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Short Communication |
From the Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients with inflammatory bowel disease (IBD) encompassing both ulcerative colitis (UC) and Crohn’s disease (CD) typically present with relapsing diarrhea. Diarrhea has been attributed to increased paracellular permeability in the epithelial lining from both the acutely inflamed and chronically damaged areas of the intestine in patients with IBD.2,3 Although data pertaining to intestinal permeability changes are not completely consistent, the majority of studies suggest that most patients with CD as well as UC have a defect in epithelial barrier function.4,5,6 Disease activity in IBD is linked to an influx of transepithelial migration of neutrophils (PMN) into the mucosal epithelium (cryptitis) and subsequently into the intestinal lumen resulting in the formation of so-called crypt abscesses. In vitro studies using human intestinal epithelial cell lines such as T84 have shown that PMN migration across epithelial monolayers occurs via a paracellular route.7 Furthermore, it is apparent that migration of PMN across epithelial monolayers causes transient and rapidly reversible perturbations of TJs.8,9
Paracellular permeability across epithelial cell monolayers is regulated primarily by TJs that encircle the apical poles of epithelial cells thereby establishing distinct microenvironments on both sides of the polarized epithelium. The TJ is comprised of a complex of proteins that are linked to the underlying actin cytoskeleton in the apical perijunctional F-actin ring. Cytoskeletal affiliations of the TJ are believed to play a very important role in regulating TJ function in diverse physiological and pathological states.10 Transmembrane proteins of TJs include occludin,11,12 junction adhesion molecule (JAM),13 and members of the claudinfamily.14 Juxtaposed to the TJ membrane is an electron dense area referred to as the terminal plaque that contains the scaffolding protein, ZO-1. ZO-1 is an important linker protein in TJs that affiliates with transmembrane protein, occludin, and other cytoplasmic proteins such as ZO-2, ZO-3, and actin.15,16
The adherens junction (AJ) is immediately subjacent to the TJ and is
important in maintaining integrity of other intercellular junctions,
thereby playing an essential role in cell-cell recognition and cell
sorting.17
The AJ protein complex includes a
single-spanning transmembrane glycoprotein, cadherin (E-cadherin in
epithelial cells), cytoplasmic proteins, and catenins
(
, ß, 
) that
associate with the underlying actin cytoskeleton.18-20
Since enhanced paracellular permeability and PMN infiltration into the intestinal mucosa are a central feature of active inflammatory bowel disease, our objective was to determine the relationship between expression of key epithelial intercellular junction proteins and PMN infiltration in mucosal biopsies from patients with active IBD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Colorectal mucosal tissue samples from patients with chronic active colitis (n = 20) were obtained from the Emory Epithelial Pathobiology Research Group frozen tissue bank (ulcerative colitis, n = 11; Crohn’s disease, n = 9). In addition, normal control colorectal mucosa consisted of biopsies obtained at colonoscopy (n = 11) and resection specimens performed for failure of medical treatment or for colon cancer (n = 18). These samples were used for determining expression of epithelial intercellular junction proteins by immunofluorescence labeling/confocal microscopy, Western blotting, and Northern blotting.
Additional archived intestinal mucosal tissue from Emory surgical pathology files was analyzed for TJ and AJ protein expression by immunohistochemistry. Formalin-fixed, paraffin-embedded samples of colorectal mucosa from patients with chronic active colitis (n = 10) and chronic inactive colitis (n = 2) (ulcerative colitis, n = 10; Crohn’s disease, n = 2) were analyzed. Controls included intestinal tissue from normal mucosa (n = 9) and mucosa showing features of collagenous colitis (n = 3) or lymphocytic colitis (n = 2).
Pathological Analysis
Hematoxylin and eosin stained sections of both frozen and permanent samples were reviewed by two gastrointestinal pathologists (A.N., S.V.W.) for the presence of chronic colitis and degree of disease activity. Histological grading of activity was based on a modified protocol by Truelove and Richards.21 Disease activity was defined as identification of PMN within colonic or ileal epithelium and was graded as mild, moderate, or severe depending on increasing numbers of PMN within the epithelium (crypt and surface epithelial cells). According to this classification, we identified 19 patients with chronic active ulcerative colitis (mild activity, n = 7; moderate activity, n = 4; severe activity, n = 8) and 11 patients with chronic active Crohn’s disease (mild activity, n = 3; moderate activity, n = 3; severe activity, n = 5).
Immunohistochemistry
Immunofluorescence Labeling of Frozen Tissue Sections
Five-µm frozen sections of colonic mucosa cut with a cryostat, were mounted on ethanol-sterilized glass coverslips, air dried, and stored at -80°C. Tissue sections were fixed in either ethanol (20 minutes at -20°C), methanol (20 minutes at -20°C), 3% paraformaldehyde (30 minutes at room temperature), or 3% buffered formalin (5 minutes at room temperature) followed by incubation with sodium borohydride to quench autofluorescence (1 hour at 4°C). Paraformaldehyde and formalin fixed tissue were permeabilized with 1% Triton X-100 for 10 minutes at room temperature. Tissue sections were incubated with respective primary antibodies to TJ and AJ proteins for 60 minutes in a humidity chamber, washed in Hanks’ balanced salt solution (HBSS), incubated with fluoresceinated secondary antibodies (Jackson Labs, PA), mounted in p-phenylenediamine glycerol (1:1) and analyzed by confocal microscopy (Zeiss laser confocal microscope, Emory University, Atlanta, GA). The following antibodies were used at concentrations of 1 to 5 µg/ml: ZO-1, ß-catenin mAb (Transduction Labs, KY), occludin and claudin-1 polyclonal antibodies (Zymed, CA). JAM mAb (clone J10.4) were generated in our laboratory.13 E-cadherin hybridoma supernatant ((clone HECD.1 (1:1600)) was used for these studies.
Immunohistochemistry of Paraffin-Embedded Tissue Samples
Formalin-fixed, paraffin-embedded tissue sections (5 µm) on glass slides were deparaffinized in xylene and rehydrated through graded alcohol solutions. Sections were incubated with 3% H2O2 for 20 minutes, and immunohistochemistry was performed on an automated instrument (Ventana ES, Ventana Medical Systems, Tucson, AZ) as previously described.22 Thus, sections were incubated with antibodies to the respective TJ and AJ proteins (see above) followed by peroxidase conjugated secondary antibodies and the bound complexes were visualized by using 3,3-diaminobenzidine as a substrate. Identical reaction times permitted accurate comparison of all samples. The slides were then counter-stained with Mayer’s hematoxylin. A negative control in which the primary antibody was omitted was included in each test run. Staining intensity was expressed as no change compared to healthy control, <50% reduction (diminished expression) and >50% reduction (strongly diminished expression).
Immunoblotting
Intestinal mucosa was stripped from the underlying submucosal tissue, harvested in HBSS+ containing 1% Triton X-100 and protease inhibitors (leupeptin, chymostatin, aprotinin; 10 µg/ml, phenylmethylsulfonyl fluoride; 1.25 mmol/L). Protein concentration in tissue lysates was determined by using the Pierce BCA protein assay (Pierce, Rockford, IL). Samples were mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer and analyzed for TJ/AJ protein expression by SDS-polyacrylamide gel electrophoreseis and immunoblots using standard protocol.23 10 µg of total protein was loaded in each lane of the SDS-polyacrylamide gel. Western blots with a pan-cytokeratin mAb were also done so as to normalize epithelial protein loading between samples.
RNA Isolation and Northern Blotting
RNA was isolated from stripped intestinal mucosa (as above) using the Trizol-method (Life Technologies, Gaithersburg, MD) according to the manufacturer’s specifications. 20 µg of total RNA was separated in a 1% denaturing formaldehyde agarose gel and transferred to a nylon membrane. [32P]dCTP (Amersham)-labeled DNA probes were generated from the entire occludin coding region by random oligonucleotide-priming using the NEBlot-Kit (NEB). Unincorporated nucleotides were removed using a G-50 spin column (Amersham-Pharmacia, Piscataway, NJ), and the probe was hybridized for one hour to the membrane-immobilized RNA at 68°C using ExpressHyb buffer (Clontech, Palo Alto, CA) according to the manufacturer’s recommendation. After washing at 50°C the membrane was developed using a PhosphoImager. To normalize for the amount of RNA, a 32P-labeled probe directed against glyceraldehyde-3-phosphate dehydrogenase was included.
Statistical Analysis
For statistical analysis Student’s unpaired t-test was used. Statistical significance was considered if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Idiopathic inflammatory bowel disease is associated with
intestinal epithelial damage, crypt architectural irregularity, and
relapsing acute inflammation characterized by transepithelial migration
of PMN. Histology of colonic mucosa from control and IBD patients was
characterized by hematoxylin and eosin staining of mucosal tissue
sections (Figure 1a
-d). Since increased
paracellular permeability has been reported in intestinal epithelium
from patients with IBD, and TJs are a primary determinant of epithelial
paracellular permeability, we initially analyzed the expression of TJ
transmembrane protein, occludin and its cytoplasmic plaque protein,
ZO-1 in colonic mucosal sections from control and UC patients by
immunofluorescence labeling and confocal microscopy. To further
characterize expression of these proteins relative to active
inflammation, tissue sections were double-labeled for occludin/ZO-1
versus the ß2- integrin CD11b/CD18
as a leukocyte specific marker strongly expressed on PMN. In control
biopsies, occludin and ZO-1 were localized to the apical region of the
lateral plasma membrane representing the region of TJs in surface and
crypt epithelial cells (Figure 1, e, f, i, j)
. In mucosal tissue from
patients with chronic active ulcerative colitis, occludin-staining was
globally down-regulated, both in epithelium with active inflammation
(Figure 1h)
and in crypts where transmigrating PMN were not identified
(Figure 1g)
. Thus, even in regions with only mild active inflammation,
occludin staining was markedly diminished (Figure 1g)
and was almost
absent in areas with increased PMN transmigration and crypt abscess
formation (Figure 1h)
. Neither in controls nor in the UC population was
there a difference in occludin expression in surface versus
crypt epithelial cells. Occludin expression in colonic tissue samples
from patients with Crohn’s disease paralleled that of UC patients.
However, as determined by immunhistochemical analysis (Table 1
) inactive IBD as well as in
other intestinal disorders such as lymphocytic colitis and collagenous
colitis, no change in occludin expression relative to control healthy
tissue was observed.
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. In
contrast to the global decrease observed for occludin, ZO-1 staining
was diminished to absent only in areas adjacent to intraepithelial PMN
(Figure 1l)
. In particular, no change in ZO-1 staining was
observed in areas of epithelial cells lacking intraepithelial PMN
(Figure 1k
and Table 1
). The pattern of ZO-1 expression in
tissue sections of patients with CD was identical to that observed in
the UC samples (data not shown). Figure 1k
highlights distinct
distribution of ZO-1 in TJs of epithelial cells that is comparable to
control mucosal samples (Figure 1, i and j)
even though PMN are seen in
the lamina propria but are not transmigrating across the epithelium. We
also examined the pattern of ZO-1 staining in endothelial cells and
observed no difference in IBD versus control tissues even in areas of
active inflammation. Occludin and ZO-1 staining in frozen sections from
IBD and control tissues was also identical to that observed in archived
paraffin embedded tissue sections stained by immunoperoxidase labeling
(not shown).
To further analyze occludin and ZO-1 protein expression in colonic
epithelium from control and IBD patients we performed Western blots
from mucosal tissue lysates of patients undergoing colonic resection.
Thus, colonic mucosa from individuals with chronic active colitis and
normal colonic mucosa (five patients each) was used for Western blots.
The mucosal tissue was mechanically separated from the underlying
submucosa. To normalize epithelial protein loading between samples,
Western blots were probed with a pan-cytokeratin mAb. Results from
these experiments demonstrated consistent and significant reduction of
occludin expression in patients with UC as well as with CD (Figure 2A)
. These findings are
consistent with the immunofluorescence results showing diffuse
down-regulation of occludin in the intestinal mucosa of IBD patients.
Levels for ZO-1, JAM, claudin-I, ß-catenin, and E-cadherin all showed
a tendency of diminished protein expression. However, these
levels did not reach significance in either CD or UC (Figure 2A)
.
Since we observed a global down-regulation of occludin in intestinal
epithelial cells of UC patients we evaluated occludin mRNA expression
in these mucosal tissue samples by performing a Northern blot
analysis. As shown in Figure 2B
, we observed marked down-regulation of
occludin in chronic active UC samples compared to controls.
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To confirm our Western blot results suggesting slight reduction in
expression of most TJ/AJ proteins with the exception of occludin, we
performed a morphological analysis of their expression patterns in
tissue samples of control and IBD patients. Archived intestinal tissue
embedded in paraffin blocks was analyzed by immunohistochemistry. We
first analyzed distribution of TJ proteins, JAM, and claudin-1 in
control and IBD tissue sections. Focal high concentrations of JAM were
seen in the apical region of the lateral membrane representing TJs of
control intestinal epithelial cells (Figure 3a)
. A smaller pool of JAM was
distributed in the lateral membrane below TJs. Analysis of JAM
expression in intestinal epithelium from patients with chronic active
UC revealed a pattern that was identical to that of ZO-1. Thus we
observed decreased intensity of JAM expression exclusively in
epithelial cells adjoining transmigrating PMN (Figure 3c)
. Non-inflamed
epithelium without transmigrating PMN even in areas adjoining crypt
abscesses showed normal JAM expression (Figure 3b)
. There was no
difference in JAM distribution between surface and crypt epithelium
(data not shown). The staining pattern of JAM again contrasts with that
of occludin which was observed to be diffusely down-regulated in both
the acutely inflamed and non-inflamed regions of the mucosa. JAM
expression in colonic tissue from patients with lymphocytic and
collagenous colitis was comparable to control normal tissue (Table 1
). In addition, JAM expression in intercellular junctions
of endothelial cells was indistinguishable in control versus IBD
intestinal mucosal tissue sections. Given the differential expression
patterns of occludin versus ZO-1 and JAM in IBD tissue
sections, we analyzed distribution of another TJ transmembrane protein,
claudin-1, by immunoperoxidase labeling. Interestingly, claudin-1
expression paralleled that of JAM in intestinal tissue sections from
control and IBD patients. Thus, claudin-1 was concentrated in TJs and a
small pool was additionally distributed in the subjacent lateral
membranes of surface and crypt epithelial cells (Figure 3d)
. Comparable
to JAM, claudin-1 expression in chronic active UC was decreased only in
areas with PMN transmigration both in the crypt epithelium (cryptitis,
crypt abscess) (Figure 3f)
and surface epithelium (data not shown). In
areas away from active inflammation (PMN transmigration), claudin-1
staining was indistinguishable from that in control tissue sections
(Figure 3e)
. Patients with chronic inactive UC as well as disease
controls such as collagenous colitis and lymphocytic colitis revealed
the same distribution of claudin-1 as normal controls. However,
claudin-1, JAM, and ZO-1 expression patterns in tissue sections from CD
patients was analogous to that observed for UC.
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Since the adherens junction (AJ) is immediately subjacent to TJs
of epithelial cells, and its function is intimately linked to that of
the TJ, we determined expression of AJ proteins in tissue sections from
IBD and control patients. Paraffin-embedded mucosal samples were
stained by immunoperoxidase labeling with antibodies to the AJ
transmembrane protein, E-cadherin, and its linker protein, ß-catenin.
E-cadherin was predominantly distributed in the lateral membrane of
epithelial cells (Figure 3g)
. In mucosal tissue samples of UC patients
with active PMN transepithelial migration, E-cadherin expression was
decreased only in the lateral membranes of epithelial cells exposed to
transmigrating PMN (Figure 3i)
. Epithelia distant to transmigrating PMN
showed a normal E-cadherin staining pattern in the lateral membranes
(Figure 3h)
. No significant differences between crypt and surface
epithelium were observed in the UC samples. Analogous to E-cadherin,
ß-catenin was also distributed in the lateral membranes of intestinal
epithelial cells of control mucosal biopsies (Figure 3j)
. In chronic
active UC, we observed markedly diminished expression for ß-catenin
that was restricted to sites of PMN transepithelial migration (Figure 3l)
. The surface epithelium and crypts lacking transmigrating PMN in UC
samples showed a staining pattern that did not differ from that of
normal controls (Figure 3k)
. Mucosal tissue sections from patients with
chronic active CD revealed an identical staining pattern to that
observed in UC (Table 1)
. In addition, expression pattern of
ß-catenin and E-cadherin in non-IBD patients such as lymphocytic and
collagenous colitis did not differ from that of normal controls.
Further analysis of E-cadherin and ß-catenin expression in UC mucosal
tissue samples by Western blot revealed a slight decrease in expression
of these proteins compared to control non-IBD tissue (Figure 2A)
. However, densitometric analyses did not reveal any
statistically significant differences.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is known that PMN can rapidly migrate across epithelia, induce transient opening of intercellular junctions and not cause appreciable morphological discontinuities.9 It has been postulated that down-regulation of TJ proteins during PMN transepithelial migration might be induced by the migrating leukocytes themselves either by stimulating epithelial cells to reorganize their TJ and AJ components or simply by mechanical forces.24 Physical migration of PMN from the basolateral to the apical surfaces of epithelial cells is preceeded by a fall in the transepithelial resistance to passive ion flow suggesting signaling events between PMN and the basolateral aspects of epithelial cells.25
Since PMN have been documented to release proteases that induce artifactual degradation of intercellular junction proteins in vascular endothelium, we examined the effect of different tissue fixatives on the staining pattern of these intercellular junction proteins. However, IBD and non-IBD tissue samples treated with four different fixatives (ethanol, methanol, paraformaldehyde, and formalin + sodium borohydride) revealed an identical pattern of TJ/AJ protein expression. Moreover, no significant differences in staining intensity of TJ/AJ proteins was observed in unfixed frozen tissue sections in which suspensions of PMN had been allowed to adhere to in high density before processing. It therefore appears that pre- and post-fixative protein degradation of TJ/AJ proteins by PMN-released proteases in tissue sections cannot account for the diminished TJ/AJ protein expression we observed in our studies.
Given that occludin was the only TJ/AJ protein that was diffusely
down-regulated in areas with and without PMN epithelial infiltration,
we determined its expression at the transcriptional level. Indeed, by
Northern blot analysis, markedly diminished RNA levels of occludin were
observed in intestinal tissue from patients with chronic active UC.
Global decreases in occludin expression in the colonic mucosa of IBD
patients would suggest that there are PMN-independent mechanisms
accounting for occludin down-regulation. A possible mechanism
accounting for altered intercellular junction protein expression in IBD
involves the influence of cytokines on epithelial cells. Indeed, it has
been shown that proinflammatory cytokines may alter barrier function
during intestinal inflammation.26
interferon- (IFN-)
as well as interleukin-4 (IL-4) are cytokines that have previously been
shown to influence epithelial barrier function and may be likely
candidates that may modulate transepithelial migration of
PMN.27,28
It has been reported that IFN- decreases
barrier function in T84 cells by diminishing ZO-1 expression
levels.29
In addition, tumor necrosis factor- (TNF-)
that is known to play a role in the pathology of active IBD decreases
epithelial barrier function and tight junction complexity of a human
intestinal epithelial cell line as analyzed by freeze-fracture electron
microscopy.30
Recently, it has been demonstrated that
expression of the human occludin promoter is down-regulated by TNF-
as well as IFN-.31
Given that both TNF- and IFN-
play an important role in the pathophysiology of intestinal
inflammation in IBD32,33
, and their influence on the
occludin promoter activity, it is reasonable to suspect that these or
other cytokines are likely to account for our observations showing
global down-regulation of occludin at the protein and transcription
level. It is conceivable that some of the cytokines that may play a
role in barrier function during inflammation are secreted by PMNs
thereby regulating junction proteins. It is well known that PMNs
secrete a variety of inflammatory cytokines after
activation.34
Further studies are therefore necessary to
clarify whether cytokines play a predominant role in diminished barrier
function and decreased tight junction expression during chronic active
IBD. In general, it is conceivable that a combination of these factors,
local effects induced by neutrophils as well as cytokine mediated
mechanisms, is responsible for diminished expression of junction
proteins in active IBD. This would be consistent with our observation
that changes in expression of different junction proteins in active IBD
are not uniform. Whether the observed changes in occludin expression
may contribute to increased paracellular permeability that has been
described in a subgroup of patients with CD and UC remains to be
determined. Impedance analysis of intestinal mucosal tissue from mice
lacking occludin expression failed to reveal differences when compared
to normal mice.35
However, these studies lacked precise
determination of paracellular permeability as defined by flux of
paracellular solutes. In addition, susceptibility of knock-out versus
wild type mice to colitis was not examined. Thus, the precise role of
occludin in regulation of paracellular permeability remains to be
determined.
In conclusion, our study provides evidence that PMN transepithelial migration during chronic active UC is associated with a diffuse global down-regulation of occludin and a reduction of other TJ and AJ proteins specifically at sites of active inflammation. Global down-regulation of occludin in IBD-patients may account for increased paracellular permeability that is observed in a subgroup of IBD patients. Further studies are necessary to elucidate factors that are responsible for differential down-regulation of occludin and other TJ and AJ proteins in IBD.
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Footnotes |
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Supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft) (Ku 1328/1–1; T.K.), the American Digestive Health Foundation (S.V.W.), the National Institutes of Health (HL54229 and HL60540 to C.P.; DK53202 to A.N.), the Crohn’s & Colitis Foundation of America (A.N.), and the Arthritis Foundation (A.N.).
Accepted for publication September 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in a highly polarized fashion. J Cell Biol 1993, 120:785-798 (TNF-) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci 1999, 112:137-146[Abstract]
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