Gut, 1990, 31, 134-138

Bacterial adhesion and disease activity inHelicobacter associated chronic gastritis

S J Hessey, J Spencer, J I Wyatt, G Sobala, B J Rathbone, A T R Axon, M F Dixon

AbstractUltrastructural examination of biopsiesshowing Helicobacter pylon associatedchronic gastritis reveals close attachmentbetween gastric surface epithelial celis and theorganism. The finding of 'adhesion pedestals',which represents a cellular response to thepresence of the organism, is analogous to theresponse ofintestinal cells to enteropathogenicE coli. Thus the development of bacterialattachment sites in H pylori associatedgastritis might be an indication of patho-genicity. We have therefore explored therelationship between the proportion oforganisms forming attachment sites and histo-logical indices of disease 'activity'. Antralbiopsies from 40 patients with Hpyloni positivegastritis were examined histologically andultrastructurally, and the percentage ofattached organisms compared with subjectiveassessments of epithelial degeneration, mucindepletion, polymorphonuclear and chronicinflammatory cell infiltration. We found a

significant increase in the proportion ofattached bacteria in cases showing histologicalepithelial degeneration, and a significantdecrease in cases showing intraepithelialpolymorph infiltration. The direct relationshipbetween bacterial attachment and cellulardegeneration lends further support to a

pathogenic effect. Reduced attachment in theface of polymorph infiltration might indirectlyreflect aspects of the immune response -

namely, blocking of adhesion by IgA, withcomplement activation and generation ofleucotactic factors.

Departments ofPathology, University ofLeeds and St James'sUniversity Hospital, andthe GastroenterologyUnit, General Infirmaryat Leeds, LeedsS J HesseyJ SpencerJ I WyattG SobalaB J RathboneAT R AxonM F DixonAddress for correspondence:Dr M F Dixon, Department ofPathology, University ofLeeds, Leeds LS2 9JTAccepted for publication10 April 1989

Since the identification and isolation of spiralorganisms from the human stomach by Warrenand Marshall' in 1983, there have been numerousreports linking such organisms, now termedHelicobacter pylori, with active chronicgastritis.>' Despite immunological,5 ultra-structural,9 ingestion'0 " and treatment'2studies, the pathogenicity of H pylon remainscontroversial.

Previous ultrastructural studies have madereference to the close association betweenH pylori and the gastric epithelium in chronicgastritis," and these epithelial attachment siteshave been compared with those formed byenteropathogenic E coli.6 The ability ofE coli toadhere to epithelial cells through a specialisedattachment site, the 'adhesion pedestal' isimportant in the initiation of intestinal disease,'3and it could be that H pylori gives rise to chronicgastritis in a similar manner. If this is so, then the

*The new nomenclature for Campylobacter pylori is Helicobacterpylon'. (Leading article Lancet 1989, ii: 1019).

development of specialised attachment sitesrepresents an important aspect of pathogenicityand their presence and frequency should berelated to the degree of gastritis.The aim of this study was to investigate the

morphological features of H pylori adhesion atthe ultrastructural level and to explore therelationship between bacterial attachment andthe severity of gastric disease.

PATIENTS

Three adjacent antral biopsies were collectedfrom each of 83 consenting patients complainingof dyspepsia, using an Olympus GIF QlOendoscope. Two biopsies were fixed in formalin,paraffin embedded, and 5[tm sections stainedwith haematoxylin-eosin (HE), Alcian Blue/periodic acid-Schiff (AB/PAS) and the modifiedGiemsa technique.'4 The latter was used todetermine H pyloni positivity. The severity ofchronic gastritis in each case was assessed by onepathologist (MFD) using the H and E and AB/PAS stained sections. In addition to assessinginfiltration of the lamina propria (interstitium)and epithelium by polymorphs, which is theconventional criterion for defining 'active'chronic gastritis, attention was also directedtowards mononuclear cell infiltration of themucosa, mucin depletion and epithelialdegeneration. Each of these five features wasgraded 0-3 (0=absence, 1=mild, etc) to reflectcurrent disease 'activity' in a broader sense.Glandular atrophy and intestinal metaplasiawere not assessed as these are considered to beindicators of duration and progress of thegastritis rather than activity.The remaining biopsy from each patient was

fixed for three hours in 3% glutaraldehyde inphosphate buffered saline (PBS), pH 7 4, andthen stored in PBS until the H pylon status wasknown. InH pylori positive cases, the tissue wasorientated and routinely processed fortransmission electron microscopy. 0-1 Iimsections were stained with uranyl acetate andlead citrate and viewed independently (by SJH)using a Phillips 420T or a Joel 1200 Ex ElectronMicroscope.

Ultrastructurally H pylon were seen eitherrandomly scattered in the mucus layer or morefrequently in close proximity to the plasmamembrane of the surface or foveolar epithelialcells. H pylon adhesion sites were defined as 'aclose attachment between bacteria and epithelialcells such that no space is visible between them'.Adherent organisms were quantified in each caseas a percentage of the total organisms present.The proportion of adherent organisms wasrelated to the grades of severity of each of the five

134

Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis

histological parameters as assessed in theadjacent formalin fixed biopsies. Statisticalanalysis was performed using the Spearman rankcorrelation and Mann-Whitney U test.A limited number of additional antral biopsies

were stained with Ruthenium-red to showsurface glycoproteins using a pre-embeddingstaining technique based on those of Luft'5 andof Fowler.'6 Three such biopsies came fromH pylon positive patients and were subjected toultrastructural examination.

ResultsOf the 83 dyspeptic patients, 45 had H pylonassociated gastritis of which 40 sets of biopsiesproved to be suitable for concurrent histologicaland ultrastructural examination.

ULTRASTRUCTUREHpylori were seen to be darkly staining flagellatedorganisms with a double unit membrane. Theouter wall was most densely stained and bore anextraneous layer of irregular, radially arrangedfine fibrils. These were often condensed betweenthe organism and epithelial cell producing anintervening 'fuzzy layer'. It was thought thatthese fibrillar structures might be pili but thiswas not confirmed by negative staining ofcultured organisms with methylamine tungstateacid. Ruthenium red stained the epithelial cellbrush border and the extraneous coat of thebacteria forming a granular deposit (Fig 1), thissuggests the involvement of a negatively chargedacidic polysaccharide in connecting the bacterialglycocalyx with that of the epithelial cell.Where H pylon were closely associated with

epithelial cells, there was loss of microvilli withflattening of the plasma membrane which oftenhad a degenerate appearance with outpouchingsand blebs. The epithelium also showed depletionofmucin granules and some cytoplasmic loss, the

Figure 1: Transmissionelectron micrograph ofruthenium red stained Hpylori adherent to theplasmna membrane ofasurface epithelial cell. Thegranular deposits representstaining ofglycosubstancescovering the bacterium andassociated with the cellglycocalyx.

Figure 2: Transmission electron micrograph ofantral amucosashowing organisms lodged in a celljunction. The adjacentcytoplasm ofthe epithelial cells shows vesiculation andaccumulation oflysozymes.

cells becoming more cuboidal with an increasednucleo-cytoplasmic ratio. Where organisms hadmigrated between cells or were concregated inniches at pit bases, intracellular oedema,vacuolation and lysosome accumulation wereobserved (Fig 2). The latter changes, however,were not seen in surface cells exhibiting bacterialattachment on their luminal aspects alone.Between 23 and 629 (mean=125-5, SEM=

24 8) H pylon were found per biopsy. The widevariations in the number of organisms present inthe ultrathin sections meant that comparisonsbased on absolute numbers of adhesion siteswould be meaningless, accordingly adhesion hasbeen expressed as a percentage of the total countof H pylon found in each case. Between 0 and41-5% (median 21-5%) of the total bacteriaformed adhesion sites. On the basis of themorphological appearances, three categories ofadhesion site were observed: the adhesionpedestal where the epithelial cell forms a plateaulike extrusion and cups the organism to varyingdegrees, as described for E coli adhesion byInman et al'7 (Fig 3a); indentation sites where theorganisms dip into the epithelial surface (Fig3b); and abutting adhesion where apart from lossof microvilli, the contour of the epithelial cell isunchanged at the point of contact with theorganism (Fig 3c). The majority (67%) ofadherent H pylori were abutting; indentationsand pedestals were less frequently seen (17% and16% respectively).

ULTRASTRUCTURE v DISEASE ACTIVITYIn comparing the proportion of adherentorganisms with histological disease activity, thethree morphological categories of adhesion sitehave been grouped together because thesignificance of the different forms of adhesion is

135

It

Hessey, Spencer, Wyatt, Sobala, Rathbone, Axon, Dixon

Figure 3a: Adhesion site with raised plasma membraneforming a plateau like extrusion - an adhesion pedestal; (b) H pylori occupying depressions in the plasmamembrane constituting 'indentation' sites; (c) Organisms abutting onto the plasma membrane which is devoid ofmicrovilli.

as yet unknown and it seems likely that they areall steps in a single adhesion process. Theproportion of adherent organisms for varyingdegrees of severity of the five histologicalparameters are shown in the Table.When the individual results for percentage

adhesion were compared with the correspondinggrades for the histological features using theSpearman rank correlation test, no significantrelationship emerged. In a two categorysituation, however, significantly increasedpercentages of adhesion were found in casesshowing epithelial degeneration (grades 1, 2 and3) when compared with those cases withoutdegeneration (p=0 013; Mann-Whitney U test).Epithelial degeneration (Fig 4) was alwayspresent when more than 20% of organisms werefound to be adherent (Fig 5). In contrast, therewas a significant decrease in the percentages ofadherent organisms when intraepithelialpolymorphs were seen (grades 1, 2 and 3)compared with the percentages found in theabsence of polymorphs (p=0-041).

Although increased adhesion was found withmore severe mucin depletion, this did not reachstatistical significance. Only one case showedabsence of interstitial polymorphs but there wasno difference in adhesion between mild (grade 1)and more severe grades (2 and 3) of interstitialinfiltration. All cases showed some increase inchronic inflammatory cells over those found in

TABLE Mean percentage ofadhesive H pylorifound with increasing severity (grade) offivehistological features

Epith Mucin Interst Intra-ep ChronicGrade degeneration depletion polymorphs polymorphs inflamm

0 10-2% [8]' 16-6% [6] 16-4% [1] 24-4% [12] [0]Absentornormal (2.70)2 (5-51) (H) (3 09) (-)

1 22-0% [14] 16-6% [161 19-3% [29] 16-2% [20] 20-7% [161Mild (4-17) (3-48) (2 39) (3 02) (3 20)

2/3 21-2% [18] 22-6(18] 19-6% [10] 19-4% [8] 18-3% [24]Moderate

/severe (2 47) (2 62) (4-14) (4 24) (2-61)

Figures in square parenthesis represent the number of patients in each category' (total =40), and thestandard error of the mean.2

normal mucosa; the percentages of adhesionfound in cases with moderate and severeinfiltration were not significantly different frommild cases.

DiscussionWhen comparing the epithelial attachment ofH pylori to that of E coli several features incommon were noted, for example the loss ofmicrovilli and membrane flattening, the loss ofcytoplasm from involved cells and the raggedirregular luminal borders of such cells. The lossof the terminal web and depletion of glycocalyxwhich is typical of E coli attachment to smallintestinal cells'8 was, however, not seen with Hpylori, but these structures are much lessdeveloped in gastric epithelial cells. Theconsistent finding of a 'fuzzy' layer, whichproved to be Ruthenium red positive, indicates arole for glycosubstances in adhesion. Recentwork has shown that oligosaccharide residues are

i;,~~~~~~~~~~WFigure 4: Antral biopsy on histology showing severe epithelialdegeneration with irregular 'budding' ofcells, heaping up ofnuclei, and mucin depletion. These changes werefoundadjacent to a focal erosion.

136

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Figure 6: H pylori adherentto the 'roof ofa largeintacellular vacuole lined byplasma membrane with wellpreserved microvilli andcontaining dense orfinelygranular material.

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involved in ligand-bacterial interactions forE coli," and after the completion of our studyEvans et a12' have shown that H pylori possesses a

fibrillar N-acetylneuraminyllactose-bindinghaemagglutinin which the authors suggest is theadhesin responsible for epithelial attachment.Interestingly their results with immunogoldstaining show that the fibrillar antigen covers theentire bacterial surface in the form of a capsulelike network, a picture analagous to that we

obtained with Ruthenium red staining.Previous ultrastructural studies on H pylori

associated gastritis have noted intracellularchanges'2' similar to those in E coli inducedenteritis,2022 including oedema, vacuolation,swelling of endoplasmic reticulum and lysosomeaccumulation. These more severe intracellulardegenerative changes were not conspicuous inthis study except where organisms had migratedbetween cells or were congregated in niches at

the bases of the gastric pits. It seems likely thattoxins released by the bacteria are responsible forthe cell damage and exert a maximal effect whenconcentrated in areas such as the intercellularspaces. That H pylorn produces cytotoxins hasnow been established'3 and Leunk et al'4 haveshown cytoplasmic vacuolation in a culturedintestinal cell line exposed to broth culturefiltrates of the organism.A feature not described in E coli adhesion was

the finding of intracellular cystic spaces whichwere partially filled with granular and amorphousmaterial and lined by microvilli bearing aglycocalyx (Fig 6). These were observed in aminority of epithelial cells in three of the 40Hpylori positive cases. They have been previouslyobserved in Helicobacter-bearing epithelium byFiocca et al,2' and could be larger versions ofthe cytotoxin-induced vacuoles described byLeunk et al.24 Similar intracellular 'cysts' areseen in some gastric carcinoma cells. Theaetiology and significance of such cysts has notbeen elucidated, but presumably they representatypical endocytic vacuoles.Although 'activity' in chronic gastritis is

conventionally defined by the presence andnumber of neutrophil polymorphs, for thepurposes of this study we have broadened theterm to include other possible responses toinfection - that is, mononuclear cell infiltration,mucin depletion and epithelial degeneration.Degeneration was recognised histologically byragged cytoplasmic margins, a high nucleo-cytoplasmic ratio, and heaping up of cells to formsyncytia-like masses (Fig 4). While these cellsinvariably show loss of microvilli, distortion ofthe plasma membrane and loss ofmucin granuleson electron-microscopy, it is surprising thathistological 'degeneration' was not generallyparalleled by intracellular degenerative changeson electron-microscopy. Despite this, we founda significantly increased proportion of H pyloriexhibiting adhesion to the gastric epithelium inthe presence ofhistological degeneration assessedindependently by a second observer. Inparticular, epithelial degeneration was alwayspresent when more than 20% oforganisms had formed adhesion sites. There aretwo possible explanations for this, either there ispreferential attachment ofH pylon to previouslydamaged cells, or H pylori adhesion plays a rolein the production of cell damage. The firstexplanation seems unlikely; H pylon' do notcolonise gastric mucosa injured by refluxgastritis26 or lymphocytic gastritisf.2 On the otherhand adhesion sites could produce damage, forexample, by providing a site for direct toxindelivery from H pylori to the epithelial cell,24 orby allowing localised luminal accumulation ofharmful substances produced by the 'anchored'organisms. Whether H pylori adhere before orafter epithelial degeneration occurs could only bediscovered by sequential studies in a suitablemodel system where the development ofcolonisation could be followed.

IMucin depletion was readily recognised in the

AB/PAS stained sections and was a consistenti feature of active gastritis. Loss ofmucin granulesI was confirmed on electron microscopy. While

more severe grades of mucin depletion were

137

138 Hessev, Spencer, Wyatt, Sobala, Rathbone, Axon, Dixon

associated with higher proportions of adherentorganisms, the differences failed to achievestatistical significance.With regard to inflammatory cells, there were

no significant changes in the percentages ofadhesion with increasing infiltration by chronicinflammatory cells and polymorphs in the laminapropria. For intraepithelial polymorphs,however, there was a significant decrease in thepercentages of adherent organisms in casesshowing epithelial infiltration. This apparentlyparadoxical finding might be a consequence ofthe local immune response. Coating oforganismsby IgA would inhibit adhesion and at the sametime opsonisation by complement activatedthrough the alternate pathway28 would bothpromote phagocytosis and generate complementproducts chemotactic for polymorphs. In thisway, one could arrive at the inverse relationshipbetween bacterial attachment and intraepithelialpolymorph infiltration which we observed.To conclude, we believe that the demon-

stration of a direct relationship between bacterialattachment and epithelial degeneration lendsfurther support to the pathogenic role ofH pylonin the development of chronic gastritis.

1 Warren JR, Marshall BJ. Unidentified curved bacilli on gastricepithelium in active chronic gastritis. Lancet 1983; i: 1273-5.

2 Drumm B, Sherman P, Cutz E, Karmali M. Association ofcampylobacter pylori on the gastric mucosa with antralgastritis in children. New EnglJ Med 1987; 316: 1557-61.

3 Marshall BJ, McGechie DB, Rogers PA, Glancy RJ. Pyloriccampylobacter infection and gastroduodenal disease. Med]Aust 1985; 142: 439-44.

4 Steer HW. The gastroduodenal epithelium in pepticulceration. ] Pathol 1985; 146; 355-62.

5 Wyatt JI, Rathbone BJ, Heatley RV. Local immune responseto gastric campylobacter in non-ulcer dyspepsia. Scand ]Gastroenterol 1988; 23 (Suppl 142): 44-9.

6 Goodwin CS, Armstrong JA, Marshall BJ. Campylobacterpyloridis, gastritis, and peptic ulceration. 7 Clin Pathol1986; 39: 353-65.

7 Chen XG, Correa P, Offerhaus J, et al. Ultrastructure of thegastric mucosa harboring campylobacter-like organisms.Am] Clin Pathol 1986; 86: 575-82.

8 Tricottet V, Bruneval P, Vire 0, Camilleri JP.Campylobacter-like organisms and surface epithelialabnormalities in active, chronic gastritis in humans: anultrastructural study. Ultrastruc Pathol 1986; 10: 113-22.

9 Bode G, Malfertheiner P, Ditshuneit H. Pathogeneticimplications of ultrastructural findings in campylobacter

pylori related gastroduodenal disease. Scand ] Gastroenterol1988; 23 (Suppl 142): 25-39.

10 Marshall BJ, Armstrong JA, McGechie DB, Glancy RJ.Attempt to fulfil Koch's postulates for pvloric campy-lobacter. Med] Aust 1985; 42: 436-9.

11 Morris A, Nicolson G. Ingestion of campylobacter pyloridiscauses gastritis and raised fasting gastric pH. Am 7Gastroenterol 1987; 82: 192-9.

12 Rauws EAJ, Langenberg W, Houthoff Hj, Zanen HC,Tytgat GNJ. Campylobacter pyloridis-associated chronicactive antral gastritis: a prospective study of its prevalenceand the effects of antibacterial and antiulcer treatment.Gastroenterologv 1988; 94: 33-40.

13 Clausen CR, Christie DL. Chronic diarrhoea in infants causedby adherent enteropathogenic escherichia coli. .7 Paediatr1982; 100: 358-61.

14 Gray SF, Wyatt JI, Rathbone Bj. Simplified techniques foridentifying campvlobacter pyloridis letter. 7 Clin Pathol1986; 39: 1279-80.

15 Luft JH. Ruthenium Red and Violet I; chemistry, purification,methods of use for EM and mechanism of action. Anat Rec1971; 171: 347-68.

16 Fowler BA. Ruthenium red staining of rat glomerulus,perfusion of ruthenium red into normal and nephrotic ratkidney. Histochemi'stw, 1970; 22: 135-62.

17 Inman LR, Cantev JR. Specific adherence of Escherichia coli(strain RDEC-l) to membranous (M) cells of the Pever'spatch in Eschericia coli diarrhoea in the rabbit.]_ C/in Invest1983; 71: 1-8.

18 Rothbaum R, McAdams AJ, Giannella R, Partin JC. Aclinicopathologic study of enterocyte adherent Escherichiacoli: a cause ofprotracted diarrhoea in infants. Gastroentrologv1982; 83: 441-54.

19 Rosenstein IJ, Stoll MS, Mizuochi T, Childs RA, HounsellEF, Feizi T. New tvpe of adhesive specificitv revealed byoligosaccharide probes in Escherichia coli from patients withurinary tract infection. Lancet 1988; ii: 1327-30.

20 Evans DG, Evans DJ, Moulds JJ, Graham DY. N-acetylneuraminyllactose-binding fibrillar hemagglutinin ofCampylobacter pylorn: a putative colonization factor antigen.Infect Immun 1988; 56: 2896-906.

21 Fiocca R, Villari L, Turpini F, Turpini R, Sokia E. Highincidence of campylobacter-like organisms in endoscopicbioposies from patients with gastritis, with or without pepticulcer. Digestionl987; 38: 231-44.

22 Cantey JR, Blake RK. Diarrhoea due to Esherichia coli in therabbit: a novel mechanism. ] Infect Dis 1977; 135: 454-62.

23 Figura N, Guglielmetti P, Rossolini A, et al. Cytotoxinproduction by Campylobacter pylon' strains isolated frompatients with peptic ulcers and from patients with chronicgastritis only. ] Clin Microbiol 1989; 27: 225-6.

24 Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR.Cytotoxic activity in broth-culture filtrates of campy-lobacter pylori.] Med Microbiol 1988; 26: 93-9.

25 Nevalainen TJ, Jarvi OH. Intracellular cysts in gastriccarcinoma. Acta Pathol Microbiol Scand [A] 1976; 84: 517-22.

26 O'Connor HJ, Dixon MF, Wyatt JI, et al. Effect of duodenalulcer surgery and enterogastric reflux on campylobacterpyloridis. Lancet 1986; ii: 1 178-8 1.

27 Dixon MF, Wyatt JI, Burke DA, Rathbone BJ.Lymphocytic gastritis - relationship to campylobacter pylorninfection. ] Pathol 1988; 154: 125-32.

28 Dao SS, Karin QN, Easmon CSF. Opsonophagocytosis ofCampylobacter pylori. ] Med Microbiol 1988; 27: 125-30.