Gut, 1991, 32, 913-917

Serum concentrations of tumour necrosis factor inchildhood chronic inflammatory bowel disease

S H Murch, V A Lamkin, M 0 Savage, J A Walker-Smith, T T MacDonald

Departments ofPaediatricGastroenterology andChild Health, StBartholomew's Hospital,West Smithfield, LondonEClA 7BES H MurchV A LamkinMO SavageJ A Walker-SmithT T MacDonaldCorrespondence to:Dr Simon Murch.Accepted for publication10 September 1990

AbstractSerum tumour necrosis factor a (TNFa) con-centrations were measured by enzyme linkedimmunoadsorbent assay in 31 normal childrenand during 65 episodes of clinical remissionand 54 episodes of relapse in 92 children withchronic inflammatory bowel disease. Anappreciable rise in TNFa was found only inchildren in relapse of ulcerative colitis andcolonic Crohn's disease. The group ofchildrenwith small bowel Crohn's disease in relapse didnot show increases of TNFa above controlconcentrations, despite an equivalent rise indisease indices. Height velocity was depressedin children with relapse of large bowel Crohn'sdisease and ulcerative colitis compared withthe equivalent condition in remission. Theimpairment of growth velocity was signific-andy greater in relapse of large bowel Crohn'sdisease and ulcerative colitis than in smallbowel Crohn's disease alone, although for thesubgroups in stage 1 puberty (prepubertal) thedifferences were not significant. Inadequategrowth in chronic inflammatory bowel diseaseis currently ascribed to inadequate nutritionand TNFa may contribute to this through itscachexia inducing effects. It may, in addition,diminish pituitary growth hormone release.These results suggest that production ofTNFa may be associated with growth failure inrelapse of colonic inflammatory bowel disease.

Tumour necrosis factor a (TNFa)/cachectin, acytokine produced mainly by activated macro-phages and monocytes, is generating increasinginterest both as a mediator of tissue maturationand local immunity and as a destructive agent

capable of causing profound cachexia and tissueinjury. The balance of beneficial and deleteriouseffects are determined by its level of productionand its interaction with other mediators.'4

Excess production ofTNFa has been found inparasitic diseases including trypanosomiasis andmalarial (in which it may be a major mediator ofcerebral involvement6), in malignancy,7 connec-tive tissue diseases,8 and septicaemia.' Highserum concentrations have been shown to beassociated with poor outcome in meningococcalsepticaemia'° and raised concentrations in cere-brospinal fluid have been shown in bacterialmeningitis in childhood."There have been no studies in which TNFa

concentrations have been measured in childhoodchronic inflammatory conditions, and it has notpreviously been implicated in growth failure.

Methods

PATIENTS

Control subjectsTwenty five of the 31 children used as controlsubjects underwent colonoscopy and bariumfollowthrough examination for investigation ofpossible inflammatory bowel disease. In all casesfull investigation was normal and a diagnosis offunctional abdominal pain or irritable bowelsyndrome was made. Biopsy specimens showedno evidence of microscopic colitis, bariumfollowthrough was normal or showed lymphoidnodular hyperplasia only (five cases) and therewas no rise in erythrocyte sedimentation rate orC reactive protein. In the other children venesec-tion was performed to investigate short stature(two cases), possible food allergy (one case), andasthma (three cases). All children were under-going venesection for other investigations.

TABLE I Median (range) tumour necrosis factor a (TNFa) and disease indices (SEM) inrelapse and remission ofchildhood inflammatory bowel disease

Erythrocytesedimentationrate (mm in C reactive TNFa

Condition No the Ist hour) protein (mg/l) (pg/ml) p*

Control subjects 31 <10(0-18)

Small bowel Crohn's disease 17 7-3 10-7 <10 NSin remission (1-97) (2-18) (0-30)

SmallbowelCrohn'sdisease 13 27-38 40-1 <10 NSin relapse (4 65) (6-88) (0-30)

Colonic Crohn's disease 25 14-4 14-1 <10 NSin remission (1.88) (2 30) (0-30)

Colonic Crohn's disease 25 23-2 37-7 16 <0 00001in relapse (2 72) (4-49) (0-60)

Ulcerative colitis 23 13-47 10-7 <10 NSin remission (2-93) (2.16) (0-18)

Ulcerative colitis 1 1 36-2 26-1 27 <0 00001in relapse (4 7) (10-45) (0-150)

Distal colitis 5 39 3 41-2 <10 NSin relapse (14-1) (20 2) (0-12)

*Probability values, by Kolmogorov-Smirnov 2 group test, comparing disease group median serumTNFa with that of control subjects. NS= not significant.

Chronic inflammatory bowel diseaseAll patients had been assigned a definite diagno-sis of Crohn's disease or ulcerative colitis on thebasis of radiological and histological findings andhad undergone colonoscopy and barium follow-through investigation in the preceding 12months. The children with Crohn's disease weredivided into two groups on the basis of colonicinvolvement. Those with disease affecting thesmall bowel up to and including the terminalileum, but with no evidence of macroscopic ormicroscopic involvement ofthe caecum or colon,were labelled as having small bowel Crohn'sdisease. Those whose disease included thecaecum or colon, whether or not there was alsosmall bowel involvement, were included in thelarge bowel Crohn's disease group.

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Murch, Lamkin, Savage, Walker-Smith, MacDonald

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Control Crohn's Crohn's Crohn's Crohn's Ulcerativen = 31 small bowel small bowel large bowel large bowel colitis

remission relapse remission relapse remissionn= 17 n= 14 n=25 n=26 n=23

DISEASE ACTIVITYPatients were assessed in the paediatric inflam-matory bowel disease clinic at two monthintervals if well and every two to four weeks ifnot. Disease activity was assessed on the basis ofreported symptoms, change in weight, clinicalexamination, and investigation, with the patientclassified as follows. (a) Fully in remission(asymptomatic, appropriate weight gain, noabdominal tenderness or masses, no activeperianal or oral disease, no appreciable rise indisease indices at last review). (b) Partiallyrelapsed (minor episodic abdominal pain with-out systemic upset; active perianal disease; poorweight gain; isolated rise in disease indices at lastreview). (c) Relapsed (recurrent moderate orsevere abdominal pain or diarrhoea >5 timesdaily with stools containing blood or mucus, orboth; appreciable weight loss; abdominal tender-ness or mass). Only those patients who wereclassified as fully relapsed or fully in remissionwere included in the study.

ASSESSMENT OF GROWTH AND PUBERTAL STAGEAll patients underwent formal auxologicalmeasurement and pubertal staging at each clinicvisit and during any admission. Standing heightwas measured by the same observer (VAL) usinga stadiombter and height velocity was calculatedover the preceding three or six months. Apubertal stage was assigned on the basis ofgenital development in boys and breast develop-ment in girls using standard ratings from stage 1

(prepubertal) to stage 5 (adult).'2

TNFa ASSAY PROTOCOLAfter venesection blood was stored overnight at4°C before being centrifuged at 2000 rpm for 10

Ulcerativecolitisrelapsen = 15

Distalcolitisrelapsen = 6

minutes and the supernatant separated. Theserum was then stored at -70°C until assayed.

Aliquots of 50 itl of serum were assayed forTNFa by the enzyme linked immunoadsorbentassay (ELISA) technique, using a commerciallyavailable assay (T Cell Sciences, CambridgeMA, USA). Results were expressed in pg/ml,with the lower limit of detection of the assaybeing 10 pg/ml.

STATISTICAL ANALYSISThe Kolmogorov-Smirnov 2 group test was usedfor comparison of TNFa and pubertal stagesbetween groups and the unpaired Student's t testfor analysis of erythrocyte sedimentation rate, Creactive protein, and height velocity.

Results

TNFa CONCENTRATION AND DISEASE ACTIVITYSerum TNFa was significantly raised abovecontrol values (30 out of 31 below 10 pg/ml,range <10-18) in only two disease states, bothassociated with colonic inflammation (Figureand Table I): ulcerative colitis in relapse (n= 11,median 27 pg/ml, range <10-150, p<0 000007)and Crohn's colitis in relapse (n=25, median 16pg/ml, range <10-60, p<0000002). Relapse ofsmall bowel Crohn's disease alone, with no

evidence of colonic involvement, was not associ-ated with a significant rise in TNFa abovecontrol values (n= 13, median <10 pg/ml, range<10-30, p>02), despite insignificant differ-ences between the small and large bowel groupsin the severity of relapse as measured by diseaseindices (see Table I: mean (SEM) erythrocytesedimentation rate 27-38 (4-65) v 23-2 (2-72)mmin 1st hour, mean (SEM) C reactive protein 40 1

Serum TNFa (pglml) in controlsubjects and in disease groups.

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Serum concentrations oftumour necrosisfactor a in childhood chronic inflammatory bowel disease

TABLE II Current age and mean (SEM) growth velocity in entire group and in patients in stageI puberty in relapse and remission groups

Growth velocity

EntireAge group Patients in stage I

Condition (years) (cm/year) puberty (cmlyear) p*

Control subjects 11-35(0-51)

Small bowel Crohn's disease in remission 13 74 6-49 6-48 NS(0.61) (0-78) (0-89, n=4)

Small bowel Crohn's disease in relapse 14-48 5 24 3-78(049) (089) (0-83, n=5)

Colonic Crohn's disease in remission 13-34 4 90 4-71 NS(0 57) (0-55) (0-69, n=9)

Colonic Crohn's disease in relapse 13-20 2-35 2-58 <0-001(0-34) (0-47) (0-71, n=1II)

Ulcerative colitis in remission 12366 5°59 530 NS(0 74) (0-48) (0-18, n= 10)

Ulcerative colitis in relapse 11-58 2-14 2 50 <0-001(0 99) (0-62) (0-98, n=9)

Distal colitis in relapse 12-4 5 64 NS(1-06) (0 74)

*Probability values, by unpaired Student's t test, comparing group mean height velocity (entiregroup) with that in subgroup with relapsed small bowel Crohn's disease. NS=not significant.

(6-9) v 37-7 (4 49) mg/I, p>02). In the smallgroup with distal colitis alone serum TNFa wasnot raised above control values despite a greaterrise in the sedimentation rate than in the groupwith relapsed colonic Crohn's disease (39-3(14 1) v 23'2 (2-72), p<005) and a similar rise inC reactive protein. Ulcerative colitis in relapsewas associated with a higher sedimentation ratethan relapsed colonic Crohn's disease (36&2 (4 7)v 23-2 (2 72), p<0007) with no significantdifference in concentrations ofC reactive proteinor TNFa.

TNFa CONCENTRATION AND CURRENT HEIGHTVELOCITYCurrent mean growth velocity was significantlylower in the groups with relapse of colonicCrohn's disease (p<0001) and ulcerative colitis(p<0 001) than in the groups with the equivalentcondition in remission (Table II). Although forthe groups with small bowel Crohn's diseasethere was no significant difference between thosein relapse and remission, analysis of the sub-groups in stage 1 puberty showed significantlylower growth in those with symptomatic relapse(p<O05).

Current growth velocity was significantlygreater in the group with relapsed small bowelCrohn's disease than in those with relapsedCrohn's colitis (p<0 0002) or ulcerative colitis(p<00005).No significant differences were found between

the groups in overall pubertal staging, althoughpatients in the group with small bowel Crohn'sdisease in relapse were significantly older (mean(SEM) age 14-48 (0-49) years) than those withcolonic Crohn's disease (13-20 (0 34) years) orulcerative colitis (11 58 (0 99) years) in relapse(p<0 05). Further analysis of the subgroups instage 1 puberty, however, showed no significantdifferences between the groups, although thenumber ofcases was small. The group with distalcolitis had less impairment of growth than thegroups with relapsed colonic Crohn's disease(p<0-02) or ulcerative colitis (p<0c0004).

Fifteen of the 48 patients with colonic diseasein remission were taking prednisolone (5-30 mg)

daily compared with 27 of the 46 with colonicdisease in relapse (7 5-40 mg), two of the 17 withsmall bowel disease in remission (5-30 mg), andthree ofthe 13 with small bowel disease in relapse(5-20 mg). No significant differences in serumTNFa or height velocity were found betweenthose taking prednisolone and those not takingcorticosteroid treatment.

DiscussionThese results show a clear difference between theconcentrations of serum TNFa in children withrelapsed colonic inflammatory bowel disease andthose with relapsed Crohn's disease affecting thesmall bowel alone. The TNFa in serum isprobably an overspill from that produced locallyby activated macrophages in the colon and istherefore presumably an underestimate of localproduction. In support of this, a similar variationbetween local and systemic TNFa concentrationhas been shown by Nadal et al," who foundconcentrations in the cerebrospinal fluid of57-20 000 pg/ml in 11 children with bacterialmeningitis, with serum TNFa detectable in onlyone.Most TNFa is produced in activated macro-

phages and monocytes, although it is producedto some extent by hepatic Kupffer cells, cerebralastrocytes, and microglial cells. '4 Activatedmacrophages and T cells are abundant in intesti-nal mucosa in inflammatory bowel disease"3 andin recent experiments it has been shown thatproduction ofTNFa is increased at the single celllevel,'4 thus showing formally that at least someof the TNFa detected in serum in inflammatorybowel disease is gut derived.

Populations of histiocytes and macrophageswith different morphology and biochemicalactivity have been found in the small bowelcompared with the colon.'5 It is thought that thisdifference reflects the requirement for antigenhandling in the small intestine as opposed toscavenging in the bacteria laden colon. In colonicinflammatory bowel disease a further distinctgroup of macrophages has been found to replacethose normally present in the colon, the popula-tion returning to normal with treatment. 15 Varia-tion in expression of surface markers on T -cellsand macrophages between colonic and smallbowel inflammatory bowel disease has morerecently been shown. 16 These results suggest thatour findings of increased serum TNFa inrelapsed colonic but not small bowel disease mayreflect fundamental differences between thepopulations of macrophages in these sites.TNFa has been shown in animals to have a

cytopathic effect on bowel mucosa," probablymediated in part by platelet activating factor,'8raising the possibility that it may be involved inthe pathogenesis of inflammatory bowel disease.Multifocal gastrointestinal infarction, mediatedby enhanced procoagulant activity, has in factrecently been proposed as a pathogeneticmechanism in Crohn's disease,'9 and raisedconcentrations of interleukin-1 have beenreported.2'0 I Production ofTNFt from activatedmacrophages, and its subsequent systemicaction, has been attenuated by administration ofnon-steroidal anti-inflammatory drugs or

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916 Murch, Lamkin, Savage, Walker-Smith, MacDonald

corticosteroids,422 although we found no signifi-cant difference in serum TNFa between patientstaking mesalazine, sulphasalazine, or predniso-lone and those taking no drugs.

Suppression of linear growth is a commonfeature ofchildhood chronic inflammatory boweldisease, affecting up to 30% of patients.23-27Although children may present with growthfailure alone, without symptoms or weight loss,28it is currently accepted that nutritionaldeficiency is the major cause ofthe growth failureand that caloric supplementation can restoregrowth.2325-27 29-32 Somatomedin C (insulin likegrowth factor 1) concentrations have been foundto be low in growth impaired children withchildhood inflammatory bowel disease and toincrease as calorie intake improves.33 Delayedgastric emptying, which may contribute to poororal intake, has also been found in malnourishedchildren with Crohn's disease.34

Although there seems to be major variationsbetween the reported studies in the amount ofcaloric supplementation required to reinstitutegrowth and the nutritional supplementation wasprovided in several of the studies by methodswhich may in themselves be therapeutic,35-37 suchas parenteral nutrition23 31 32 or elemental diet,27 itis clear that restoring caloric intake, even using apolymeric non-elemental diet29 or by aggressiveoral nutrition25 may lead to improvement oflinear growth velocity in many patients.The possible contribution of growth hormone

deficiency to the growth suppression of chronicinflammatory bowel disease has been more con-troversial. After the report by McCaffery et a126 ofimpaired growth hormone response to insulininduced hypoglycaemia in 11 of 14 growthretarded patients, subsequent studies have failedto show consistent abnormalities of growthhormone production.38" These studies may becriticised on the grounds that growth hormonereserve was not retested in remission and thatgrowth hormone concentrations in the normalrange were considered appropriate, althoughraised plasma concentrations are normally foundin protein-calorie malnutrition.4' In the onereport in which growth hormone reserve wasretested it was found to be low in relapse andin. ~~~~42normal in remission.The value of surgical resection in stimulating

growth, particularly in children in early puberty,has been emphasised by recent reports.445 Suchpronounced improvement of growth velocity,after resection of affected bowel, suggests thatone or more mediators may be produced locallywhich have an inhibitory effect upon growth.

Although it is not possible to infer such directinhibition from these results, TNFa couldpotentially affect growth by its adverse effects onappetite, nutrition, and growth hormone pro-duction. Its effects on nutrition are profound,with chronic production causing wasting despiteunlimited intake.' In vitro studies have showncatabolic changes at the cellular level.47 48Administration of recombinant TNFa toanimals and humans causes delayed gastricemptying,49 profound anorexia, weight loss, anda redistribution of body proteins with depletionof skeletal muscle protein and increase in hepaticacute phase protein synthesis.50 5' Hepatic

expression of albumin mRNA was decreased by90% after TNFa administration to mice.52 Inaddition, TNFa has been shown in vitro to affectanterior pituitary hormone release,53 specificallydiminishing release of growth hormone.54Many of the children in this study were

passing through puberty, or alternatively suffer-ing pubertal delay because of their disease. Themost unusual finding was the relatively goodgrowth of the group with relapsed small bowelCrohn's disease, and it seems likely that moremembers of this group were undergoing theirpubertal growth spurt, despite the similarities inoverall pubertal staging between the groups. Themean age of this group was significantly greaterthan the others and only this subgroup in stage 1puberty had a lower mean height velocity thanthat of the entire group. Further study ofgrowthand TNFa production in prepubertal childrenwith chronic inflammatory bowel disease isrequired.With the potential advent of therapeutic

monoclonal anti-TNFa antibodies in conditionswhere TNFa may be playing a pathological part,it is clear that further work is required to deter-mine the site of TNFt production in chronicinflammatory bowel disease and to establishwhether its excess production does play a part inmucosal inflammation. In our study the highestserum TNFca (150 pg/ml) was found in a girl inwhom toxic dilatation of the colon was con-sidered possible (she settled on conservativemanagement) and it will be important to studyTNFca production in cases where this diagnosis iscertain. Should TNFa be found to have a role inthe pathogenesis of this life threatening compli-cation (and its credentials for direct bowel walldestruction are well established from animalwork),'7 18 then therapeutic monoclonal anti-TNFa antibodies may present an importantalternative to the radical surgery currentlyrequired. Their possible future use in less seriousrelapse of disease will be more difficult to assess,and it is important that those looking afterchildren with chronic inflammatory boweldisease should aim for precise localisation of theaffected bowel and stringent monitoring ofgrowth.

This work was supported by the Crohn's in Childhood ResearchAssociation and the Wellcome Trust.

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