apoptosis of crypt epithelial cells in ulcerative colitis

JOURNAL OF PATHOLOGY, VOL. 180: 152-159 (1996)

APOPTOSIS OF CRYPT EPITHELIAL CELLS IN ULCERATIVE COLITIS

MICHIKO IWAMOTO, TAKEHIKO KOJI*, KAZUYA MAKIYAMA, NOBUYUKI K O B A Y A S H I ~ AND PAUL K. NAKANE"

Depurtments of Medicine I1 rind *Histology and Cell Bin fogy, Nugusaki University School of Medicine, 1-12-4 Sakrirnoto, Nugusuki 852, Japan; 'r Depurtment of Parasitology, Yumaguchi University of Medicine, Ubeshi 755, Jupan

SUMMARY

In the colon of ulcerative colitis (UC) patients, apoptotic bodies have been recognized in routine histopathological preparations. To investigate the extent of the apoptosis, colonic biopsies were examined from involved and uninvolved areas of untreated active UC and from normal areas in patients with colonic polyps, utilizing various markers of apoptosis. The markers included DNA breaks detected by TUNEL, Fas (CD95/APO-1) and Fas ligand (Fas-L) localized by immunohistochemistry, electron microscopic features of apoptosis, and laddering of extracted DNA. Apoptosis marker positive cells were found mainly on the luminal epithelium of the normal colon and were present in active UC in crypts of involved and uninvolved areas of the colon, in addition to the luminal epithelium. The DNA extracted from active UC colon electrophoresed as a ladder. These findings suggest that the loss of epithelial cells in active UC occurs mainly by apoptosis in crypts of involved and adjacent uninvolved areas and that the FaslFas-L interaction is a mediator of the apoptosis.

KEY worms-ulcerative colitis; colon; apoptosis; epithelial cell; Fas; Fas ligand; TUNEL,; cell death

INTRODUCTION

Ulcerative colitis (UG) is one of the major chronic digestive diseases encountered in clinical practice. The aetiological agents have not been identified, but it is generally agreed that am interaction of genetic disposi- tion, exogenous triggers, and endogenous anomalies leads to the onset of the disease (see reviews in refs 1-3). Mucosal alterations and inflammation result as a consequence of this interaction.

During progression of the disease, a noticeable histological anomaly is the gradual reduction in the size of crypts,' which has been attributed to an accelerated rate of turnover of the epithelium. 1+6 However, the presence of apoptotic bodics in the crypt epithelium in active UC, as recognized by routine histopathological examination,' suggests that this loss might be due to apoptosis of epithelial cells. Although the apoptotic body is a classical hallmark of cells undergoing apoptosis,8 it forms momentarily in the pathway of apoptosis; only a fraction of cells undergoing apoptosis may be recognizedx-] by the presence of the apoptotic body and it is often difficult to discriminate from the nucleus of a granulocyte in colonic tissue.'

To examine the extent of apoptosis in active UC, mucosal epithelium of colonic biopsies taken from involved and uninvolved areas of untreated active UC, and from normal areas in patients with colon polyps, was examined utilizing various current histochemical methods for the detection of cells undergoing apoptosis.

It was found that there were more cells with the markers of apoptosis in crypts of the involved and adjacent uninvolved areas of untreated active UC than in crypts of the normal colon. These findings suggest the

Addressee for correspondence: Paul K. Nakane, Department of Histology and Ccll Biology, Nagasaki Univesity School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan.

CCC 0022-341 7/96/1001 9-08 c) 1996 by John Wiley & Sons, Ltd.

the loss of colonic epithelium in active UC occurs mainly by apoptosis of crypt epithelial cells and that a mediator of the apoptosis is a Fas/Fas-L interaction. Further- more, it is suggested that intervention directed towards the apoptotic process (see review in ref. 12) may prove beneficial in the therapy of UC.

MATERIALS AND METHODS

Matevials Colonic biopsies of active inflammatory lesions were

obtained from 45 active UC patients who had received no prior drug therapy. Sometimes biopsies were also obtained from uninvolved areas several centimetres away from involved areas towards the descending colon. In addition, normal areas adjacent to polyps were excised from 43 patients with colonic polyps. In all cases, the diagnosis was made from the clinical history, endo- scopy, and histopathological examination. At the time of biopsy, many of the specimens were divided into three groups. The first group was fixed in 10 per cent formalin, embedded in paraffin, sectioned semi-consecutively at 5 ,urn in thickness, and used for histological, histochemical, and imunohistochemical observations. The second group was fixed in 2 per cent glutaraldehyde and processed for routine electron microscopy. The third group was homogenized for DNA extraction.

Immunohistochemistvy

The indirect peroxidase-labelled antibody methodJ3 was used. Sections were deparaffinized in warm toluene and hydrated in a graded ethanol series. Endogenous peroxidase activity was then inactivated by immersion in methanol containing 0.3 per cent hydrogen peroxide (H202). For the first antibodies, rabbit anti-Fas (R-anti- Fas) (Kaken, Yokohama, Japan), rabbit anti-Fas ligand

Received 7 August 1995 Acceptc>d 3 April I496

APOPTOSIS IN ULCERATIVE COLITIS 153

(R-anti-Fas-L), and mouse monoclonal anti- proliferating cell nuclear antigen (Dako, Copenhagen, Denmark) were used. R-anti-Fas was made against synthetic peptide 104-1 12, a portion of the extracellular domain,14 and R-anti-Fas-L was made against synthetic peptide 41-56, a portion of the intracellular domain.15 Their specificities were confirmed by Western blot analysis. l6.I7 For the second antibodies, peroxidase-labelled goat anti-rabbit IgG (MBL, Nagoya, Japan) and peroxidase-labelled rabbit anti-mouse IgG (Amersham, London, U.K.) were used for those sections treated with rabbit antibodies and for those sections treated with mouse antibodies, respectively. As immunohistochemical controls, some sections were reacted with normal rabbit IgG (Miles Scientific, Naperville, IL, U.S.A.) in place of the rabbit antibodies and with normal mouse IgG in place of the mouse monoclonal antibodies. The sections were incubated in a solution containing 3,3'- diaminobenzidine 4-HC1 (DAB) and H202 and were counter-stained with methyl green.

In situ DNA end-labelling (TUNEL) TUNEL was carried out essentially as described else-

where.'* The sections were incubated in a solution containing DAB and H,02 or in the DAB solution with Ni and Co ions.l8 Some stained sections were counter- stained with methyl green. In the stained sections where one found the entire depth of a crypt, TUNEL-positive cells and TUNEL-negative cells were counted in about the lower two-thirds of the crypts, from the base to the lip, but not in the luminal surface. From the counts, the total number of epithelial cells in each crypt section, the percentage of TUNEL-positive cells in each crypt section, the average numbers of cells per crypt section, and the average percentage of TUNEL-positive cells per crypt section were obtained.

Electron microscopy The glutaraldehyde-fixed tissues were processed using

a routine method and examined with a JEOL 1210 transmission eiectron microscope.

DNA size analysis High molecular-weight DNA was extracted from the

homogenate as descn'bed else~here. '~ Aliquots (1Opg per lane) of the DNA were electrophoresed in 1.5 per cent agarose gel and stained with ethidium bromide.

RESULTS Normal colon

The baso-lateral surfaces of epithelial cells near the mouths of crypts and on the luminal surface were positive for Fas (Fig. la) and Fas-L (Fig. lb). Fas (Fig. Id), as found by Moller et a1.,20 and Fas-L (Fig. le) were found near or on the baso-lateral surfaces of the epithelial cells. The nuclei of some of these Fas and Fas-L- positive cells were either condensed or fragmented in

routine haematoxylin and eosin-stained sections and were labelled with biotin after TUNEL (Figs lc and If). The cytoplasm of epithelial cells in the lower two-thirds of crypts was free of Fas (Fig. la) and Fas-L (Fig. lb). The nuclei in these zones were also free of biotin after TUNEL (Fig. lc). Proliferating cell nuclear antigen (PCNA)-positive nuclei were found mainly in the bottom third, as reported by others.6 In the lamina propria, one or two mononuclear cells per field were positive for Fas and Fas-L.

Morphologically normal colon taken j iom uninvolved areas of active UC colon

Most of the crypts presented essentially the same marker profile as observed in the normal colon, but in some crypts, some of epithelial cells in the lower two- thirds were positive for Fas (Fig. 2a) and Fas-L (Fig. 2b). Nuclei labelled with biotin after TUNEL (Fig. 2c) were found in most, if not all, of the Fas and Fas-L- positive cells, but not all Fas and Fas-L-positive crypt epithelial cells were positive by TUNEL. None of the Fas/Fas-L/TUNEL-positive cells was inimuno-positive for PCNA. PCNA-positive nuclei were found in the middle thirds of crypts, in addition to the bottom third. Near the mouths of those crypts and on the luminal surface, the profile of marker positive cells was similar to that of normal colon. Occasional mononuclear cells in the lamina propria were positive for Fas and Fas-L.

Involved area of active UC colon

Most crypts were shallow or occluded. Some crypts were totally devoid of goblet cells. Fas (Fig. 3a) and Fas-L (fig. 3b)-positive epithelial cells were found scattered singly or in groups in the walls of the crypts. In some crypts, the positive cells were grouped in a crescent; in some, the positive and negative cells were admixed; and in some, most of the cells were free of the antigens. Nuclei labelled with biotin after TUNEL were found in a pattern similar to that of the Fas and Fas-L-positive cells (Fig. 3c), but none was immuno- positive for PCNA. In general, PCNA-positive nuclei were scattered within the crypts, although there was a tendency for them to be concentrated at the base. There were numerous infiltrating mononuclear cells in the adjacent lamina propria and occasional cells were Fas, Fas-L, and/or TUNEL-positive. All immunohistochemical controls, as well as those for TUNEL, were negative.

Frequency of nuclear TUNEL-positive epithelial cells in the crypts

The results are summarized in Table I. There were significantly more epithelial cells per crypt section of the normal colon than in the uninvolved areas of active UC (51-09 f 0-78 per crypt section vs. 43.43 f 0.85 per crypt section, P<O.OOl), and more epithelial cells per crypt section of the uninvolved area of active UC than of the involved area (43.43 +045 per crypt section vs. 36.29 f 1.32 per crypt section, P<O-OOl). Conversely, there were significantly more TUNEL-positive cells per

154 M. IWAMOTO ET AL

Fig. 1 - (a f) Normal colonic biopsy. (a) lmmunohistochemically stained for Fas. (b) Imrnunohistochemically stained for Fas-L. (c) Stained by TUNEL. The sections were incubated in a solution containing DAB, H,O, and N i and Co ions. The bar indicated 40pm. (d) A portion of the luniinal surface of a. Immunohistochemically stained for Fas. (e) A portion of the luminal surface of b. Immunohistochemically stained for Fas-L. (0 A portion of the luminal surface ofc. Stained by TUNEL. Thc bar indicates 20pm. Fas and Fas-L were found mainly on baso-lateral surfaces of epithelial cells in the luminal surface. TUNEL-positive cells were i n the luminal surface. Occasional cells in the lamina propria immediately below the luminal surface were positive by TUNEL (arrow)

crypt section of the involved area of active UC than of the uninvolved area (2.96 i 0.30 per crypt section vs. 2.58 f 0.25 per crypt section, P<0.05), and more TUNEL-positive cells per crypt section of the uninvolved area of active UC than of the normal colon (2.58 i 0.25 per crypt section vs. 0.55 f 0.08 per crypt section, P<O.OOI) . When the percentages of TUNEL- positive cells in a given crypt section were obtained and averaged, the order was the same as that of the numbers of TUNEL-positive cells per crypt section.

Electron microscopy

Cells with prominent features of apoptosis, such as condensation of nuclei and cytoplasm, as described by other^,^ were frequently found in the luminal surface,

but rarely in the crypt epithelium of normal colon. In the uninvolved and involved areas of active UC, apoptotic cells at various stages were frequently found. Some were at an early phase of apoptosis, with condensed chromatin at the periphery of the nucleus and with buds (Fig. 4a), whereas some were at a later phase, where the cells were fragmented (Fig. 4b) or ingested by neighbouring cells.

Electrophoresis of extieacted DNA DNA extracted from involved and uninvolved areas

of active UC electrophoresed as a ladder (Fig. 5 ) with a high molecular weight band. From the size markers (marker), the ladder consisted of a series of DNA fragments in multiples of approximately 180-200 bp.

APOPTOSIS IN ULCERATIVE COLITlS 155

Fig. 2-(a-c) Biopsy of uninvolved area of active UC. (a) Immunohistochemically stained for Fas. (b) Immunohistochemically stained for Fas-L. (c) Stained by TUNEL. The sections were incubated in a solution containing DAB and H,O,. Fas and Fas-L were found on baso-lateral surfaces of epithelial cells in crypts. Some of the Fas and Fas-L-positive cells were also positive by TUNEL. The bar indicates 40 pm

The ladder could be recognized in the DNA from the controls as faint bands when the micrographs were image-enhanced, but in an ordinal photograph, the ladder was not discernible.

DISCUSSION

In normal colonic crypts, it is generally agreed that columnar epithelial cells proliferate in the lower por- tions, migrate upwards along the walls as they differen- tiate, and finally are sloughed off or removed at the luminal surface.46 Prior to their removal, the epithelial cells in the luminal surface were thought to undergo apoptosis, since in these areas there are many apoptotic b ~ d i e s , ~ , . ~ ' a morphological hallmark of apoptosis designated by Kerr et Some of these cells show electron microscopic (EM) features of apoptosis.2' Nuclear DNA strand breaks, a biochemical marker of apop to~ i s '~ recognized by TUNEL,") are concentrated in the nuclei of some of these cells.lO.l I

By utilizing these luminal surface epithelial cells as a positive control of apoptosis, we examined whether they were associated with the recently described FasI4 and Fas-LI5 and whether they were positive by TUNEL. Fas (CD95/APO-1) is a cell surface receptorL4 whose engagement with Fas-L,I5 a TNF like ligand, results in the triggering of a cellular apoptotic programme.22 Their

co-localization in a given cell should be further evidence that the cell is on an apoptotic pathway. In this study, the presence of Fas and Fas-L on TUNEL-positive luminal surface epithelial cells (Figs la-lf) was demon- strated. This suggests that both Fas and Fas-L are products of the epithelial cells;I5 that an autocrine or paracrine type of interaction between Fas and Fas-L is more likely involved in the apoptotic process; and that Fas and Fas-L serve as additional markers of apoptosis in colonic epithelium.

By utilizing the above markers, we then studied the state of apoptosis of the crypt epithelium in active UC. In involved areas of active UC, marker-positive cells were located deep in the crypts when the crypt was open to the lumen, as noted on haematoxylin and eosin- stained sections by Lee,7 and were abundant in occluded crypts as well as in the luminal surface epithelium. Unexpectedly, the marker-positive cells were also found in the lower two-thirds of crypts in biopsies taken from uninvolved areas of active UC (Figs 2a-2c). In the active UC colon, not all Fas/Fas-L-positive crypt epithelial cells were positive by TUNEL and it is suggested that the appearance of Fas and Fas-L precedes that of the DNA breakages in the pathway of apoptosis.

Statistically, the incidence of nuclear TUNEL-positive cells per crypt section of involved areas of active UC over that of normal colon was about 5.4 times (2.96 per cent/0.55 per cent Table I), a ratio slightly higher than

156 M. IWAMOTO ET A L

Fig. 3-(a-c) Biopsy of involved area of active UC. (a) Immunohistochemically stained for Fas. (b) Immunohistochemically stained for Fas-L. (c) Stained by TUNEL. The sections were incubated in a solution containing DAB and HZO2. Fas, Fas-L, and TUNEL positive epithelial cells were found in the luminal surface (arrows) and crypts. Occasional cells in the lamina proprin wcrc positive for Fas, Fas-L or by TUNEL (arrow-heads). The bar indicates 40pm

Table I-Numbers of apoptotic cells in crypts of normal colon and in uninvolved and involved areas of active UC

Histological No. of cellskrypt No. of TUNELkrypt %I of TUNEL/celk diagnosis f SE f SE in crypt f SE

Normal colon 51.09 f 0.78 (1) 055 i 0.08 (4) 1.1 f 0.2 (7)

UC (involved) 36.29 f 1.32 (3) 2.96 + 0.30 (6) 9.0 f 0.9 (9) UC (uninvolved) 43.43 i 0.85 (2) 2.58 i 0.25 ( 5 ) 6-2 f 0.7 (8)

______~ ~~ ~

In each group, 100 crypt sections were examined and TUNEL-positive cells and TUNEL-negative cells presenl in each cypt section were counted. The sum of TUNEL-positive and TUNEL-negative cells in each crypt section was designated as the number of cells in each crypt section. (1-3): Mean of the number of cells in each crypt section. ( 6 6 ) Mean of the number ol' TUNEL-positive cells in each crypt section. (7-9) Mean of the perccntagcs of TUNEL-positive cclls in each crypt section. By Student t-test, P values were obtained. The P values between the groups are (1) vs. (2) P<0.001. (2) vs. (3) P<0.001, (I) VS. (3) P<0.001, (4) VS. ( 5 ) P<0.001, ( 5 ) VS. (6) P<0,05, (4) VS. (6) i"<0~001, (7) VS. (8) P<0.001, (8) VS.

(9) P<042, and (7) vs. (9) P<0,001.

the apoptotic body counts (2 --4 times) previously An attempt was made to establish a correlation reported.' This difference was expected, since the DNA between the incidence of nuclear T U N EL-positive crypt strand breaks detected by the TUNEL method persist epithelial cells and that of PCNA-positive crypt epithe- longer than the apoptotic body.Io The percentage of lial cells. However, the intensity of inmunostaining for TUNEL-positive cells in involved areas of active UC PCNA varied among specimens and we were unable to colon was about 8.2 times more than that of normal establish the incidence or the percentage of PCNA- colon (9.0 per centA.19 per cent Table I). positive cells per crypt section with confidence, although


Fig. &(a) Electron micrograph showing an early stage of apoptosis in the crypt of an involved area of active UC colon. In this cell (Ap), chromatin is condensed at the margins of the nucleus and many buds (b) are found, yet its cytoplasm contains intact cellular organelles and the plasma membrane is intact, with its desmosomal attachments (arrows). The bar indicates 2 pm. (b) Electron micrograph showing a later stage of apoptosis in the crypt of an involved area of active UC colon. An epithelial cell (Ap) has shrunk, pulled away from its neighbours, and fragmented. The fragments (arrows) contain intact cellular organelles. The bar indicates 2pm.

there was an impression that the frequency of PCNA in crypts of active UC was higher than in normal colon as reported by 0 the1- s .~~~

The DNA extracted from the involved and uninvolved areas of active UC colon electrophoresed as clear distinct ladders (Fig. 5). Since the DNA was extracted

158 M. IWAMOTO ET A L .

Fig. 5-Electrophoresis of extracted DNA. Marker: size markers. Control: DNA extracted from normal areas of colon with polyps. UC-invol.: DNA extracted from involved areas of active UC colon. UC-uninvol.: DNA extracted from uninvolved areas o f active UC colon. IOpgllane of extracted DNA was electrophoresed a t 50 V for 60 min usins a mini-slab. DNA extracted from involved areas (UC-invol.) and from uninvolvcd arcas (IJC-uninvol.) electrophoresed as a ladder with a high molecular weight band. T h e ladder consisted o f a series of DNA fragments in multiples of approximately 180-200 bp. The ladder was not rcadily observed in DNA from normal areas o f colon with polyps (control)

from total mucous membrane, some of the fragmented DNA originated from cells undergoing apoptosis in the lamina propria, but few cells were positive by TUNEL in the lamina propria and it may be assumed that the majority of fragmented DNA originated from epithelial cells undergoing apoptosis. These results suggested that the absolute counts of apoptotic crypt epithelial cells, the percentages of apoptotic crypt epithelial cells, and the number of DNA breaks in involved and uninvolved areas of active UC colon were higher than in normal colon.

Apoptosis that occurs in an orderly fashion at the luminal surface epithelium of normal colon is in accord- ance with programmed cell death. Our finding that apoptosis takes place in an irregular manner in the walls of crypts in involved and uninvolved areas of active UC epithelium merits some discussion. Various kinetic studies indicate that the life span of UC colonic epithelial cells is shortened to about half that of normal epithelial cell^.^,^ Hence, it is possible that the onset of apoptosis in UC epithelial cells occurs earlier than usual and that they die prior to reaching the luminal surface. Whether the hastened onset of apoptosis results from the intrinsic nature of the epithelium, or is induced by environmental factors, remains to be established. Cur- rently, most of the suspected intrinsic aetiological factors for UC" 2h are not linked with the onset of epithelial cell apoptosis. On the other hand, some of environmental factors suggested for the onset of UC are

known to induce apoptosis, including free and interfer~n-gamma,~~ and some of them may be involved with the early onset of apoptosis in the crypt epithelium in UC.

In conclusion, the death of epithelial cells by apoptosis is an early event during the onset of UC, although the prime factor that initiates the process is not as yet defined. Regardless of the aetiology, the utilization of anti-apoptotic agents such as N-acetyl-L-cysteine and vitamin E3* as therapeutic agents for UC may prove worthy of consideration.

ACKNOWLEDGEMENTS

We thank Mr Takashi Suematu and Mr Masahiro Harada for their excellent technical assistance and Ms Cynthia R. Nakane for language improvements.

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