7 Clin Pathol 1996;49:717-720

Immunocytochemistry of mucosal changes inpatients infected with the intestinal nematodeStrongyloides stercoralis

H B Coutinho, T I Robalinho, V B Coutinho, J R Almeida, J T 0 Filho, G King,D Jenkins, Y Mahida, H F Sewell, D Wakelin

Centro de PesquisasAggeuMagalhaes/FIOCRUZ,Caixa Postal No 7471,CEP: 50670-420,Recife, BrazilH B CoutinhoT I RobalinhoV B Coutinho

Department ofGastroenterology,Federal University ofPernambuco, BrazilJ R AlmeidaJ T 0 Filho

Department ofPathology, UniversityofAberdeenG King

Department ofPathology, UniversityofNottinghamD Jenkins

Department ofMedicine, UniversityofNottinghamY Mahida

Department ofImmunology,University ofNottinghamH F Sewell

Department of LifeScience, University ofNottinghamD Wakelin

Correspondence to:Professor D Wakelin,Departnent of Life Science,University of Nottingham,Nottingham NG7 2RD.

Accepted for publication4 June 1996

AbstractAim-To investigate the immunopatho-logical changes in duodenal tissues in-duced by strongyloidiasis and to relatethese to degrees of clinical severity.Methods-Tissues taken from 21 patientsshowing mild, moderate or severe symp-toms of strongyloidiasis, and from non-infected controls, were sectioned andstained immunocytochemically for IgA,secretory component (SC) and HLA-DR.Immunopathology was assessed bychanges in numbers, intensity and distri-bution of stained cells.Results-Parasitised individuals showedvillous atrophy and crypt hyperplasia.There was notable infiltration of thelamina propria by IgA positive plasmacells and ofthe epithelium by intraepithe-lial lymphocytes. Infection was also asso-ciated with increased expression of SCand decreased expression of HLA-DR inepithelial cells. Changes in all parameterscorrelated with degree of clinical severity.Conclusions-Profound mucosal changesare induced by strongyloidiasis. Some areanalogous to those seen in coeliac disease,but others seem quite unusual. It is likelythat these changes are functionally relatedto the immunopathophysiological conse-quences of infection seen in patients withsevere disease.(7 Clin Pathol 1996;49:717-720)

Keywords: strongyloidiasis, unusual mucosal changes,immunopathology.

The parasitic nematode Strongyloides stercoralisinfects millions of people in tropical andsubtropical regions as well as travellers fromdeveloped countries. Infections are acquiredwhen larvae penetrate the skin and migrate tomature in the small bowel. The interest ofstrongyloidiasis to clinicians lies in the natureof the associated pathology, the potential forlong term debilitating illness' and exacerbationof infection by immune suppression.2 Theadult worms (parthenogenetic females) liveamong the villi, laying eggs which hatch torelease larvae that pass out with stools.Infection can be maintained within the patient,sometimes for decades, by autoinfection,whereby larvae reinvade without leaving thebody. In immunocompromised individuals thisprocess can produce a massive, life threateninghyperinfection involving many organs.2 Inimmunocompetent patients pathology is re-

stricted to the small bowel and varies in sever-ity according to the numbers ofworms present.Almeida3 categorised the symptoms associatedwith strongyloidiasis into mild, moderate andsevere, on the basis of levels of infection andclinical assessment of the degrees of malaise,abdominal pain, vomiting, and diarrhoea.Patients with severe symptoms can showuncontrolled vomiting and a bloody mucusdiarrhoea, and may suffer from dehdration andmalnutrition. These three categories are simi-lar to those described on the basis ofhistopathology.4To our knowledge no reported studies have

been made of the duodenal pathology associ-ated with strongyloidiasis in immunocompe-tent patients using immunocytochemical tech-niques that permit identification and analysisofimmune or inflammatory cells. The purposeof this initial study was to look for correlationsbetween intestinal immunopathology andclinical symptoms of strongyloidiasis. A varietyof monoclonal and polyclonal antibodies wasused to examine immunological aspects of thehistopathological changes seen in Brazilianpatients presenting with the categories ofsymptoms defined above. Our data haverevealed some pathological changes that arenovel for an intestinal infection and others thatare strikingly similar to inflammatory bowelconditions, particularly coeliac disease.

MethodsTISSUESDuodenal biopsy material was available from alarge number of patients with strongyloidiasisregistered at the Gastroenterology OutpatientClinic of the Federal University of Pernam-buco Hospital. For the purposes of this study21 patients were selected, of whom seven(three women and four men; age range 19-67years) had mild symptoms, seven (four womenand three men; age range 21-36 years) hadmoderate symptoms and seven (one womanand six men; age range 29-51 years) had severesymptoms, the latter having been treated at theUniversity Hospital intensive care unit; none ofthe patients had the hyperinfection syndrome.Selection was based on strict criteria. Patientswho had recently received immune suppressivetreatment were excluded from the study, aswere patients with other intestinal infections.Particular care was taken to screen for giardia-sis; none of the 21 individuals studied had thisinfection. Control tissues were taken from twouninfected patients presenting with unrelated

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conditions in Recife and at the Queen's Medi-cal Centre, University of Nottingham.

IMMUNOHISTOLOGYBiopsy specimens from the upper small intes-tine were taken during endoscopy, fixed in 10%formalin, dehydrated in ethanol, clearedin xylene, and embedded in paraffin wax.Sections cut at 5 gm were mountedon slides previously treated with a 2% 3-amino-propyltrimethoxysilane solution in acetone for10 minutes. After mounting, the sections weredried at 37°C for 48 hours.

Dewaxing of sections, endogenous peroxi-dase blockade and immunostaining by theStreptavidin biotin complex technique formonoclonal and polyclonal antibodies wasperformed as described previously.5 Negativecontrols for the monoclonal system comprisedparallel sections treated with Tris bufferedsaline in place of primary antibody. For thepolyclonal system, negative controls wereprovided in similar sections by substitution ofprimary antibody with a dilution of normalrabbit immunoglobulin fraction. The primaryantibodies used were: anti-IgA rabbit polyclo-

.,

Figure 1 Immunocytochemistry of duodenal tissues stainedfor HLA-DR, secretory component (SC) or IgA. APC = antigen presenting cells; IEL =intraepithelial lymphocytes; LP = lamina propria. Panel 1, normal tissue: SC expressed in crypt cells but not in enterocytes on the villi;panel 2, normaltissue: apical HLA-DR in enterocytes (arrowed) and HLA-DR positive APC in the LP (arrowed);panel 3, moderate strongyloidiasis: HLA-DRexpression on enterocytes occurs primarily at the villus tip; panels 4-6, severe strongyloidiasis: panel 4, infiltration of epithelium by IEL (arrowed) and ofLP by IgA positive cells; panel 5, strong expression ofSC in enterocytes and crypts; panel 6, virtual absence of HLA-DR positive enterocytes and APC (onearrowed). Parasites are present in the intervillous space (W) and in the epithelium of the crypts (C).

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Mucosal immunopathology in strongyloidiasis

Table 1 Mucosal immunopathological changes associated with severe strongyloidiasis

Mucosal IgA positive HLA-DR HLA-DRGroup morphology cells in LP Stainingfor SC positive enterocytes positive APC

Uninfected Normal Few Enterocytes negative Many Many (LP of villi)Crypt cells positive

Severe symptoms Villous atrophy Many Enterocytes positive Few Few (LP of villi)Crypt cells positive Many (villus tip

and epithelium)

APC = antigen presenting cell; LP = lamina propria; SC = secretory component.

nal (Behring, Lewes, Sussex, UK; ORCI 05)diluted 1 in 2000 for 30 minutes; anti-HLA-DR mouse monoclonal (Dako, HighWycombe, UK; MO 746) diluted 1 in 40 for 60minutes; anti-secretory component (SC)mouse monoclonal (Dako; AO 187) diluted 1 in1000 for 15 hours at 4°C. All sections werecounterstained with Harris's haematoxylin forfive seconds.

ResultsNORMAL DUODENUMThe material studied represented tissue col-lected from the duodenal bulb close to thepylorus, in which Brunner's glands occupiedthe submucosa and mucosa. In stained sec-tions there were no indications of any his-topathological changes in the mucosa. Few IgApositive plasma cells were observed in thelamina propria of the villi or in the looseconnective tissue surrounding the intestinalcrypts and Brunner's glands. SC was detectedin the Golgi region and the cell membrane ofthe crypt cells but was absent from enterocyteson the villi (fig 1, panel 1). It was also seen inthe cell membrane and weakly in the cytoplasmof the Brunner's glands. HLA-DR was ex-pressed in the form of linear small granules inthe apical pole of the enterocytes lining theintestinal villi (fig 1, panel 2). NumerousHLA-DR positive cells with a morphologyequivalent to macrophage and dendritic cells(antigen presenting cells (APC)) were presentin the lamina propria of the villi.

PATIENTS PRESENTING WITH MILD SYMPTOMSThe villi were shorter and broader than in con-trols and the lamina propria was infiltrated bylymphocytes and plasma cells, IgA positiveplasma cells being concentrated in the laminapropria. Brunner's glands and crypt cells werepositive for SC. The enterocytes lining thedeepest portion of the villi also expressed SC.Enterocytes expressing HLA-DR occupied theupper two thirds of the villi. In the lamina pro-pria, HLA-DR positive APC were concen-trated in the medium and upper regions of thevilli.

PATIENTS PRESENTING WITH MODERATESYMPTOMSAgain, the villi were noticeably shorter andbroader than in the normal controls. Thelamina propria was oedematous and infiltratedby lymphocytes and IgA positive plasma cells.The latter were also present in the looseconnective tissue around the Brunner's glandsand crypt cells. The Golgi regions of the cryptcells were intensely positive for SC, as were theenterocytes lining the villi, with the exception

of those located at the tip. Brunner's gland cellswere also positive for SC. HLA-DR wasexpressed only by the enterocytes located closeto the tip of the villi. APC were reduced in thelamina propria and were concentrated at thetip of the villi (fig 1, panel 3).

PATIENTS PRESENTING WITH SEVERE SYMPTOMSIn some cases the flattening of the duodenalmucosal surface resulted in the complete lossof villi. A most striking feature was the infiltra-tion of the epithelial layer by large numbers ofintraepithelial lymphocytes and by a significantnumber ofHIA-DR positive APC (fig 1, panel4). Subepithelial aggregates of histiocytes wereapparent and there were focal lymphoepitheliallesions infiltrating and surrounding the base ofthe crypts. The oedematous lamina propriawas infiltrated by numerous IgA positiveplasma cells and a few eosinophils. IgA positiveplasma cells were also concentrated among thecrypts and Brunner's gland secretory units.Both the crypt cells and the enterocytes liningthe villi were SC positive, the stain being moreintense than observed in patients with lesssevere symptoms. SC was also identified in thecell membrane and diffusely in the cytoplasmof Brunner's gland cells. Only a few groups ofisolaced enterocytes were HLA-DR positive.HLA-DR positive APC in the lamina propriawere greatly reduced (fig 1, panels 5 and 6).Eggs and larvae of the parasite were commonlypresent in the crypts, the eggs located betweenthe epithelial cells and basal membrane, thelarvae mainly located in the crypt lumen.However, there was little or no inflammatoryreaction immediately around these stages (fig1, panel 5).The major changes associated with severe

strongyloidiasis are summarised in table 1.

DiscussionThe data presented add to existing knowledgeof pathological changes in strongyloidiasis.Some changes-for example, the villous atro-phy, crypt hyperplasia and IgA positive plasmacell infiltration, are similar to those in otherparasitic infections-for example, giardiasis,6as well as in coeliac disease7 and wereproportional to the degree of clinical severityobserved. Other changes are unusual anddeserve particular comment. These include thereciprocal changes in HLA-DR and SCexpression on enterocytes and the epithelialinfiltration by intraepithelial lymphocytes andHLA-DR positive cells, which are unusualimmunpathological responses to gastrointesti-nal infection. Histopathology similar to thatseen in severe strongyloidiasis occurs in maras-mus and kwashiorkor, associated with protein

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malnutrition. Malnutrition in strongyloidiasishas been described8 9 and was recorded in ourpatients with severe symptoms. Infection itselfmay cause reduced food intake, but the patho-logical changes in the intestine must also con-tribute to impaired nutrition.The association of IgA positive plasma cells

with the Brunner's glands and the expressionof SC in these glands was a novel observation,suggesting that these structures may act as aroute by which IgA enters the intestinal lumen.This observation has been confirmed subse-quently in greater detail. ' In this study, the IgApositive lamina propria cell population in-creased proportionately with the severity of thesymptoms so that, in patients with severesymptoms, infiltration of IgA positive plasmacells into the lamina propria of the villiextended to the intestinal crypts and the aciniof the SC positive Brunner's glands. SC isessential for IgA transport into the intestinallumen. In its dimeric form IgA is secreted byplasma cells near the intestinal crypts, and cap-tured by specific SC receptors synthesised bythe crypt cells. After intracellular transport, inthe form of sIgA, antibody is delivered into thecrypt lumen." 12 As well as an increasedexpression in Brunner's glands in patients withstrongyloidiasis, there was a progressive in-crease in SC positive cells towards the apex ofthe villi so that, in those with the severestsymptoms, the villous epithelium was predomi-nantly SC positive. The increased IgA secre-tion elicited by infection would require agreater number of epithelial cells able to trans-port the IgA into the gut lumen. Our findingsare compatible with the noticeably increasedamounts of IgA in duodenal secretions ofpatients with strongyloidiasis."'The altered distribution of HLA-DR posi-

tive enterocytes in strongyloidiasis followed apattern opposite to that of SC positive cells. Inthe normal intestine, the entire epithelial liningof the villi was HILA-DR positive, but ininfected patients, as symptoms became moresevere, there was a progressive reduction. Insome patients with moderate symptoms onlythe upper third of the villi had HLA-DR posi-tive cells and these disappeared completely inpatients with the most severe symptoms. How-ever, the reduced HLA-DR staining did notalways parallel the clinical severity of disease.These unusual, reciprocal changes in expres-

sion of SC and HLA-DR suggest that, withincreasingly severe pathology, there is a gradualsubstitution of enterocytes capable of antigenpresentation by cells that can deliver increasingamounts of sIgA into the intestinal lumen. Thissubstitution must reflect influences uponnormal enterocyte differentiation. Enterocytesare derived by mitosis of crypt cells and movetowards the apex of the villi to detach at theapical extrusion zone as they differentiate."' In

strongyloidiasis, it seems that cell turnover isnot accompanied by differentiation, expressionof SC along the length of the villi remainingsimilar to that in the original crypt cells andHLA-DR expression being much restricted. Ifthis interpretation is correct it can be assumedthat the absorptive capacity of enterocytes mayalso be reduced, which may contribute to themalabsorption associated with this infection.'5The changes in cellular differentiation re-

flected in expression of SC and HLA-DR arepresumably mediated by factors (probablycytokines) released from a variety of cells,including epithelial cells, in response to infec-tion. Data from experimental models ofintestinal helminthiasis would suggest that Tlymphocytes corresponding to the T helpersubset should play an important role in theimmunopathological responses to Strongyloidesinfection, and that interactions between Thland Th2 cells may determine the balancebetween resistance and susceptibility.'6

This work was supported by research grants from the BritishCouncil/FACEPE Agreement, from the Conselho Nacional dePesquisas, Bank of Brazil Foundation, and from the EuropeanUnion.

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4 De Paola D, Braga-Dias L da Silva JR. Enteritis due toStrongyloides stercoralis. Am J Dig Dis 1962;7: 1086-98.

5 Coutinho HB, King G, Sewell HF, Tighe P, Coutinho VB,Robalinho TI, Carvalho AB. Immunocytochemical studyof Peyer's patches follicular-assocaited epithelium in themarsupial Didelphis albiventris. Dev Comp Immunol 1993;17:537-48.

6 Nash TE. Giardia lamblia and giardiasis. In: Warren KS, ed.Immunology and molecular biology ofparasitic infections. 3rdedn. Oxford: Blackwell Scientific Publications, 1993:157-69.

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10 Coutinho HB, Robalinho TI, Coutinho, VB, Amorin AM,Almeida JR, Filho JTO, et al. Immunocytochemicaldemonstration that human duodenal Brunner's glandsmay participate in intestinal defence. J Anat 1996;189:193-7.

11 Scott H, Brandtzaeg P, Solheim BG, Thorsby E. Relationbetween HLA-DR-like antigens and secretory component(SC) in jejunal epitheium of patients with coeliac diseaseor dermatitis herpetiformis. Clin Exp Immunol 1981;44:233-8.

12 Brandtzaeg P, Prydz H. Direct evidence for an integratedfunction of J chain and secretory component in epithelialtransport of immunoglobulins. Nature 1984;311:71-3.

13 Bezjak B. Immunoglobulin studies in strongyloidiasis withspecial reference to raised serum IgE levels. AmJ Trop MedHyg 1975;24:945-8.

14 Leblond P, Stevens CE The constant renewal of the intesti-nal epithelium in the albino rat. Anat Rec 1948;100:357-78.

15 Milner PF, Irvine RA, Barton CJ, Bras G, Richards R.Intestinal malabsorption in Strongyoides stercoralis infes-tation. Gut 1965;6:574-81.

16 Locksley RM. Th2 cells: help for helminths. J Exp Med1994;179:1405-7.

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