterstained with Mayer’s hematoxylin.
515
Fig. 1. Localization of mouse antiserum results in uniform staining
of the stored granule mass in all goblet cells in the crypts and on the
villi. x 240.
Fig. 2. Localization of R801 is indistinguishable from antiserum. All
goblet cells have their stored granule mass completely labeled. x 240.
ter and counterstained with Mayer’s hematoxylin.
Slides were coverslipped and photographed on a n
Olympus Vanox microscope.
RESULTS
Immunocytochemistry of antisera from mice immunized for spleen-harvest demonstrated uniform localization in the mucin granule mass of goblet cell
throughout the epithelium, including those deep
within the crypt (Fig. 1).The two fusions performed
with immunized mouse spleens and mouse myeloma
cells produced 11 antibody-producing hybridoma cell
lines. Of these 11, i t was found that six of these cultures produced antibody that uniformly labeled secretory granules in all villous goblet cells and virtually all
crypt goblet cells except those near the crypt base (Fig.
21, a staining pattern indistinguishable from that of
mouse antisera (Fig. 1).The other five produced antibodies against epitopes that demonstrated a large degree of heterogeneity between surface and crypt goblet
cells, between adjacent crypts, and between secretory
granules in a single goblet cell. None of the antibodies
localized within the brush border.
R803 localizes within the apical granule mass of all
surface goblet cells and most crypt cells except those
found deep within the crypt (not illustrated). The majority of goblet cells had patches of stain dispersed
throughout the granule mass. Additionally, there were
goblet cells that contained labeled mucin granules
within only the center of the granule mass.
Immunocytochemical localization of R805 reveals
that the majority of goblet cells contain mucin granules
that are immunoreactive with this antibody. Within
the crypts and on the villi the majority of the goblet
cells have their apical granule mass labeled uniformly.
Interspersed among the uniformly labeled cells exists a
subpopulation of goblet cells that contain few or no
immunoreactive granules (Figs. 3, 4).Interestingly, i t
is only the centrally stored granules in those partially
stained cells that react to R805, suggesting that there
is spatial segregation of glycoproteins that contain this
antigenic determinant.
R807 labels the majority of goblet cells both on the
villi and within the crypts; however, the granules do
not label uniformly. In the goblet cells, R807 localizes
sparsely but uniformly within the granule mass of
crypt goblet cells (Fig. 5). but in some villous cells i t
localizes more heavily within the peripherally located
granules (Fig. 6). This staining pattern suggests that
there are organizational distinctions between the immature crypt goblet cells and the more mature surface
goblet cells.
In a smaller fashion R804 stains both villous and
crypt goblet cells irregularly (not illustrated). In the
Figs. 3-8.
MONOCLONAL ANTIBODIES AGAINST RAT MUCINS
517
crypts some of the goblet cells are stained uniformly, crypts, and sulphated mucins in colonic crypts. Biobut many of them have only a portion of their granules chemical analyses of human colonic (Podolsky and Isstained. This results in the partially stained cells hav- selbacher, 1983) and small intestinal mucins (Wesley
ing a “mottled” granule mass. On the villi, the majority et al., 1985) a s well as r a t colonic mucins (Lamont and
of the goblet cells are uniformly stained; the minority Ventola, 1980) have demonstrated multiple mucin speof villous goblet cells contain both labeled and unla- cies. The differences that defined a fraction as a mucin
species, however, were more numerous and more subtle
beled granules.
All crypts contain a subpopulation of goblet cells that than was suggested by histochemical studies. Previous
are labeled by R808, but rarely are all cells stained. studies in human colon have demonstrated, by using
Cells a t the base of the crypt are either unstained or monoclonal antibodies, that distinct subpopulations of
contain very little immunoreactive material. Goblet goblet cells exist (Podolsky et al., 1986). Results in this
cells in the middle to upper crypt vary greatly in the study strongly suggest that, in rat ileum, goblet -cells
amount of labeling within the granule mass. Uni- exist as a heterogeneous population. This variability,
formly labeled goblet cells are found adjacent to those however, is not as dramatic a s suggested by histochemthat are either partially unstained or unstained (Fig. ical studies. Although a large degree of antigenic
7). At the villous base, cells that are only partially similarity of mucin granules stored in the mucosa is
stained have the labeled granules located either within evident, specific differential staining patterns are dethe central region or a t the apex of the granule mass monstrable, not only within the epithelium as de(Fig. 8). On the upper region of the villus all cells are scribed by Podolsky and co-workers (Podolsky et al.,
uniformly labeled. Thus, it appears that the reactivity 1986), but also within distinct subpopulations of granto R808 increases from the base of the crypt to the tip ules within a single cell.
Goblet cells arise by mitosis from multipotential
of the villus.
stem cells a t the base of the crypt and migrate to the
DISCUSSION
villus tip, where they die and are sloughed into the
Histochemical studies on intestinal goblet cells have lumen (Chang and Leblond, 1971). During the migrasuggested that goblet cell mucins in both human colon tion the cells mature and undergo profound morpho(Filipe, 1969) and rat colon (Thomopoulos et al., 1983) logic changes that ultimately produce the characterisare heterogeneous due to variable carbohydrate con- tic, highly polarized shape (Radwan et al., 1990). Due
tent. These studies demonstrated that not only are mu- to the common ancestry, i t would be reasonable to ascins present a s acidic, neutral, and sulphated classes, sume that goblet cells within a crypt would synthesize
but also t h a t they are distributed unequally along the the same glycoprotein(s); however, there are marked
length of the intestinal tract. Acidic mucins predomi- immunologic differences a s recognized by monoclonal
nate on the upper villus and colonic surfaces, neutral antibody between adjacent cells. Four of the 11 antimucins on lower villus surfaces and in intestinal bodies produced did not stain stored mucins within all
goblet cells of a n individual crypt; i t thus appears then
that although goblet cells within a crypt may have
originated with the ability to produce the same products, the synthetic mechanism within a n individual
Fig. 3. R805 localization in crypt goblet cells. The majority of goblet
cell can vary which chemical structures it will incorcells in the crypt label uniformly. A small subpopulation of goblet
cells exist within the epithelium that have only the centrally stored porate into its apical granule mass.
Not only do there appear to be different subpopulagranules labeled (arrows). x 500.
tions of goblet cells in the mucosa, there also appear to
Fig. 4. R805 localization in villous goblet cells. On the villus, the
be immunologically distinct populations of mucin granmajority of goblet cells contain uniformly stained mucin granules.
ules within a single goblet cell. Under baseline condiInterspersed among these are goblet cells that only contain labeled
granules within the center ofthe apical granule mass (arrows). X 500. tions goblet cells preferentially secrete peripherally located granules; centrally located granules appear to
Fig. 5. R807 localization in crypt goblet cells. Virtually all goblet
not
turn over, and remain a static population. In concells within the crypt are labeled sparsely, with labeled glycoproteins
trast, during a n accelerated secretory event, such a s
threaded through the granule mass. An occasional unlabeled goblet
stimulation by cholinergic agents or luminal irritants,
cell (asterisk) is found in the lower to middle crypt. X 500.
mucus release is accomplished by compound exocytosis
Fig. 6. R807 localization in villous goblet cells. All goblet cells on
of predominantly centrally stored granules (Specian
the villus are found to contain immunoreactive glycoproteins. Only a
and Neutra, 1980,1982). In the present study the proportion of the total stored glycoproteins are labeled and are intermixed with the unlabeled glycoproteins. While labeled granules are duction of a n antibody that preferentially localizes
found throughout the granule mass, the antibody localizes more
within the central granule mass or along the periphery
heavily within the peripheral granules. X 500.
suggests that mucin granules secreted under baseline
conditions to maintain the mucus layer are antigeniFig. 7. R808 localization in crypt goblet cells. Within the crypt,
cally different than mucin granules that are secreted in
there exists a distinct subpopulation of goblet cells that are entirely
nonreactive to R808 (asterisks). This subset of goblet cells coexists response to a stimulus. Previous studies on human cowith one that is uniformly labeled with the antibody. In addition, a n
lonic mucins have demonstrated that, under baseline
intermediate group containing only a portion of their stored mucin
conditions, mucosal explants maintained in organ culgranules labeled can be found. X 500.
ture preferentially secrete certain mucin species into
Fig. 8. R808 localization in villous goblet cells. At the base of the
the media while retaining other species in the epithevillus the population of goblet cells that are intermediate between
lium
(Smith and Podolsky, 1987). These data suggest
those labeled and those unlabeled. In these two goblet cells only the
centrally and apically located granules are immunoreactive to R808. that intestinal goblet cells, under defined physiological
conditions, secrete mucins with a specific structure and
x 500.
518
M.G. OLIVER AND R.D. SPECIAN
chemistry t h a t will be functionally advantageous to
the organism.
In conclusion, we have demonstrated that rat goblet
cells, as has been previously documented for human
goblet cells, constitute a heterogeneous population
based on the products that they synthesize. Additionally, antigenically distinct granules can be found
stored within a single goblet cell, with the granules
segregated into structurally, and perhaps, functionally
distinct subpopulations.
ACKNOWLEDGMENTS
The authors thank Ms. Kimberly Robinson for her
technical assistance and Dr. Jerry W. Pickering for his
invaluable guidance in establishing a hybridoma facility. This study was supported by NIH grant #DK
33720 and a grant from the Cystic Fibrosis Foundation.
LITERATURE CITED
Bjerknes, M., and H. Cheng 1981 Methods for isolation of intact epithelium from the mouse intestine. Anat. Rec., 199565-574.
Chang, W.W.L., and C.P. Leblond 1971 Renewal of the epithelium in
t h e descending colon of the mouse. Am. J . Anat., 131t73-100.
Filipe, M.I. 1969 Value of histochemical reactions for mucosubstances
in the diagnosis of certain pathological conditions of the colon and
rectum. Gut, 10577.
Florey, H.W. 1962 Secretion and functional of intestinal mucus. Gastroenterology, 43r326-329.
Gefter, M.L., D.H. Margulies, and M.D. Scharff 1977 A simple method
for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet., 3t231-236.
Lamont, J.T., and A S . Ventola 1980 Purification and composition of
colonic epithelial mucin. Biochim. Biophys. Acta, 626t234-343.
Nakane, P.K., and G.B. Pierce 1966 Enzyme-labeled antibodies: preparation and application for the localization of antigens. J . Histochem. Cytochem., 14t929-93 1.
Oliver, M.G., S.S. Wiggins, and R.D. Specian 1990 Tissue preparation
for immunocytochemical localization of goblet cell mucin. Trans.
Am. Microsc. SOC.,IO9r205-212.
Podolsky, D.K., D.A. Fournier, and K.E. Lynch 1986 Human colonic
goblet cells: Demonstration of distinct subpopulations defined by
mucin-specific monoclonal antibodies. J. Clin. Invest., 77t12631271.
Podolsky, D.K., and K.J. Isselbacher 1983 Composition of human colonic mucin. Selective alteration in inflammatory bowel disease.
J . Clin. Invest., 72t142-153.
Podolsky, D.K., and K.J. Isselbacher 1984 Glycoprotein composition
of colonic mucosa. Specific alterations in ulcerative colitis. Gastroenterology, 87t991-998.
Pool, Chr.W., R.M. Buijs, D.F. Swaab, G.J. Boer, and F.W. Van Leeuwen 1983 On the way to a specific immunocytochemical localization. In: Immunohistochemistry. A.C. Cuello, ed. John Wiley and
Sons, New York, pp. 1-46.
Radwan, K.A., M.G. Oliver, and R.D. Specian 1990 Cytoarchitectural
reorganization of rabbit colonic goblet cells during baseline secretion. Am. J. Anat., I89:in press.
Sibley, D.A., and R.D. Specian 1990 Intestinal peroxidase, a secretory
product of intestinal goblet cells. J. Cell Biol., lIlt441A.
Smith, A.C., and D.K. Podolsky 1987 Biosynthesis and secretion of
human colonic mucin glycoproteins. J. Clin. Invest., 80t300-307.
Specian, R.D., and M.R. Neutra 1980 Mechanism of rapid mucus secretion in goblet cells stimulated with acetylcholine. J. Cell Biol.,
85t626-640.
Specian, R.D., and M.R. Neutra 1982 Regulation of goblet cell secretion. I. Role of parasympathetic stimulation. Am. J . Physiol.,
242(Gastrointest. Liver Physiol. 5itG370-379.
Specian, R.D., and M.G. Oliver 1990 Rat intestinal mucins: a n immunocytochemical study. Trans. Am. Microsc. SOC.,I09t213-222.
Streefkerk, J.G. 1972 Inhibition of erythrocyte pseudoperoxidase activity by pretreatment with hydrogen peroxidase following methanol. J. Histochem. Cytochem., 202329-831.
Thomopoulos, G.N., B.A. Schulte, and S.S. Spicer 1983 Light and
electron microscopic cytochemistry of glycoconjugates in t h e rectosigmoid colonic epithelium of the mouse and rat. Am. J . Anat.,
168t239-256.
Wesley, A,, J. Forstner, R. Qureshi, M. Mantle, and G. Forstner 1983
Human intestinal mucin in cystic fibrosis. Pediatr. Res., 17:6569.
Wesley, A,, M. Mantle, D. Man, R. Qureshi, G. Forstner, and J. Forstner 1985 Neutral and acidic species of human intestinal mucin:
evidence for different core peptides. J. Biol. Chem., 260t79557959.
Документ
Категория
Без категории
Просмотров
9
Размер файла
790 Кб
Теги
antibodies, intestinal, mucins, variation, monoclonal, granules, intracellular, localized, rat
1/--страниц
Пожаловаться на содержимое документа