Analysis of enteropathy induced by tumour necrosis factor α

ANALYSIS OF ENTEROPATHY INDUCED BY TUMOUR NECROSIS FACTOR a Paul Garside,

Campbell

Bunce, R. Christopher Tomlinson, Allan MCI. Mowat

Buford

L. Nichols,*

Tumour necrosis factor (TNF)-a has been implicated in the pathogenesis of experimental and clinical enteropathy, but its exact role is unknown. We show here that a single dose of TNF-cw causes significant small intestinal pathology in normal adult mice, which develops within 15 minutes, persists for up to 48 hours and is enhanced by interferon-y (IFN-y). The enteropathy consists of villus atrophy and crypt hyperplasia and is therefore similar to that found in immunologically mediated enteropathies such as graft-versus-host reaction (GvHR). TNF-a is also cytotoxic to an intestinal crypt cell line in vitro. Thus, a direct action of TNF-cY on crypt cells may be involved in its enteropathic effects in vivo. Together, these findings indicate that TNF-(U alone, or in concert with other cytokines, may be an important effector molecule in immunologically mediated intestinal pathology and may ultimately provide a target for specific immunotherapy for clinical enteropathies.

Cytokines are critically important both in normal immune responses and in many forms of immunopathology, including enteropathies associated with local cell mediated immunity.l-3 Although this raises the possibility that immunotherapy targeted at specific cytokines could be used in a variety of diseases, cytokine-dependent phenomena usually involve complex interactions between a number of different mediators. Thus it will be essential to identify the exact role of individual cytokines in immunopathology. Tumour necrosis factor (TNF)-a is a wellcharacterized inflammatory mediator which has been implicated in several aspects of immunopathology, including experimental and clinical enteropathies. Enhanced production of TNF-ol from activated macrophages is a feature of experimental murine GvHR4 and antibodies to TNF-a prevent the intestinal consequences of this disease in mice.3 Furthermore, children with inflammatory bowel disease and bone marrow transplant (BMT) patients who later go on to

develop graft-versus-host disease (GvHD) have raised serum levels of TNF-cx~~~ and increased numbers of TNF-a-secreting cells have been demonstrated in intestinal biopsies from patients with inflammatory bowel disease.1 Although these findings suggest that TNF-a could be an important target for the treatment of intestinal disease, the exact role of TNF-a in enteropathy is not known. Here we have examined whether TNF-a itself can act as an effector molecule in enteropathy by examining intestinal architecture in normal mice treated with recombinant TNF-a. In addition, we have used an intestinal crypt cell line to determine whether TNF-(Y can have direct effects on enterocytes in vitro and have investigated whether IFN-y can synergise with the pathological activities of TNF-(-w.

RESULTS Effects of TNF-a

From the Department of Immunology, University of Glasgow, Western Infirmary, Glasgow, Gil 6NT and *Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas, USA. Received 9 July 1992; accepted for publication 20 August 1992 @ 1993 Academic Press Limited 1043-4666/93/010024+07 $08.0010 KEY

24

WORDS:

IFN-y/intestinelmice/pathology/TNF-a

on Mucosal Architecture

in vivo

Depletion of TNF-a in vivo prevents the villus atrophy and crypt hyperplasia which normally occurs in the intestine of mice with GvHR3 and our first experiments investigated whether TNF-(Y itself would produce similar lesions when administered to normal animals. Injection of adult mice with TNF-ok produced dose-dependent intestinal damage 6 hours later (Fig. 1). At the highest dose (lOW), this consisted CYTOKINE,

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TNF-a

of significant villus atrophy and crypt hypertrophy compared with controls. Mice given 104U TNF-ol also had significant crypt hypertrophy, but had no significant change in villus length, while 103U TNF-a produced no intestinal pathology. All further studies used 105U TNF-a to induce enteropathy. We next performed detailed time-course studies to investigate the evolution of the pathology. In the first experiments, we found that mice given TNF-a 6-48 h previously had significant crypt hypertrophy compared with controls and this was of identical intensity at all times examined (Fig. 2A). Significant villus atrophy was also present 6 and 24 h after injection, but by 48 h, villus lengths were now significantly longer than those in controls, suggesting that mucosal

T

800

Combined Effects of TNF-(u and IFN- y on Mucosal Architecture

(ii)

125

t

Dose

3 of TNF-u

Figure 1. Dose-dependent induction by administration of TNF-IX.

(log

i 25

repair was in progress (Fig. 2A). As enteropathy was already fully developed at the earliest time point examined in these experiments (6 h), we carried out additional studies to investigate how rapidly TNF-a induced intestinal pathology and to explore whether it would be possible to determine if TNF-(Y had its primary effects on the crypt or villus. These experiments showed that significant crypt hypertrophy was apparent 15 minutes after injection of TNF-(Y and this remained until the study was terminated at 2 h (Fig. 2B). In contrast, significant villus atrophy was not detectable until 60 minutes after injection and was not fully developed until 120 min (Fig. 2B). The findings of these morphometric studies were confirmed by histological analysis, which revealed marked villus blunting which was maximal l-2 h after injection (Fig. 2C). Subsequent ultrastructural examination of these tissues has revealed evidence of apoptosis of crypt epithelial cells, focal necrosis and haemorrhage and loss of interstitial fluid (Garside, Nichols & Mowat, unpublished observations). These findings have been confirmed in several subsequent experiments and indicate that TNF-cx itself is capable of inducing a pattern of intestinal pathology similar to that found in immunologically mediated enteropathy.

** ItI *

T

and enteropathy

u)

of enteropathy

A 5 in normal

mice

Mucosal architecture in the jejunum of TNF-a-treated or control (CBA x BALBlc) Fl mice 6 h after a single i.p. injection of 103, 104 or 1OsU TNF-a/animal. Bars represent mean villus (i) and crypt (ii) lengths f ISD for 5 mice/group. *P < 0.05; **P < 0.02.

IFN-)I has also been implicated in intestinal GvHR* and as IFN-y and TNF-(-Y frequently have synergistic effects,T-9 it was of interest to determine whether IFN-7 would enhance the enteropathic effects of TNF-ok. In this experiment, we also examined whether the cytokine-induced morphological changes in crypt architecture were accompanied by alterations in epithelial cell proliferation. As before, mice given 105U TNF-a alone had significant villus shortening 24 h after injection and had evidence of crypt cell pathology, with a significant increase in CCPR and a degree of crypt lengthening compared with controls, although this feature did not attain statistical significance in the experiment shown (Fig. 3). M’ ice g iven 105U EN-y alone also had mild villus shortening compared with controls, which was statistically significant but was not as marked as that seen with TNF-CL Again, there was no significant crypt hypertrophy (Fig. 3). Mice given both TNF-a and IFN-y had much more severe intestinal damage compared with that seen after either cytokine alone. The villus atrophy in these animals was significantly greater than that in mice given TNF-a or IFN-?/ alone, while animals given both cytokines how had marked and highly significant

26 I Garside

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Figure TNF-a.

ZA-D.

Time

course

of the development

of enteropathy

in normal

mice

following

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5, No.

1 (January

administration

x BALB/c)Fl mice 6, (A) Mucosal architecture in the jejunum of TNF-a-treated or control (CBA 48 h after a single i.p. injection of 1OW TNF-a. Bars represent mean villus (i) and crypt (ii) lengths for 5 mice/group. **P < 0.01; ***P < 0.001. (B) Mucosal architecture in the jejunum of TNF-u-treated control (CBA x BALB/c)FI mice 15, 30, 60 and 120 min after a single i.p. injection of 1OsU TNF-a. represent mean villus (i) and crypt (ii) lengths f 1SD for 4 mice/group. *P < 0.05; **P < 0.02; ***P (C) (D) Histological appearance of jejunum of TNF-a treated (C) or control (D) mouse, 60 min after i.p. injection of lOsU TNF-cu (H&E x 100).

of

24 and f 1SD or Points < 0.05. a single

1993: 24-30)

TNF-a

crypt hypertrophy, as well as retaining the significant increase in CCPR. Thus, IFN-)I markedly enhances the enteropathy induced by TNF-(r in vivo.

and enteropathy

i 27

(Fig. 4). Identical results have been obtained in four further experiments. IFN-y was unable to kill either cell type in this assay and did not enhance the effects of TNF-(-Y (data not shown)

Effects of TNF-(Y and IFN- y on Numbers of Intraepithelial Lymphocytes (IEL) In Vivo We also examined the effects of TNF-a and combined administration of TNF-a and IFN-?/ on the density of IEL in the jejunal mucosa. An increase in the numbers of these cells is one of the characteristics of several forms of enteropathy. However, in several separate experiments, we observed no changes in the numbers of these cells following cytokine treatment (data not shown). Toxicity of TNF-cy for Intestinal Vitro

Epitheliul

Cells In

The results above show that TNF-(r can induce intestinal damage in vivo. However, as this cytokine has effects on many different cell types,*SlO~l~ its enteropathic activity could be an indirect result of these other actions. We therefore investigated whether TNF-cx could have direct effects on enterocytes. To do this, we used the RIE cell line, a non-transformed epithelial cell line which appears to represent the undifferentiated crypt cells12 that have been proposed as the targets of immune attack in viva. As shown in Fig. 4, TNF-(Y showed a dosedependent ability to kill actinomycin D treated RIE cells in vitro. Indeed, the RIE cell line was as sensitive to the effects of TNF-(-w as the L929 fibroblasts which are the conventional targets for the TNF bioassay

T *P
L

r

T

20

0 .I)07

0.192 TNF

Figure

4.

Toxicity

of TNF-cs

120

3000

(u/ml)

for RIE and L929 cells in vitro.

Dose dependent lysis of target cells pre-treated with actinomycin D, assessed by uptake of amido black 48 h after treatment. Results shown are % killing for triplicate cultures, calculated using Triton X to obtain complete lysis.

* P < 0.05

L

Figure 3. enteropathy administration IFN--,J.

1 1 12.x

11.7

u

u

TNF-a

IFN-y

J Control

4.8

concentration

TNF-a

+ IFN-1

Induction of by combined of TNF-cu and

Mucosal architecture in the jejunum of (CBA x BALB/c) Fl mice 24 h after a single i.p. injection of 1OW TNF-ol and/or 1OW IFN-y and of controls. Bars represent mean villus and crypt lengths f lSD, while arrows show the CCPR for 5 mice/group.

28 I Garsideetal.

DISCUSSION The results presented here demonstrate that a single dose of TNF-(w induces an enteropathy in normal mice which appears within 15-60 min and includes both villus atrophy and crypt hypertrophy. In addition, TNF-ar can be directly cytotoxic to enterocytes in vitro. IFN-y enhances the enteropathic effects of TNF-a in vivo and together, these findings are further evidence that TNF-a alone, or in concert with other cytokines, may play an important role in immunologically mediated enteropathy. These studies were initiated because of experimental evidence that TNF-a plays an important role in immunologically mediated enteropathies, such as intestinal GvHR. Several features of this condition, such as epithelial cell apoptosis,3 are characteristic of the actions of TNF-ol, and increased expression of mRNA for TNF-o is found in the gut of mice with GvHRis. More significantly, the villus atrophy and crypt hyperplasia of murine GvHR can be prevented by in-vivo treatment with specific antibodies against TNF-a. 3 However, previous work has not distinguished whether TNF-a itself is an effector molecule of intestinal pathology or whether it merely plays an immunoregulatory role during the inductive phase of GvHR. The current studies support the former hypothesis, by demonstrating the rapid onset of villus atrophy and crypt hyperplasia in normal mice given TNF-a. Although others have reported intestinal damage and inflammation following administration of TNF-ok, these changes have not been quantified previously and specific alterations in individual aspects of mucosal architecture have not been identified.14J5 Furthermore, the continuous administration regimes used in some previous reports have many systemic effects, raising the possibility that pathological changes in organs like the gut are merely secondary to a more generalized debility or infection. We show here that a single i.p. injection of TNF-a rapidly produces an enteropathy which is fully developed within 60 min, coinciding with the physiological gut dysfunction following TNF-cx treatment, which has been described previously.15 As the pattern of enteropathy we observed is similar to that found in intestinal GvHR, our study supports the view that TNF-o may play a direct effector role in disorders of this type. The mechanisms by which TNF-a produces enteropathy remain to be elucidated. As one feature of GvHR which TNF-a did not reproduce was an increased IEL count, this finding suggests that TNF-a is not acting via this particular population of mucosal lymphocytes. Nevertheless, TNF-a is a multipotent cytokine which has inflammatory effects on many cell types, including vascular endothelium,

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NK cells,16 mast cells,17 macrophagesis and other accessory cells. 19 In addition, it can stimulate the release of several other cytokines.ls Although we cannot exclude a potential role for some of these indirect activities, our in-vitro studies showed clearly that TNF-a can have direct effects on enterocytes. These findings are the first demonstration of a direct action of TNF-ok on non-transformed enterocytes, but are consistent with the direct effects TNF-a has on normal tissue cells from other organs, such as the pancreas and thyroid,9 as well as tumour cell lines derived from the intestine.7 It seems likely that TNF-a also interacts with other mediators to cause enteropathy in vivo. IFN-y is required for intestinal GvHR in mice* and we demonstrate here that IFN-y synergises with the enteropathic effects of TNF-o in normal mice. These results are consistent with the synergy between these mediators which occurs in many other systems, including their ability to increase expression of class II MHC antigens and secretory component by colonic carcinoma cells in vitro.7 As TNF-c~ had no effect on enterocytes in vitro unless the cells had been treated with antinomycin D, we propose that the usual role of endogenously produced TNF-a in vivo may be to exacerbate cellular damage which has been initiated by other mediators, such as IFN-y. It will be important to investigate the exact interrelationships between TNF-cx and other cytokines which have been reported to be important in enteropathy, including IFN-7.2 The source of the TNF-(w which might be involved in intestinal damage is unknown. Recent evidence indicates that products of activated macrophages, such as IFN-or/p,*0 nitric oxide*’ and TNF-o1,4 play an important role in immunologically mediated enteropathy and thus it is tempting to speculate that TNF-ol, IFN-(-w/p and nitric oxide are all released by local macrophages which have been activated by IFN-y, as has been proposed for TNF-a.4 Alternatively, TNF-(r may originate from IFN-activated NK cells or from alloreactive donor T cells themselves. Finally, it has been shown that intestinal Paneth cells produce mRNA for TNF-a.13 Distinguishing between these potential sources of enteropathic TNF-a will require direct analysis of protein or mRNA expression at the mucosal level, and such studies are in progress. The results of our studies in vivo and in vitro support the view that crypt epithelial cells are the primary target of enteropathic cytokines such as TNF-a. Thus the earliest change in mucosal architecture which occurred in mice given TNF-(Y was an increase in crypt length, with significant alterations in villus architecture not being found until later. Similarly, mice given a low dose of TNF-a had crypt hypertrophy, but no villus atrophy. This pattern is identical to that found during intestinal GvHR,

TNF-a and enteropathy

underlining the pivotal role of self-renewing crypt cells as targets for the immune response in vivo. This idea is also supported by our findings that TNF-(-w interacted directly with RIE crypt cells in vitro. In conclusion, our results support the view that TNF-ol may be an important mediator of clinical enteropathies associated with a local cell-mediated immune response, including coeliac disease, Crohn’s disease, parasite infections and GvHD after bone marrow transplantation. Expanded numbers of mucosal TNF-ol secreting cells have already been found in inflammatory bowel disease1 and further studies of this kind, together with more detailed dissection of the exact targets of TNF-or and its interactions with other mediators may help develop specific immunotherapy for these disorders.

MATERIALS

AND METHODS

Mice

/ 29

BRL) were plated into flat-bottomed 96-well tissue culture plates (Costar, Northumbria Biologicals, Cramlington, Northumberland, UK) and incubated for 24 h at 37°C in 5% CO,. Serial dilutions of rTNF-cc in culture medium containing 2 pg/ml actinomycin D were then added to the

plate in a volume of 10 ~1 and the cells incubated for a further

18 h, before

being fixed in 10% formalin

in O.lM

sodium acetate for 30 min and then washed with PBS. The stain associated

with viable

cells was eluted by the addition

of 100 ~150 mM NaOH and the plate read on an automatic plate reader at 620 nm. Assays were performed in duplicate and the results expressed

as percentage

killing,

calculated

using cells cultured with 10% Triton-X-100 (Sigma Ltd, Poole, Dorset, UK) to obtain 100% lysis and with RPM1 164015% NCS as negative controls.

Assessment of Mucosal Architecture Villus and crypt lengths and crypt cell production rates (CCPR) were measured by microdissection of Feulgenstained samples of (Schiff Reagent, Sigma) jejunum as described in detail previously.*lJ3 In order to determine CCPR, mice were killed at intervals of 20-90 minutes after

injection of 7.5 mg/kg colchicine (Sigma) i.p. to produce

Adult (CBA x BALBlc) Fl mice were obtained from Harlan Olac (Bicester, UK) and maintained under standard conditions until firrt being used aged 6-8 weeks.

metaphase arrest. each specimen.

Ten crypts and villi

were examined

in

Cell Lines

Statistics

The Rat Intestinal Epithelial (RIE) cell line’2 was obtained initially from Dr K. Brown, (Institute of Animal Physiology and Genetics Research, Cambridge, UK) and then maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) Gibco BRL, Paisley, UK) containing 5% newborn calf serum (NCS) (Gibco BRL), 2mM glutamine, lOOU/ml penicillin/lOO~g/ml streptomycin (Gibco BRL). They were subcultured when confluent by

Groups of means and standard deviations were compared by Student’s t-test, while crypt cell production

treatment with 0.05% trypsin/0.02% EDTA (Gibco BRL) and, in all experiments, the cells were used at an early passage (<20). L929 fibroblasts were maintained in RPM1 1640 (Gibco BRL) supplemented with 5% NCS and also subcultured after trypsinization.

Recombinant human TNF-ct was kindly supplied by Dr G. Adolf, BASF Knoll Ag., Austria. Recombinant rat IFN-7 was kindly supplied by Dr van der Meide,

Institute of Applied

Acknowledgement Supported by MRC grant G8823030. Campbell Bunce was supported by a Wellcome Trust Vacational Scholarship.

REFERENCES 1. MacDonald

Cytokines

TN0

rates were compared by covariance analysis.

Radiology and Immunology,

Cooke gamma

A (1990). production

‘IT, Tumour measured

Hutchings

P, Choy M-Y,

Murch S,

necrosis factor-alpha and interferonat the single cell level in normal and

inflamed human intestine. Clin Exp Immunol X1:301-305. 2. Mowat A MCI (1989) Antibodies to IFN-y prevent immunologically mediated intestinal damage versus-host reaction. Immunology 68:18-23.

in

murine

graft-

The Netherlands. These materials contained ~2.7 pgimg endotoxin, as determined by the Limulus lysate assay. Mice were injected i.p. with cytokines diluted in 0.2 ml sterile phosphate-buffered saline (PBS) containing 2% newborn calf serum (NCS) while controls received diluent alone.

3. Piguet P-F, Grau GE, Allet B & Vassalli P (1987) Tumour necrosis factoricachectin is an effector of skin and gut lesions of the acute phase of graft-vs-host disease. J Exp Med 166:128&1289.

Assessment of Cytotoxic Effects of TNF-cy In Vitro

5. Murch SH, Lamkin VA, Savage MO, Walker-Smith JA & MacDonald TT (1991) Serum levels of tumour necrosis factor-alpha in childhood chronic inflammatory bowel disease. Gut 32:913-917. 6. Symington FW, Pepe MS, Chen AB & Deliganis A (1990) Serum tumor necrosis factor alpha associated with acute graft-versus-host disease in humans. Transplantation 50:518-521.

Cytotoxicity against cell lines was assessed using a TNF-u bioassay. Briefly, 75~1 of a cell suspension containing 2.5 x 105 L929 or RIE cells/ml RPM1 1640/5% NCS plus penicillin/streptomycin, glutamine and Fungizone (all Gibco

4. Nestel, FP, Price KS, Seemayer TA & Lapp Macrophage priming and lipopolysaccharide-triggered tumour necrosis factorcv during graft-versus-host disease. 175:405-413.

WS (1992) release of J Exp Med

30 I Garside

et al.

CYTOKINE,

Vol.

5, No.

1 (January

1993: 24-30)

7. Kuale D, Brandtzaeg P & Lvhaug D (1988) Up-regulation of the expression of secretory component and HLA molecules in a human colonic cell line by tumour necrosis factor-alpha and gamma interferon. Stand J Immunol28:351-357.

15. Patton JS, Peters PM, McCabe J, Crase D, Hansen S, Chen AB & Liggitt (1987) Development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-a in rodents. J Clin Invest 80:1587-1596.

8. Stein B, Synergistic gamma Exp Cell

16. Ostensen ME, Thiele DL & Lipsky PE (1987) Tumor necrosis factor-a enhances cytolytic activity of human natural killer cells. J Immunol 138:4185-4191.

Scharfeller K, Heckmann M, Hatamochi A, Mauch C, Reithmuller G, Ziegler-Heitbrock HW & Krieg T (1989) effects of tumor necrosis factor alpha and interferon on collagen synthesis of human skin fibroblasts in vitro. Res 181:40%419.

9. Weetman AP & Rees AJ (1988) Synergistic effects of recombinant tumour necrosis factor and interferon-gamma on rat thyroid cell growth and Ia antigen expression. Immunology 63:285-289. 10. Symington factor and gamma human keratinocytes. 92:798-805.

FW

(1989) interferon The Journal

Lymphotoxin, are cytostatic of Investigative

tumor necrosis for normal Dermatology

11. Scheurich P, Thoma B, Ucer U & Pfizeumaier Immunoregulatory activity of recombinant human tumor factor (TNF)-a: induction of TNF receutors on human T TNF-o‘mediated enhancement of T cell responses. J 138:178&1790. 12. Blay J & Brown epitheliod cell line derived tion of cytokeratin filaments. 8:551-60.

K (1987) necrosis cells and Immunol

KD (1984) Characterization of an from rat small intestine: demonstraCell Biology International Reports

13. Keshav S, Lawson L, Chung LP, Stein M, Perry VH & Gordon S (1990) Tumor necrosis factor mRNA localised to Paneth cells of normal murine intestinal epithelium by in situ hybridisation. J Exp Med 171~327-332. 14. Piguet P-F (1990) Tumour necrosis factor and graft-versushost disease. In: Burakoff SJ, Deeg HJ, Ferrara J, Atkinson K (eds) Graft-vs-host disease. Immunology, Pathophysiology and Treatment, Marcel Dekker, New York, pp 255-276.

17. Young JDE, Liu CC, Butler G, Cohn ZA (1987) Identification, purification and characterisation cell-associated cytolytic factor related to tumor necrosis Nat1 Acad Sci USA 84:9175-9179. 18. Gordon necrosis factor-a

C & Wofsy on immune

D (1990) function.

& Galli SC of a mast factor. Proc

Effects of recombinant J Immunol144:175~1758.

19. Le J & Vilcek J (1987) Tumour necrosis factor interleukin 1: cytokines with multiple overlapping activities. Invest 56:234-248. 20. Garside P, Felstein (1991) The role of interferon pathology in mice. Immunology

tumor and Lab

MV, Green EA & Mowat A. MCI a/p in the induction of intestinal 74:279-283.

21. Garside P, Hutton AK, Severn A, Liew FY & Mowat A. MCI Nitric oxide mediates intestinal pathology in graft-vs-host disease. European Journal of Immunology 22:2141-2145. 22. MacDonald IT & Ferguson A (1977) Hypersensitivity reactions in the small intestine. III. The effects of allograft rejection and of graft-versus-host disease on epithelial cell kinetics. Cell Tissue Kinetics 10:301-312. 23. Mowat A. MCI & Ferguson A (1981) Hypersensitivity reactions in the small intestine. 6. Pathogenesis of the graftversus-host reaction in the small intestinal mucosa of the mouse. Transplantation 32:238243.