Gut, 1986, 27, 405-410

Human Peyer's patches: an immunohistochemicalstudyJO SPENCER, TERESA FINN, AND P G ISAACSON

From the Department of Histopathology, University College London School of Medicine, London

SUMMARY We have used immunoperoxidase techniques to characterise the Peyer's patches inhuman terminal ileum. The mantle zones of the B cell follicles in human Peyer's patches weresurrounded by B cells which did not express surface IgD but which mostly expressed surfaceimmunoglobulin of the IgM and/or IgAl isotype. Few cells expressing surface IgG or IgA2 weredetected. Cells with cytoplasmic immunoglobulin of all isotypes except IgD were present in thedome regions of the Peyer's patches as well as in the lamina propria. There was little evidence oftraffic of immunoglobulin synthesising cells across the high endothelial venules. T cells were seento surround the lymphoid follicles. They were most concentrated on the serosal aspect around thehigh endothelial venules. Cells with macrophage-like morphology were present in both thelamina propria and the dome region of the follicles; those in the lamina propria containinglysozyme and those in the dome region S100 protein. The results are discussed in relation to thegeneration and dissemination of antibody producing cells in human gut.

A wealth of information is available which definesthe processes involved in the activation, maturationand migration of B cells resulting from immuno-logical stimulation of the guts of laboratory animals.Some of the B cells activated in the Peyer's patchesmigrate to the mesenteric lymph nodes where theymature into antibody producing cells.' These cellsthen enter the blood through the thoracic duct andlocalise in the lamina propria of the gut where theybecome plasma cells secreting antibody predomi-nantly of the IgA isotype.23 Sminia and Plesch4have supported these observations in rats usingimmunohistochemistry.

It is tempting to apply these theories on thedevelopment of antibody producing cells in the gutdirectly to human tissue, but there are differencesbetween the gut associated lymphoid tissue of manand laboratory animals even on a fundamental level.First, the Peyer's patches themselves are distributeddifferently. Peyer's patches in untreated humantissue are macroscopically invisible and they aredistributed in small clusters throughout the gastro-intestinal tract, being most concentrated in theterminal ileum.5 The Peyer's patches of mice andAddress for correspondence: Dr J M Spencer, Department of Histopathology,University College London School of Medicine, University Street, London,WCIE 6JJ.Received for publication 21 June 1985.

rats, however, are found in approximately 10 clustersalong the length of the ileum and in sheep they aredensely concentrated in the terminal ileum.7 Inthese species the Peyer's patches are clearly visiblemacroscopically on the serosal surface of the bowel.Secondly the synthesis of IgA in human tissue is notrestricted to the mucosae to the extent seen in rats.Unlike rats which synthesise mostly dimeric IgA,man synthesises both monomeric and dimeric formsof two subclasses of IgA, the monomeric form notapparently being involved in the protection ofmucosal surfaces because of its inability to combinewith secretory component. Because of these dif-ferences between the gut associated lymphoid tissueof laboratory animals and man we have made animmunohistochemical study of the Peyer's patchesin human ileum the priority being to look forevidence of the maturation sites of antibody syn-thesising cells generated in the Peyer's patches andthe migratory route of these B-cells to the laminapropria.

Methods

TISSUE COLLECTION AND PROCESSINGTerminal ileum was collected from seven righthemicolectomy specimens resected at laparotomyfor adenoma or carcinoma of the colon. The patients

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Spentcer, Finnt1, ands1 Isaacson

were four women and three men ranging in age from17-91 years. Specimens of macroscopically normalileum were fixed in 10% formalin containing 2%acetic acid8 and routinely processed and embeddedfor paraffin sections. Tissue was also snap frozen inliquid nitrogen for frozen sections. Sections ofterminal ileum were stained using haematoxylin andeosin. Blocks that contained Peyer's patch follicleswere selected for immunohistochemistry.

IMMUNOHISTOCHEMISTRYThe sources and the specificities of the antibodiesused throughout this study are shown in the Table.

Paraffin sections for immunostaining were de-waxed, rehydrated, and subjected to controlledtrypsin digestion for optimal staining.'" Frozensections were air dried for half an hour and fixed infresh acetone for half an hour immediately beforestaining.With the polyclonal primary antibodies a peroxi-

dase anti-peroxidase technique was used, and themonoclonal primary antibodies were used with aperoxidase conjugated secondary antibody. Peroxi-dase activity was shown using the 3,3-diamino-benzidine reagent. 12 The precise details of thetechniques have been published elsewhere.'3

Results

MORPHOLOGICAL OBSERVATIONS

The most prominent feature of Peyer's patchfollicles was the follicle centre which contained large

centroblasts with euchromatic nuclei and mediumsized centrocytes with irregularly shaped hetero-chromatic nuclei. The follicle centre was surroundedby a mantle with indistinct borders composed ofsmall lymphocytes which merged into a mixedpopulation of cells in the dome region of the follicle.In addition to some small lymphocytes, the domeregion contained plasma cells, macrophages

Table Antibodies used in this study

Monoclonalantibodies Source Specificity

Coultcr CloncCoultcr CloncScward LaboratoricsScward Laboratorics

Bcckton DickinsonScra Lab

t

antiAseraDakopattsDakopattsDakopatts

b-chains-chainsal -chainsu2-chainsT ccilsSupprcssor T ccilsHclper ccilsLangcrhans ccils,intcrdigitating ccilsand cortical thymocytesB cclls othcr thanfolliclc centrc ccilsIILA-D rcgion antigen

y-chainLysozymcCow 511(f brain protcin

b AN J'V VJ2AFig. 1 Paraffin section of Peyer's patch Jfrom terminalileum. (a) The follicle centre is surrounded by a mantle oJsmall lymphocytes which has indistinct borders. The mamitlezone merges with a population of cells of intermnediate sizeand irregular nuclear outlines. These cells are illustrated in(b) where they extendfrom the dome region of the follicleinto the overlying epithelium. Haematoxylin anid eosin,(a) x 125, (b) x280, orig mag.

IgDIgMIgA IIgA2T1T4Lcu3aNal/34

KB6I (9)

IB5 (1(0)PotlytlonatllIgGLysozymcSII)()

Antiscra wcrc kindly provided by *Drs P Bevcrlcy and.tDr T Adams of the Impcrial Canccr Rcscirch Fund. London,tDr K Pulford of the John Raidcliffc Hospital. Oxford.

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Human Peyer's patches: an immunohistochemical study

c

Fig. 2 Serialfrozen sections ofa Peyer's patch immunostained using (a) the monoclonal antibody KB6J which recognisesan antigen on B cells other than follicle centre B cells, (b) anti-IgD, (c) anti-IgAI, (d) anti-IgM. B cells recognised by KB61are present in excess of the mantle cells which express surface IgD. Most of the B cells which surround the mantle zoneappear to express slgM andlor sIgA l. Immunoperoxidase (x 100, orig. mag).

and cells with irregularly shaped heterochromaticnuclei similar to the centrocytes in the follicle centre(Fig. 1). These cells were also present infiltratingbetween the epithelial cells of the dome, but not theepithelium of the adjacent crypts. They were alsocontinuous with the zone of cells on the serosalaspect of the follicle which contained the highendothelial venules.

IMMUNOHISTOCHEMICAL OBSERVATIONSFrozen sectionsCells with surface immunoglobulin (sIg) were stu-died in cryostat sections of Peyer's patches. Clearrings of immunostaining were seen around theoutside of cells with slg rather than the thicker morediffuse rings of staining which surrounded the nucleiof cells with cytoplasmic immunoglobulin (clg).The mantle zone was composed of cells with

sIgD and sIgM but this was diminuitive when com-pared with the total number of B cells presentin the Peyer's patches outside the follicle centre(Fig. 2 a,b). Cells with sIgM and sIgAl surroundedthe mantle zone extending both into the dome

region and between the dome epithelial cells andinto the T cell zone (Fig. 2 b,c,d). Cells with sIgA2and sIgG could be seen occasionally in the Peyer'spatches.The follicle centres contained network staining of

immunoglobulin of all isotypes except IgD. Thisimmunoglobulin bound to the follicular dendriticreticulum cells masked any positivity which mayhave been present on the follicle centre cellsthemselves.

Helper and suppressor T-cells were present in theT-cell zones extending around the follicles and intothe mixed cell zones, the ratio of helpers tosuppressors being approximately 7:1 but this variedbetween individuals. T helper cells were observed inthe follicle centres.The antibody Nal/34 recognised only occasional

cells between the follicles in Peyer's patches. Nocells were stained in the dome regions.

Paraffin sectionsMost of the cells with clg were observed in the domeregions of the Peyer's patches and the adjacent

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Spencer, Finn, and Isaacson

lamina propria, although some cells with cIg couldoccasionally be seen within the follicle centres. Itwas not possible to detect cells with slg in paraffinsections.

Cells with cIgAl, cIgA2, cIgM and cIgG (in orderof decreasing frequency) were all present in thedome regions of the follicles. Some of these cellswere clearly plasma cells. No cells with clg werepresent in the epithelium (Fig. 3).Very few cells with cytoplasmic immunoglobulin

were seen in the T-cell zones in association with thehigh endothelial venules and there was no evidenceof a gradient of cells with cytoplasmic immunoglo-bulin from this area to the lamina propria.The lamina propria contained cells synthesising

immunoglobulin of all isotypes, cells synthesisingIgAl and IgA2 being the most abundant. The

Fig. 3 Paraffin sections of Peyer's patches immunostainedusing anti-IgAl . Cells with cytoplasmic IgAl are present inthe dome ofthe Peyer's patch and between the crypts but notin the dome epithelium. Immunoperoxidase (x200, orig.mag).

frequency of cells with cIgG appeared to decreasewith increasing distance from the follicle.There were many cells in the Peyer's patches

expressing HLA-DR antigens which extended intothe dome epithelium. Some of these cells resembledthe centrocytes in the follicle centres and othersresembled macrophages, having long cytoplasmicprocesses. The antilysozyme antibody recognisedcells with cytoplasmic processes in the laminapropria but it failed to stain cells in the dome regionsof the Peyer's patch follicles with equal intensity(Fig. 4). There appeared to be a qualitative differ-ence in staining of cells with similar morphologybetween these two areas. The antibody to S100protein also stained cells with cytoplasmic processesbut in contrast with the lysozyme containing cellsthese were concentrated in the dome regions of thePeyer's patch follicles, decreasing in frequency withincreasing distance from the follicles (Fig. 4).

Discussion

Peyer's patch follicles as described in this reportclosely resemble the lymphoid follicles in humanappendix with the exception of the B cells in thedome regions of the follicles. The population of Bcells which lack sIgD which surround the follicles inthe Peyer's patches is much less well represented inthe appendix. In the appendix these cells can only beclearly identified when they show their characteristictendency to infiltrate the dome epithelium. Unlikefollicles in the appendix the dome regions of thePeyer's patch follicles contain cells with cytoplas-mic Ig.

Antigen presentation is thought to occur in thedome regions of Peyer's patches and to involve cellswith dendritic processes.14 15 Cells recognised byNal/34 and lysozyme were rarely seen in the dome.A population of cells with cytoplasmic processeswhich contained S100 protein was shown to have thedistribution which would be expected of cellsresponsible for antigen presentation and they maybe involved in this process. These cells may beanalogous to the T6 and C3b-receptor negative,HLA-DR positive cells described in skin and lymphnode by Ralfkaier et al. 16The distribution of cells with slg and clg in human

gut associated lymphoid tissue contrasts sharply withthat observed in similar studies of animal tissue.Sminia and Plesch used immunohistochemistry tocharacterise the cells expressing slg and clg in thePeyer's patches of rats.4 These authors showed thatalthough cells with slg of all isotypes were present inthe Peyer's patches of rats, only cells with cIgM andcIgG could be found in the dome regions of thefollicles. Cells with cIgA were detected in the T-cell

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Human Peyer's patches: an immunohistochemical study

Fig. 4 Serial paraffin sections ofa Peyer's patch immunostained using antisera to (a) lysozyme and (b) S100 protein.Lysozyme containing macrophages are present in abundance between the crypts (Lysozyme releasedfrom the macrophagesresults in generalised staining of this area), but not in the dome region. In contrast with this, cells with cytoplasmic processescontaining SfOO protein are concentrated in the dome area. (x200, orig. mag).

zones presumably extravasating into the gut associ-ated lymphoid tissue. This supports experimentalevidence that IgA secreting B cells activated in thePeyer's patches mature in the mesenteric lymphnodes, enter the blood via the thoracic duct andreturn to the gut through the high endothelialvenules in the T cell zones of the Peyer's patches.The B cells in human Peyer's patches differ fromthose in rat Peyer's patches both in the isotypes ofimmunoglobulin that they express and also thedistribution of cells with slg and clg. In human tissueonly cells with sIgM and sIgAl were clearly andconsistently present in the Peyer's patches, cellsexpressing other isotypes were comparatively rare.Cells with clg of all isotypes other than IgD,however, could be observed in the dome regions ofhuman Peyer's patches. The presence of cells withcIgA in the dome regions of human Peyer's patchesis of particular importance. In human, unlike rattissue, cells fated to synthesise IgA in the lamina

propria may mature in the dome regions of thePeyer's patches together with the cells synthesisingother isotypes.4 The study of Crago et al,'7 showedthat although lymphoid tissue associated withhuman mucosae contained a higher proportion ofcells synthesising both IgA2 and J-chain thanlymphoid tissue elsewhere, mesenteric lymph nodesappeared to be no different to lymph nodes else-where in the body. This again suggests that inhuman tissue, cells synthesising IgA may mature inthe mucosa rather than in the mesenteric nodes.

There is little evidence of traffic of immunoglobu-lin synthesising cells within the T cell zones ofhuman Peyer's patches. If the immunoglobulinsynthesising cells generated in the Peyer's patchesreach the lamina propria via the lymphatics and theblood as shown in laboratory animals they appearnot to return to the gut via the high endothelialvenules, but they may use an alternative route suchas the capillary network.

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410 Spencer, Finn, and Isaacson

The results of this study provide grounds to doubtthat B cells activated in normal human gut associ-ated lymphoid tissue behave in the same way asthose generated in that of laboratory animals. Thereis little evidence as yet from immunocytochemicalstudies of human gut associated lymphoid tissue thatB cells migrate via the mesenteric lymph nodes, thethoracic duct lymph and the blood to reach thelamina propria. The Peyer's patches which arediffusely distributed in the human ileum may be ableto seed the lamina propria with antibody producingcells directly.

This work was supported by MRC Grant G8222346CA.

References

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2 Craig SW, Cebra JJ. Peyer's patches: an enrichedsource of precursors for IgA-producing immunocytes inthe rabbit. J Exp Med 1971; 134: 188-200.

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4 Sminia T, Plesch BEC. An immunohistochemical studyof cells with surface and cytoplasmic immunoglobulinsin situ in Peyer's patches and Lamina propria of ratsmall intestine. Virchows Arch [Cell Pathol] 1982; 40:181-9.

5 Cornes JS. Number, size, and distribution of Peyer'spatches in the humian small intestine. Part 1. Thedevelopment of Peyer's patches. Gut 1965; 6: 225-33.

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10 Epenetos AA, Bobrow LG, Adams TE, Collins CM,Isaacson PG, Bodmer WF. A monoclonal antibodythat detects HLA-D region antigen in routinely fixed,wax embedded sections of normal and neoplasticlymphoid tissue. J Clin Pathol 1985; 38: 12-17.

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13 Isaacson P, Wright DH. Immunocytochemistry oflymphoreticular tumours. In: Polak JM, Van NoordenS, eds. Immunocytochemistry. Practical applications inpathology and biology. Bristol: John Wright, 1983:249-73.

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16 Ralfkaier E, Stein H, Plesner T, Hou-Jensen K, MasonD. In situ immunological characterisation ofLangerhans cells with monoclonal antibodies: compari-son with other dendritic cells in skin and lymph nodes.Virchows Arch [Pathol Anat] 1984; 403: 401-12.

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