J Clin Pathol 1981 ;34:759-763

Evidence for an immune complex vasculitis inneonatal necrotising enterocolitisELIZABETH S GRAY, DAVID J LLOYD, STANLEY S MILLER,ALAN I DAVIDSON, NICOLA J BALCH, CHARLES HW HORNE

From the Department ofPathology, University of Aberdeen, the Department of Neonatal Paediatrics,Aberdeen Maternity Hospital and University of Aberdeen, and Surgical Unit, Royal Aberdeen Children'sHospital

SUMMARY Despite many attractive hypotheses, neonatal necrotising enterocolitis (NNEC) remainsa disease of unknown aetiology. By means of the immunofluorescent direct sandwich technique wehave demonstrated immune complex deposition in the small vessels of the diseased bowel wall. Webelieve this is the first report of evidence of an immunological reaction occurring in this disease.

In 1979 Tait and Kealyl described in detail thehistopathological features of neonatal necrotisingenterocolitis (NNEC), a potentially fatal conditionpredominantly affecting low birth weight preterminfants in modern intensive care nurseries. Thedisease consists of a fulminating necrosis of theintestinal tract together with production of gaswithin the bowel wall (pneumatosis intestinalis), thelatter presumably being due to invasion of the tissuesby gas producing bacteria.Although a variety of hypotheses has been

proposed, the aetiology, despite much research, isstill unknown. Interest has centred recently on thebacterial flora of the affected bowel, especially on therole of the clostridia.2 However, in 1977, Lake andWalker3 studying immune defence mechanisms inneonatal gut, in particular the role of secretory IgAin the regulation of antigen absorption, suggestedthat as the neonatal gut had such a precarious defenceagainst excessive absorption of antigen, NNEC

Accepted for publication 19 November 1980

could be due to an altered host defence. Unfortunatelythey could offer no clinical or laboratory evidence tosupport this hypothesis.We report here four patients with NNEC, in three

of whom we found evidence of an immune reaction.To the best of our knowledge we believe these to bethe first patients with NNEC in which immuno-logical features have been demonstrated.

Patients and methods

The four infants were patients requiring treatment inthe Special Care Nursery in the Aberdeen MaternityHospital. Brief clinical details are supplied inTable 1.

Before fixation in 10% neutral buffered formalin,fresh tissue was obtained from the acutely diseasedbowel of three infants with NNEC requiringemergency surgical intervention. In one (case 1)further material was obtained at necropsy and inanother (case 3) only necropsybiopsieswere available.In all four patients portions of necrotic and of less

Table 1 Immunoglobulins, complement (C3) and fibrin deposition within the small vessels of diseased bowels inNNEC using the immunofluorescent technique

Case No Clinical summary Outcome IgG IgA IgM C3 Fibrin

Gestational age (wk) Age at biopsy (days)

1 32 6 died 0 0 0 + + + +(9 days)

2 37 8 alive 0 0 + + +3 29 12 died 0 + ++ ++ ++

(12 days)4 30 12 alive 0 + ++ ++ ++

O to ++ + = arbitrary scale of fluorescence intensity.759

on May 22, 2021 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.34.7.759 on 1 July 1981. Dow

nloaded from

760

obviously involved intestine were snap-frozen andthe presence of IgG, IgA, IgM, C3, and fibrindetected in the cryostat-cut sections by the directimmunofluorescent technique with commerciallyprepared fluorescein-labelled antibody (BurroughsWellcome Ltd and Hoechst Pharmaceuticals). Afterfixation, tissue was processed and 5 ,um sections cutand stained by the haematoxylin and eosin (H & E)method.

Results

Our immunopathological findings are shown inTable 1. The intensity of the fluorescence is scoredon an arbitrary scale 0 to + + +.

In three of the infants (cases 2, 3, 4) there wasdeposition of IgM (Fig. 1) C3, and fibrin within thelumina and walls of the small vessels of the diseasedbowel both in the totally necrotic areas and insections less severely involved (even macroscopically

Fig. 1 IgM deposition within the vessel wallsdemonstrated by fluorescein-labelled anti-IgM

Gray, Lloyd, Miller, Davidson, Balch, Horne

normal bowel showed ulceration on histologicalexamination). IgA was present also in two patients(cases 3, 4), while IgG was detected in none of theinfants.A more detailed immunopathological examination

was possible from necropsy specimens in two patients(cases 1, 3) and in addition to diseased bowel,tissue was obtained from histologically normalbowel, the larger mesenteric vessels, and fromkidney. The findings are shown in Table 2 withneither case demonstrating immunoglobulin norcomplement deposition in the glomeruli. In case 3the immunoglobulins and complement were detect-able only in the vessels of diseased bowel but notin normal bowel nor in larger mesenteric vessels.In case 1 no immunoglobulins were found in anytissue although complement and fibrin were presentin the smaller vessels of all the sections of bowel,and also in the larger mesenteric vessels.

Examination of the sections stained by the H & Emethod showed in all cases the typical histologicalappearances of NNEC. Although vascular changesare not considered a notable feature, we did findevidence of vascular damage in cases 2, 3, and 4.Some of the small vessels in the damaged bowel wallshowed perivasculitis (Fig. 2), fibrinoid necrosis(Fig. 3), and thrombosis with vessel wall damage andhaemorrhage (Fig. 1). In case 1, fibrin depositionwas noted in the vessels and there was no associatedinflammatory reaction.

Discussion

The association of immunoglobulin, complement,and fibrin (cases 2, 3, 4, Table 1) within the lumenand walls of blood vessels is generally accepted asindicating intravascular immune complex deposition(ICD). The presence of complement and fibrin inonly case 1 may indicate that complement wasbeing activated within the vessel by a non-immuno-logical process-that is, via the alternative pathway.

In each of the three neonates with evidence oflocal immune complex composition the antibodyinvolved was of the IgM class, the intensity of thefluorescent staining being most marked in the

Table 2 Immunoglobulin, complement (C3) and fibrin deposition within the vessels of tissue from different sites attime of necropsy

Site of biopsy Case I Case 3

IgM C3 Fibrin IgM C3 Fibrin

Necrotic bowel 0 + + LA±++Normal bowel 0 + - 0 0Larger mesenteric vessels 0 + 0 0Renal glomeruli 0 0 0 0 0 0

0 to + + + = arbitrary scale of fluorescence intensity.

on May 22, 2021 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.34.7.759 on 1 July 1981. Dow

nloaded from

Evidence for an immune complex vasculitis in neonatal necrotising enterocolitis

Fig. 2 PerivasculitisoJ small vessels in thebowel wall.Haematoxylin andeosin x 256

Fig. 3 Fibrinoidnecrosis of bowel wallarteriole. Haematoxylinand eosin x 650

"older" infants. As maternal IgM cannot cross theplacental barrier this IgM must be fetal or neonatalin origin. IgG was not detected presumably becauseIgG antibody appears later in the immune response.The presence ofIgM and the absence of IgG indicatea recent exposure to the unknown antigen. IgA waspresent, in addition to IgM, in two of the threepatients. IgA, just detectable in cord blood, is

produced in neonates after about one week inresponse to antigenic stimuli.We have demonstrated ICD in the small vessels of

three patients with NNEC (Table 1) and, in the onlycase with ICD to come to necropsy, have shown thatthis is a localised deposition (Table 2).Two different immunological mechanisms could

produce this particular pattern of ICD. One is the

761

on May 22, 2021 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.34.7.759 on 1 July 1981. Dow

nloaded from

Gray, Lloyd, Miller, Davidson, Balch, Horne

Auer phenomenon in which circulating antigenantibody complexes are preferentially deposited inareas of tissue damage, and the other is the Arthusreaction. While the Auer phenomenon cannot becompletely disregarded, the failure to demonstratehistological evidence of circulating complexesimplies that this is an Arthus reaction. Experi-mentally the Arthus reaction is produced by inject-ing antigen, usually intradermally, into an alreadysensitised animal. Antigen/antibody complexes formin the vessels and their walls at the site of the injection-that is, where there is antigen excess. The histo-logical sequelae to this are perivasculitis, oedema,acute inflammation, haemorrhage, and necrosis,such as we have observed in our cases of NNEC.

It is known from studies of antimilk antibodiesin the human neonate that antigen passes moreeasily through the intestinal mucosa in neonatesthan in adults.4 There is also good experimentalevidence that, when the neonatal mucosal barrier toantigen is damaged by vascular insufficiency, orileus, massive antigen diffusion occurs.5 In humanneonates it has been suggested that vascular in-sufficiency of the intestine may be caused by avariety of clinical situations including hypoxia,6hyperviscosity,7 plasticisers from PVC catheters,8and stress,9 all conditions associated with anincreased incidence ofNNEC.We suggest that an Arthus type reaction develops

in NNEC due to a localised breakdown in theintegrity of the mucosa, allowing antigen from thebowel lumen to be absorbed in large quantities soprovoking a specific immune response. If antigencontinues to enter the interstitium, then with theformation of circulating antibodies, immune com-

Fig. 4 Fibrindeposition and ruptureof an arteriole in bowelwall. Haematoxylinand eosin x 256

plexes will be deposited in this area of antigen excess.Having demonstrated this immunological pheno-

menon how significant is it ? It could simply be anepiphenomenon, as are many of the antibodiesdetected in human disease. However,9 there ishistological evidence to suggest that an Arthus typereaction may have a role in the evolution of at leastsome, although probably not all, cases of NNEC.

Firstly, ICD in vessels is followed by a peni-vasculitis and, although vasculitis has not beenconsidered previously to be a feature of NNEC, wehave shown it to be present in a proportion of ourcases. Secondly, ICD in vessels leads to fibrin andplatelet aggregates and this certainly is a strikingfeature in many cases of NNEC. Thirdly, in 1959,Goldgraberand Kirsner,-'Owhileattempting to producean experimental animal model for ulcerativecolitis andCrohn's disease,9 induced an Arthus reaction in thecolons of rabbits sensitised to egg albumin. He failedto produce the chronic inflammatory condition ofulcerative colitis but did produce an acute lesioncharacterised by oedema, haemorrhage, and tissuenecrosis of the colon. His detailed histologicaldescriptions and excellent photomicrographs showmany similar features to those present in NNEC.Fourthly, in 1965, Hermann,"1 on the basis of histo-logical observations of NNEC, suggested that themasses of fibrin in the small vessels together withtissue necrosis, bore a striking resemblance to alocalised Shwartzman reaction. There was someexperimental but never any clinical evidence tosupport this interesting theory. However, if theantigen entering the bowel wall was an endotoxinthen an immunological reaction could be producedas well as a Shwartzman reaction, although the

762

on May 22, 2021 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.34.7.759 on 1 July 1981. Dow

nloaded from

Evidence for an immune complex vasculitis in neonatal necrotising enterocolitis

time scale is admittedly different. Indeed it may bethat the intravascular activation of complement incase 4 was due to a Shwartzman reaction.

In conclusion we have demonstrated for the firsttime vascular ICD occurring in the damaged bowelsof three neonates with NNEC. While this may be anepiphenomenon we believe there is both immuno-logical and histopathological evidence to supportour hypothesis that at least in some cases of NNEC,immune complexes have a role in the pathogenesisof the disease possibly by increasing the vasculardamage and tissue ischaemia.

We thank Mr GD Milne for his excellent technicalassistance.

References

Tait RA, Kealy WF. Neonatal necrotising enterocolitis.J Clin Pathol 1979;32:1090-9.

2 Pedersen PV, Halveg AB, Hansen FH, Christiansen ED.Necrotising enterocolitis of the newborn-is it gasgangrene of the bowel? Lancet 1976 ;ii :715-6.

3 Lake AM, Walker WA. Neonatal necrotising enterocolitis;a disease of altered host defence. Clin Gastroenterol 1977;

6 :463-80.4 Rieger CHL, Rothberg RM. Development of the capacity

to produce specific antibody to an ingested food antigenin the premature infant. J Pediatr 1975;87:515-8.

5 Walker WA, Isselbacher KJ. Uptake and transport ofmacromolecules by the intestine: possible role in clinicaldisorders. Gastroenterology 1974 ;67 :531-50.

6 Lloyd JR. The etiology of gastrointestinal perforation inthe newborn. JPediatr Surg 1979;4:77-84.

7 Leake RD, Thampoulos B, Nieberg R. Hyperviscosityassociated with necrotising enterocolitis. Am J Dis Child1975;129:1192-4.

8 Rogers AF, Dunn PM. Intestinal perforation, exchangetransfusion, and PVC. Lancet 1969;ii:1246.

9 Mizrahi A, Barlow 0, Berdon W, Blanc WA, SilvermanWA. Necrotising enterocolitis in premature infants.JPediatr 1965 ;66:697-706.

10 Goldgraber MD, Kirsner JB. The Arthus phenomenonin the colon of rabbits. Arch Pathol 1959;67:556-71.

Hermann RE. Perforation of the colon from necrotisingcolitis in the newborn: report of a survival and a newetiologic concept. Surgery 1965;58:436-41.

Requests for reprints to: Dr Elizabeth Gray, Departmentof Pathology, University of Aberdeen, Foresterhill,Aberdeen AB9 2ZD, Scotland.

763

on May 22, 2021 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.34.7.759 on 1 July 1981. Dow

nloaded from