Gut 1994; 35: 1037-1041

Detection of interferon gamma mRNA in themucosa of patients with coeliac disease by in situhybridisation

M Kontakou, R P Sturgess, R T Przemioslo, G A Limb, JM Nelufer, P J Ciclitira

AbstractIn situ hybridisation has been used tostudy interferon gamma (IFNy) mRNAexpression in the small intestine ofpatients with coeliac disease. Sectionsof jejunal biopsies were obtained fromfive patients with treated and fivewith untreated coeliac disease and fivedisease controls. These sections werehybridised with radiolabelled specificDNA oligonucleotide probes. The laminapropria of untreated coeliac diseasepatients contained a significantly in-creased number of IFN-y producing cellscompared with controls but there was nosignificant difference between the coeliacpatients treated with a gluten free dietand controls. The results suggest thatIFN,y may play a part in the immuno-pathogenesis of coeliac disease.(Gut 1994; 35: 1037-1041)

The Rayne Institute,St Thomas's Hospital,LondonM KontakouR P SturgessR T PrzemiosloG A LimbJ M NeluferP J Ciclitira

Correspondence to:Professor P J Ciclitira,Gastroenterology Unit,UMDS, The RayneInstitute, St Thomas'sHospital, London SE1 7EH.

Accepted for publication29 November 1993

Coeliac disease is characterised by gluteninduced damage to the small intestinalmucosa, which exhibits villous atrophy, cryptcell hyperplasia, and infiltration of the laminapropria with lymphocytes and plasma cells.'-3There is also an increased ratio of intraepi-thelial lymphocytes to small intestinal surfaceenterocytes.4 5 Another important feature ofcoeliac disease is the enhanced expressionof class II major histocompatibility complexantigens.6 7 In most cases these changes revertto normal on removal of gluten from the diet.It has been suggested that coeliac diseaserepresents an abnormal cellular immuneresponse in genetically susceptible people tothe ingestion of cereal peptides, which are as

yet unidentified. During this immune responsemany cytokines which are capable of initiatingcellular and molecular changes are released.

Interferon gamma (IFN-y) is producedduring immune reactions, mainly by antigen,mitogen, or lectin stimulated T lymphocytesand also by large granular leukocytes. IFN-yhas the ability to induce expression of majorhistocompatibility complex by human entero-cytes in vitro89 and to activate macrophagesand natural killer cells.

Studies of the role of IFN-y in other

enteropathies such as Crohn's disease suggestthat there is an increase in the number ofIFNysecreting cells compared with controls.'0Conflicting results have been reported,however, on interleukin 2 and IFN-y expressionin the mucosa of patients with Crohn's diseaseand ulcerative colitis."I-3 These results were

mainly attributed to the insensitivity of thevarious methods used.14 In coeliac disease,detection of IFNy in paraffin embeddedsections of normal and disease mucosawas achieved by an immunohistochemicaltechnique.15 It was reported that a relativelylarge proportion of both intraepithelial andlamina propria lymphocytes in normal smallbowel produce IFNy. The proportion oflymphocytes that produced IFN-y seemed todecrease in untreated coeliac disease patientsand returned towards normal in patients on agluten free diet. The same study, however,reported increased expression of class II majorhistocompatibility complex antigens on thesurface enterocytes of untreated coeliacmucosa, usually associated with increasedlevels of IFNy. It was suggested that althoughIFN-y is probably produced in increasedamounts in coeliac disease, it may also besecreted at a higher rate so that less stainableproduct is available for detection. To evaluatethis theory cytokine production in tissuesshould be assessed by localising not onlyprotein expression but also mRNA levels.'6 17We have used a radioactive in situ hybridisa-

tion technique to detect and localise mRNAexpression of IFNy in jejunal biopsiesspecimens from patients with coeliac disease,in order to study the role of this particularcytokine in the pathology of the disease.

Methods

PATIENTS AND BIOPSY SPECIMENSPeroral jejunal biopsy specimens were takenfrom adult patients with coeliac disease on anormal diet (n=5, mean age=45 years,range= 34-56), on a gluten free diet (n= 5,mean age=49-4, range=30-66), and fromcontrols (n=5, mean age=56, range=31-75)as part of their diagnostic investigations. Thecontrols were subsequently diagnosed ashaving the irritable bowel syndrome and allhad normal jejunal morphology. The diagnosisof coeliac disease was made according toESPGAN criteria.'8 All treated patientshad been on a gluten free diet for at leasttwo years and had normal or near normalvillous architecture. The biopsy specimenswere obtained using a Quinton multiplebiopsy instrument. Tissue specimens wereimmediately orientated, embedded in optimalcutting temperature compound (Cryo-M-bed;Brights, Huntington, UK) snap frozen inliquid nitrogen cooled isopentane, and storedin liquid nitrogen until processed. Cryostat

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Kontakou, Sturgess, Przemioslo, Limb, Nelufer, Ciclitra

TABLE I Oligonucleotids with DNA sequencescomplementary to interferon gamma mRNA used inthis study

IFNy.1 5"AAA GAG TTC CAT TAT CCG CTA CATTCG AAT3

IFN-y.2 5'GTA TTG CTT TGC GTT GGA CAT TCAAGT CAG3'

IFNy.3 5'ACC CAA AAC GAT GCA GAG CTG AAAAGC CAA3

sections were cut at 5 ,um on a Bright open-topcryostat at - 20°C and collected onto glassslides that had been washed with distilledwater rendered RNase free by diethylpyro-carbonate treatment19 and precoated withpoly-L-lysine. Sections were briefly fixed inacetone for five minutes and stored at -200C.

PROBES AND LABELLING METHODDNA oligonucleotide probes were preparedin-house using a Beckman synthesiser. IFN-ymRNA detection was achieved using a mixtureof three probes, each 30 bases long, withcDNA sequences coded by three differentexon regions of the IFN-y gene (sequencepublished by Gray and Goeddel).20 Theprobes used in this study and their sequenceare listed in Table I. Specificity of the probeswas confirmed by northern blotting of mRNAcoding for IFN-y, isolated from blood mono-

nuclear cells that had been cultured in thepresence of 1 ,ug/ml phytohaemmaglutinin or

10 ,ug/ml concanavalin A according toManiatis et al.19 Computer analysis of thesequences was achieved using the DNASISprogram.Four pmol of each oligonucleotide were

labelled by the addition of an ot 35S dATP'tail' to the 3' termini using terminal deoxy-nucleotidyl transferase, according to the sup-plier's protocol (Promega). After the labellingreaction the probes were purified by passingthem through a prepacked Sephadex G-50DNA grade column (Pharmacia), and theirspecific activity was measured using a

Rackbeta liquid scintillation counter. Thespecific activity of the probes was not less thanI X 108 cpm/Ipg.

PRETREATMENT AND HYBRIDISATIONThe method used was a modification of thatpreviously described by Hamid et al.21 Allsolutions used were made up with RNase freedistilled water. Sections, kept at -200C, were

air dried for at least one hour before fixationwith freshly prepared 4% (w/v) paraformal-dehyde in phosphate buffered saline pH 7-4 for15 minutes, followed by two washes in 15%(w/v) sucrose in phosphate buffered saline for10 minutes each. Pretreatments also involvedincubation with 0-3% (v/v) Triton-X-100 inphosphate buffered saline for 10 minutes,followed by incubation at 37°C with 1 pug/mlproteinase K for 30 minutes, postfixation in0 4% (w/v) paraformaldehyde for 10 minutesand treatment with 0 1 M triethanolamine con-

taining 0.25% (v/v) acetic anhydride for 10minutes. The sections were prehybridised in a

mixture of 2Xsaline-sodium citrate buffer, 5O0%

(v/v) formamide and 1 XDenhardt's solutionat 37°C for one hour and then hybridisedwith 2 ng of labelled probe overnight at 37°Cin a solution containing: 2xsodium citratebuffer, 50% (v/v) formamide, 04 mg/ml dena-tured sheared salmon sperm DNA and 10%(w/v) dextran sulphate (MW 500 000). Post-hybridisation stringency washes were carriedout at 39°C, for 30 minutes each, in 2Xsodiumcitrate buffer, 1 X sodium citrate buffer+ 50%formamide and 01 xsodium citrate buffer.Sections were dehydrated serially in 60%,90%, and 95% ethanol and dried at roomtemperature.

CONTROLSA sense sequence complementary to theIFN-y.l probe was used as a negative control.An oligo dT probe was also used for the detec-tion of total mRNA and served as a positivecontrol. To establish whether the hybridsformed were indeed RNA/DNA, some sectionswere treated with either DNase-free RNase(negative hybridisation signal) or RNase-freeDNase (no effect on hybridisation) after theproteinase K treatment. Sections were incu-bated at 370C with either of the two enzymes ata concentration of 600 U/ml for 30 minutes.

AUTORADIOGRAPHYDried sections were dipped in Ilford K5photographic gel emulsion that had beendiluted 1:1 in prewarmed distilled water, leftto dry in a dark room for approximately twohours, and then incubated at 4°C in a light-proof box containing silica gel for five days.Slides were developed in 44 g/l Dektol solution(Kodak) for two minutes, washed brieflyin distilled water and fixed in 30% (w/v)sodium thiosulphate at 4°C for 15 minutes.Finally, the slides were thoroughly rinsed inrunning tap water and counterstained withMayer's haematoxylin.

QUANTITATION AND STATISTICAL ANALYSISThe number of positive cells within the laminapropria were counted per unit area by usingan eye piece graticule (1 mm2) under X400magnification. At least five consecutive areas ofthe lamina propria (within and below the villigoing to the deeper lamina propria) werecounted for each section and for each specificprobe a minimum of two sections wereprepared and scored blindly for each patient.Each investigation with specific probes wasrepeated twice for all patients and the meantaken. The results were expressed as the meannumber of positive cells per unit area (0-0025mm2). The standard deviation of the mean wascalculated for each group and the Student's ttest was used to compare results for differentpatient groups.

Morphometric measurements were per-formed by an eyepiece micrometer underX 200 magnification. For each section aminimum of five measurements of villousheight to crypt depth ratio (V/C) were

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Detection of interferon-gamma mRNA in the mucosa ofpatients with coeliac disease by in situ hybridisation

,B' _Z...'-, {1. .ft__. .4-. v -M

Figure 1: Jejunal biopsy specimen from an untreated coeliac disease patient showing thedistribution of cells expressing mRNA for IFNy. Positive cells are abundant in the laminapropria but not the epithelium (original magnification X200).

made and the mean and standard deviationcalculated.

Results

IFN-y MRNA EXPRESSIONIn situ hybridisation, using 35S-labelledantisense oligonucleotide probe for IFNy,showed that numerous cells infiltrating thejejunal mucosa contained IFNy mRNA.Positively stained cells were found mainly inthe superficial lamina propria, and occasionallyin the deeper lamina propria. There was no

staining of the intraepithelial lymphocytes orsurface enterocytes. The results of one

untreated patient and one with normalmucosa, are shown in Figures 1 and 2 respec-tively. A similar label intensity was obtained inthe mucosa of the other patients within thegroups. The number of IFNy expressing cellsin the lamina propria of the untreated coeliacdisease patients was increased significantly

Figure 2: Distribution ofIFNy mRNA expressing cells in normaljejunal mucosa. Fewerpositive cells are seen compared with untreated coeliac mucosa (Fig 1), but still very clearlyidentified in the lamina propria (original magnification x200).

(p<0-001) compared with treated patients andcontrols. The difference in the numbers ofIFN-y mRNA expressing cells between treatedcoeliac patients and controls was not signifi-cant. These results are summarised in Table II,together with the morphometric analysis foreach group. The untreated coeliac patientsshowed a high degree of villous atrophy (ratioof villous height to crypt depth £ 1), while thetreated patients had near normal mucosalmorphology (normal ratio of villous height tocrypt depth between 3-5).

IN SITU HYBRIDISATION CONTROLSThe absence of hybridisation signal, in allpatient groups, with the 35S-labelled senseprobe (Fig 3) indicated that the binding of theIFNy antisense probe was specific. Treatmentwith RNase, but not DNase, before hybridisa-tion also resulted in failure of hybridisationwith labelled sense and antisense probe,indicating that the hybridisation was indeed onmRNA. Hybridisation with oligo dT probesfor assessment of total mRNA was detected inall biopsies.

DiscussionIn this study we have shown that IFNy isexpressed by lamina propria cells, increasinglyso in active coeliac disease, by assessing thepresence of cytoplasmic messenger RNA.IFN-y is known to be produced mainly byactivated T lymphocytes and most of thosefound in the lamina propria are of the CD4+(helper/inducer) subset.22 Although thephenotype of the positive cells was not identi-fied in this study, the localisation of these cellsin the lamina propria indicates that IFNy isprobably expressed by CD4+ T cells. Recentstudies on activated T lymphocytes in coeliacdisease reported the induction of proliferativeactivation of CD8+ (suppressor/cytotoxic)intraepithelial lymphocytes but non-prolifera-tive activation of lamina propria CD4+T cells.23 It was suggested that gluten specificCD4+ lamina propria T cells probablyproduce cytokines that induce epithelial cryptcell hyperplasia as well as local proliferation ofintraepithelial lymphocytes. This agrees withreports in Crohn's disease that IFNy andinterleukin 2 are produced by activated CD4+cells. 14 The same report suggested that there isa continuing delayed hypersensitivity responsewithin the mucosa in Crohn's diseasewhich may be responsible for the pathologicaldamage of the mucosa. In mice, IFN-y issynthesised by the TH1 subpopulation ofCD4+ cells, which have been shown to cause adelayed hypersensitivity response, partiallyascribed to the production of IFN-y, inresponse to local presentation of antigen.24

IFN-y, as well as tumour necrosis factor a,has been shown to be a capable effectormolecule in immune destruction of selftissues,25 and to reduce the epithelial functionof the human colon carcinoma cell line HT29cl 19A.26 Both cytokines can destroy targettissues of known autoimmune responses,

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1040 Kontakou, Sturgess, Przemioslo, Limb, Nelufer, Ciclitra

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. MX,w

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. ...i .3 .V_?ll-?

44i

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ....'.... ... ....>j;;_ _.Figure 3: Jejunal biopsy from an untreated coeliac disease patient showing absence ofhybridisation signal with the sense IFNy probe, used as a negative control (originalmagnification X200).

individually or in combination, as was shownin an animal model of diabetes where a combi-nation of both cytokines destroyed isolatedpancreatic a islet cells in vitro.27 IFNy alsoinhibits the proliferation of rodent intestinalepithelial cell lines28 and decreases epithelialtight junction permeability.29 30 In the presentstudy the treated coeliac disease patients hada near normal mucosal morphology, assupported by a normal villous height to cryptdepth ratio, and the number of IFN-yproducing cells showed no significant differ-ence from the controls. It is therefore possiblethat in coeliac disease there is an associationbetween the observed damage to the villi andincreased IFN-y production. This hypothesisshould be confirmed by studying the effect ofIFNy on epithelial differentiation and prolifer-ation in human jejunum organ culture.A possible cellular immunopathogenic

mechanism involving the intestinal mucosa incoeliac disease has been described in a recentreview.7 Short chain toxic peptides producedby the digestion of gluten are absorbed acrossthe small intestinal epithelium, bound to HLAclass II molecules, and presented bymacrophages in the lamina propria to antigenspecific CD4+ T cells. As a result, cytokinessuch as IFN-y are produced which couldlead to increased expression of HLA class IIantigens on surface enterocytes9 and adhesionmolecules within the lamina propria.31Damage to enterocytes may occur via the localproduction of toxic mediators, both within the

TABLE II Number of interferon gamma mRNA (IFNy)expressing cells in the lamina propria/00025 mm2 anddegree of villous atrophy (expressed as villous height tocrypt depth ratio) in each of the patient proups studied

IFN-y mRNA ViUlous height toexpressing cells crypt depth ratio

Group No (mean (SD)) (mean (SD))

Coeliac disease:Untreated 5 18-58 (2-71) 0-88 (0-16)Treated 5 5-0 (1-83) 3 62 (0-19)

Controls 5 4-1 (2 63) 4-12 (0 33)

lamina propria and the epithelium, and also bydirect cytotoxic mechanisms.The initial event in the action of the inter-

ferons is their binding to specific receptors onthe cell surface, present on most cells. After thebinding, the interferon receptor complexesare internalised by receptor mediatedendocytosis.32 For this reason, the localisationof the IFNy product by immunohistochemicalmethods has been very difficult to interpret,because it will also identify target cells as wellas any complexes passively uptaken by phago-cytes and mucosal epithelial cells.33 Cytokineproduction can also be affected by competitiveinhibition of cytokine receptor by specificand non-specific receptor antagonists. Thismechanism has been well described for inter-leukin 1.34 One or more of the mechanismsdescribed above could explain the conflictingresults between the present study using in situhybridisation and a previous report usingimmunohistochemistry.15 Although, mRNAlevels can also be misleading as some cytokinesare regulated post-transcriptionally, in situhybridisation remains a very powerful tech-nique enabling the localisation of the cellularsynthesis of various cytokines. The results ofthe present study support the hypothesis thatthe primary events in the immunopathogenesisof coeliac disease occur in the lamina propriaand suggest that IFNy is probably associatedwith the damage to the villi that is observed inthis condition.

The authors wish to thank the St Thomas's Research(Endowments) Committee, the Nutricia Research Fund, andthe Jean Shanks Research Foundation.

1 Marsh MN. Mucosal pathology in gluten sensitivity. In:Marsh MN, ed. Coeliac Disease. Oxford: BlackwellScientific Publications, 1992: 136-91.

2 Marsh MN. The morphology and immunopathology of thejejunal lesion in gluten-sensitivity. Eur J GastroenterolHepatol 1991; 3: 163-8.

3 Ciclitira P, Ellis H, Wood G, Howdle P, Losowsky M.Secretion of gliadin antibody by coeliac jejunal mucosalbiopsies cultured in vitro. Clin Exp Immunol 1986; 64:119-24.

4 Spencer J, MacDonald T, Walker-Smith J, Ciclitira P,Isaacson P. Changes in intra-epithelial lymphocyte sub-populations in coeliac disease and enteropathy associatedT-cell lymphoma (malignant histiocytosis of theintestine). Gut 1989; 30: 339-46.

5 Brandtzaeg P, Halstensen T, Kett K, Kracci P, Kvale D,Rognum T, et al. Immunobiology and immunopathology ofhuman gut mucosa: humoral immunity and intraepitheliallymphocytes. Gastroenterology 1989; 97: 1562-84.

6 Kagnoff MF. Genetic basis of coeliac disease: role of HLAgenes. In: Marsh MN, ed. Coeliac Disease. Oxford:Blackwell Scientific Publications, 1992: 215-38.

7 Ciclitira P, Sturgess R. Clinicopathologic mechanisms inceliac disease. Curr Opinion Gastroent 1992; 8: 262-7.

8 Sollid L, Gaudemack G, Markussen G, Kvale D,Brandtzaeg P, Thorsby E. Induction of various HLAclass II molecules in a human colonic adenocarcinomacell line. ScandjImmunol 1987; 25: 175-80.

9 Sturgess R, Hopper L, Spencer J, Hung C, Nelufer J,Ciclitira P. Effects of interferon--y and tumour necrosisfactor-a on epithelial HLA class-II expression on jejunalmucosal biopsy specimens cultured in vitro. Scand JGastroenterol 1992; 27: 907-11.

10 Fais S, Capobianchi M, Pallone F, Di Marco P, BoirivantM, Dianzani F, et al. Spontaneous release of interferon yby intestinal lamina propria lymphocytes in Crohn'sdisease. Kinetics of in vitro response to inteferon y in-ducers. Gut 1991; 32: 403-7.

11 Kusugami K, Yongman K, West G, Fiocchi C. Intestinalimmune reactivity to interleukin 2 differs among Crohn'sdisease, ulcerative colitis, and controls. Gastroenterologzy1989; 97: 1-9.

12 Ouyang Q, El-Youssef M, Yen-Lieberman B, SapatnekarW, Youngman K, Kusugami K, et al. Expression of HLA-DR antigens in inflammatory bowel disease mucosa: roleof intestinal lamina propria mononuclear cell-derivedinterferon gamma. Dig Dis Sci 1988; 33: 1528-34.

13 MacDonald T, Hutchings P, Choy M, Murch 5, Cooke A.Tumour necrosis factor-alpha and interferon-gamma-

on Decem

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http://gut.bmj.com

/G

ut: first published as 10.1136/gut.35.8.1037 on 1 August 1994. D

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Detection of interferon-gamma mRNA in the mucosa ofpatients with coeliac disease by in situ hybridisation 1041

production measured at the single cell level in normal andinflamed human intestine. Clin Exp Immunol 1990; 81:301-5.

14 Breese E, Braegger C, Corrigan C, Walker-Smith J,MacDonald T. Interleukin-2 and interferon-y-secreting Tcells in normal and diseased human intestinal mucosa.Immunology 1993; 78: 127-31.

15 Al-Dawoud A, Nakashabendi I, Foulis A, McMowat A.Immunohistochemical analysis of mucosal gamma-inter-feron production in coeliac disease. Gut 1992; 33:1482-6.

16 Dallman M, Montgomery R, Larsen C, Wanders A, WellsA. Cytokine gene expression: analysis using northern blot-ting, polymerase chain reaction and in situ hybridisation.Immunol Rev 1991; 119: 163-79.

17 Ogilvie A, Wood N, Dickens E, Wojtacha D, Duff G. In situhybridisation. Ann Rheum Dis 1990; 49: 434-9.

18 Meeuwisse GW. Diagnostic criteria in coeliac disease(European Society of Paediatric Gastroenterology). ActaPaed Scand 1970; 59: 461-3.

19 Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: alaboratory manual. New York: Cold Spring HarborLaboratory Press, 1989: 7.4.

20 Gray PW, Goeddel DV. Molecular biology of interferon-gamma. In: Lymphokines. New York: Academic Press Inc,1987.

21 Hamid Q, Azzawi M, Ying S, Maqbel R, Wardlow A,Corrigan C, et al. Expression of mRNA for interleukin-5in mucosal bronchial biopsies from asthma. J Clin Invest1991; 87: 1541-6.

22 MacDonald T. T cell-mediated intestinal injury. In: MarshM, ed. Coeliac disease. Oxford: Blackwell ScientificPublications, 1992: 293-304.

23 Halstensen T, Brandtzaeg P. Activated T lymphocytes inceliac lesion: non-proliferative activation (CD25) ofCD4+ oLI3 cells in the lamina propria but proliferation(Ki-67) of u/P and -y/8 cells in the epithelium. Eur JImmunol 1993; 23: 505-10.

24 Mosmann T, Coffman R. Heterogeneity of cytokine secret-ing patterns and functions of helper T cells. Adv Ihnnunol1989; 46: 111-7.

25 Deem R, Shanahan F, Targan S. Triggered human mucosalT cells release tumour necrosis factor-alpha and inter-feron-gamma which kill human colonic epithelial cells.Clin Exp Immunol 1991; 83: 79-84.

26 Hiribarren A, Heyman M, L'Helgouac'h A, Dejeux J.Effect of cytokines on the epithelial function of the humancolon carcinoma cell line HT29 cl 19A. Gut 1993; 34:616-20.

27 Cambell I, Iscaro A, Harrison L. IFNy and tumour necrosisfactor a. cytotoxicity to murine islets of langerhans.J3Immunol 1988; 141: 2325-31.

28 Cerf-Bensussan N, Quaroni A, Kurnick J, Bhan A.Intraepithelial lymphocytes modulate Ia expressionby intestinal epithelial cells. _7 Immunol 1984; 132:2244-52.

29 Madara J, Stafford J. Interferon-y directly affects barrierfunction of cultured intestinal epithelial monolayers.J Clin Invest 1989; 83: 724-7.

30 MacDonald T, Spencer J. Evidence that activated T cellsplay a role in the pathogenesis of enteropathy in humansmall intestine. J Exp Med 1988; 167: 1341-9.

31 Sturgess R, Kontakou M, Spencer J, Hooper L, MakgobaM, Ciclitira P. Effects of Interferon--y and Tumour necro-sis factor-ot on ICAM-1 expression on jejunal mucosalbiopsies cultured in vitro. Gut 1993; 34 (1): S31.

32 Oppenheim J, Ruscetti F, Faltynek C. Cytokines. In: StitesD, Terr A, eds. Basic and clinical immunology. Appletonand Lange, 1988: 78-100.

33 Stamp G, Poulsom R, Chung L, Keshav S, Jeffery R,Longcroft J, et al. Lysozyme gene expression inInflammatory Bowel Disease. Gastroenterology 1992; 103:532-8.

34 Seckinger P, Lowenthal J, Williamson K, Dayer J,MacDonald H. A urine inhibitor of interleukin 1 activitythat blocks ligand binding. 7Immunol 1987; 139: 1546-9.

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