Gut 1994; 35: 1582-1589

Lymphoid and non-lymphoid cells in theepithelium and lamina propria of intestinalmucosa of pigs

H J Rothkotter, T Kirchhoff, R Pabst

AbstractThe jejunum and ileum of 5 day old andadult normal pigs and of 45 day old germfree pigs were used to study the lympho-cyte pools in the epithelium and laminapropria by sequential treatments withEDTA, four hours, and 12 hours of colla-genase treatment. In adult animals theincubation of the jejunal wall with EDTAresulted in mean (SD) 26-8 (10.9) X 106intraepithelial lymphocytes per g oftissue. The ileal wall gave lower cell yields.After complete digestion of the laminapropria by collagenase a further yieldof 35-2 (10.2)X106/g lymphocytes wasachieved. The separation of the gut wallfrom 5 day old pigs resulted in a 10-foldlower total lymphocyte yield, and thetissue was totally digested after fourhours of collagenase treatment. Manyeosinophils and mast cells were found inthe suspensions from adult animal tissuesafter the collagenase treatment; 4-7 X 106/gand 4 8X 106/g, respectively. The suspen-sions after 12 hour collagenase incubationcontained up to 30% plasma cells. Almostall cells isolated by EDTA incubation wereCD8+ T cells. After collagenase incuba-tion CD4+ and CD8+ T lymphocytes werefound in all animal groups, and in adultanimals up to 20% surface Ig+ cells wereharvested. When the incorporation of thethymidine analogue bromodesoxyuridinewas used to study the lymphocyte produc-tion in vivo 3 to 7% lymphocytes in theepithelium were labelled 24 hours later(lamina propria T lymphocytes about1%). In this study lymphoid as well asnon-lymphoid cells have been analysed inmucosal cell suspensions. The absolutecell yield per gram of mucosal tissue is abasis to estimate the pool sizes ofintraepi-thelial and lamina propria lymphocytes.(Gut 1994; 35: 1582-1589)

Centre ofAnatomy,Medical School ofHanover, Hanover,GermanyJ Rothkotter,T Kirchhoff,R Pabst

Correspondence to:Dr H J Rothk6tter, Centre ofAnatomy 4120, MedicalSchool of Hanover, D-30623Hanover, Germany.Accepted for publication9 February 1994

Lymphocytes in the epithelium and laminapropria of the intestinal mucosa play an

important part in the induction and main-tenance of intestinal immune reactions and inthe development of oral tolerance.' 2 They are

dynamic cell pools with subsets of T andsurface or cytoplasmic immunoglobulin posi-tive cells, or both occurring with different inci-dences during the antenatal and postnataldevelopment of enteric function.3 5 Mucosallymphocytes have often been studied, eitherusing immunohistology or suspensions of

separated cells.6 7 Intraepithelial lymphocytescan be isolated easily by incubation of the gutmucosa with EDTA8 9 whereas the separationof lamina propria lymphocytes requires tissuetreatment by enzymes.'0 1 Although the sub-sequential isolation of intraepithelial andlamina propria lymphocytes is a standardmethod in mucosal immunology, there are stillseveral questions concerning these techniques.A remarkable cell loss has been seen afterpurification steps (gradient centrifugation,filtration),12 13 which might cause an artificialenrichment of particular cell subsets. The yieldof lymphoid cells isolated from the epitheliumand lamina propria is often not indicated or itremains unclear how the lymphocytes weredifferentiated in the suspensions. It is alsodebatable whether lamina propria cell suspen-sions are representative of all the subcompart-ments of this microanatomically complex partof the gut wall, when the tissue is not digestedtotally to isolate all lymphoid cells from themucosa. Furthermore, many studies have dealtsolely with mucosal T cells, and very few dataare available on the percentages of Ig isotypepositive lymphocytes or plasma cells inmucosal cell suspensions.'2-14 Finally, obviouslimitations in obtaining human mucosal tissuemean that only few comparative data exist oncell yield and composition in different parts ofthe small intestine.

This study was performed to elucidate thesedifferent aspects. The omnivorous pig waschosen as the experimental animal because itsgastrointestinal tract is comparable with that ofhumans in several aspects, and pigs have beenused to examine the postnatal development ofintestinal enzymes.'5 Similar patterns of thepostnatal appearance of lymphocytes in thelamina propria have been seen in humans andpigs.3 4 For these reasons, pigs were used forcell separation experiments with the followingaims: isolation of all cells in the mucosa bycomplete digestion of the gut tissue; character-isation and quantification of the subsets of Tand Ig+ lymphoid cells and the non-lymphoidcells in the resulting suspensions; and analysisof the steps in the separation process at whichparticular cell types are released from thetissue. In this model, it was also possible tostudy the influence of age and entericmicrobial antigens on the cell compositionsand separation kinetics in using conventionaland germ free reared pigs of different ages. Inaddition, the percentage of newly formedlymphocytes in the mucosal cell suspensionswas determined by incorporation of thethymidine analogue bromodesoxyuridine

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Lymphoid and non-lymphoid cells in the epithelium and lamina propria of intestinal mucosa ofpigs

(BrdU). This was of particular importancebecause little is known about the origin, cellturnover, and the life span of the lymphocytes inthe epithelium and lamina propria, andthe data that have been published are contro-versial.6 1619 The results obtained are a basis forcomparison with cell separation experiments innormal and diseased human gut mucosa.

MethodsIn the first group normal 5 day old GermanLandrace piglets (n=6) were examined. Thesecond group consisted of germ free GermanLandrace piglets (n=6, 45 days old), whichwere obtained from Professor Dr Amtsberg,Department of Microbiology, VeterinarySchool of Hanover. The rearing conditions ofthese pigs have been described previously.20Adult Gottingen minipigs (n=8, 14 monthsold) were used as a third group. The in vivoproliferation of intraepithelial and laminapropria lymphocytes was studied in anotheradult group of minipigs (n=6, 9 months ofage). These animals received a single intra-venous injection of 20 mg BrdU/kg bodyweight (Sigma, Deisenhofen, Germany) andwere tested one day later. All animals werekilled by an overdose of thiobarbiturate(Trapanal, Byk Gulden, Konstanz, Germany).Standard representation parts of the jejunum(50 cm distal to the duodenojejunal flexure)and of the ileum (10 cm proximal to thecontinuous ileal Peyer's patch) without macro-scopically visible Peyer's patch tissue wereremoved and examined immediately.The mucosal cell separation used is a

modification of methods described earlier.10 21After intensive rinsing with Cal+and Mg++free Hank's balanced salt solution (SeromedBiochrom, Berlin, Germany) the gut sampleswere opened. If present, lymphoid follicles andPeyer's patches were excised and discarded.The weight and area of the sample weremeasured. Between 5 and 10 g of gut tissuewere used for the incubation. After stripping,the mucosa was cut into small pieces(0 5 cmx05 cm). All separation steps wereperformed in a shaking waterbath (120 rpm) at37°C. Each sample was incubated twice forone hour in a conical flask containing 25 mlCa++ and Mg`+ free Hanks's balanced saltsolution supplemented with 1 26 mmol EDTAand 0-94 mmol dithiotreitol (Sigma,Deisenhofen, Germany) and antibiotics (100IU/ml penicillin, 100 ,ug/ml streptomycin, 20pug/ml gentamycin, 0 5 ,ug/ml amphotericin,Seromed) to remove the epithelium and toisolate intraepithelial lymphocytes. In contrastwith earlier studies, the enzymic digestion ofthe connective tissue was performed in twosteps to isolate cells from the lamina propria.Firstly, the tissue was incubated in 25 mlRPMI 1640 containing 5% fetal calf serum,20 mmol HEPES (both Seromed), 0- 15 mg/mlcollagenase (no 103586, lot 11888023-940-225 U/mg, Boehringer, Mannheim,Germany), 0 1 mg/ml DNAse (Boehringer),0- 1 mg/ml trypsin inhibitor (Sigma), and anti-biotics (see above) for four hours at 370C.

Secondly, the incubation was repeated usingnew medium of the same composition for 12hours overnight at 37°C. The next morningmany of the gut samples of the adult pigs werecompletely digested, in some experiments theremaining tissue fragments were pushedthrough a steel mesh (200 ,um pore size) andthe resulting suspension was collected. Aftereach step, the cells in the supernatant were fil-tered through a stainless steel sieve and col-lected in 50 ml centrifugation tubes. Thesuspensions were washed twice (400 g, 10min) and resuspended in RPMI 1640 contain-ing 0-1 mg/ml DNAse. The cells were thentransferred to a known amount of RPMI 1640supplemented with DNAse. In some experi-ments, representative tissue fragments wereexamined by routine histological tests at thedifferent stages of the separation procedure.The cells of the two EDTA incubations werepooled as the first fraction (EDTA fraction).The second fraction was obtained after theshort four hour collagenase treatment, and the12 hour overnight incubation resulted in thethird cell fraction. To exclude possible detri-mental effects of the incubation procedures onthe lymphoid cells, in one experiment ficollhypaque separated blood lymphocytes and intwo further experiments cells from mesentericlymph nodes were incubated using the sameseparation procedure. The effects of thesetreatments on the control suspensions wereanalysed by comparing their phenotype beforeand after the incubation steps using flowcytometry.Lymphocyte counts were performed in a

haemocytometer using a phase contrast micro-scope at X500 magnification and the yields oflymphocytes per gram (g) of tissue calculated.Giemsa stained cytospots were used to deter-mine the percentage of small lymphocytes,large lymphocytes, granular lymphocytes,plasma cells, eosinophils, and mast cells.Without further purification, the lymphocytesubpopulation indices were determined in thecell suspensions by flow cytometry (FACScan,Becton Dickinson, Heidelberg, Germany)using monoclonal antibodies against piglymphocyte surface antigens (CD2 (pan Tcells in pigs), CD4, CD8, antipig IgA, antipigIgM, macrophages (mAb 74-22-15),22 23 witha phycoerythrin labelled second stage antibody(goat antimouse b-phycoerythrin, Dianova,Hamburg, Germany). In forward/side scatterdot plots, a lymphocyte gate was determinedfor each suspension. The cells in the gatepositive for each antibody were determinedusing fluorescence histograms.A modification of the method described by

Westermann et al 24 was used to detect theincorporated BrdU and the lymphocyte sub-sets in the proliferation study. On cytospots,the subsets were determined using CD2, CD8,antipig IgA, and antipig IgM monoclonal anti-bodies, a secondary antibody (Z259, Dako,Hamburg, Germany), and the alkaline phos-phatase anti-alkaline phosphatase technique(APAAP D65 1, Dako) with fast blue dye.The DNA was then denaturated with form-amide/sodium hydroxide. The incorporated

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after 4 hour.c 40 collagenase0) S //after 12 hour

30 collagenasx

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(D10

0Jejunum Ileum Jejunum Ileum

5 days 14 months

Figure 1: Lymphocyte yield per gram ofjejunal and ilealmucosa in S day old and adult (14 month) normal pigs.The lymphocytes were isolated by twoxone hourEDTAtreatment and by a short (four hour)and an overnight (12hour) incubation with coUlagenase. The data are shown inthis and the subsequent illustrations as mean (SD). Thecellyield was calculated based on lymphocyte counts madeby phase contrast microscopy.

BrdU was determined using a monoclonalantibody against BrdU (anti-BrdU, BectonDickinson, Heidelberg, Germany) and theimmunoperoxidase method (Z259 and PAPP850 complex, Dako). The cells positive foreach subset were counted on the cytospots andeach checked for BrdU incorporation.For statistical analyses of results, mean (SD)

of the data were calculated. The Mann-Whitney U test was used and p<005 wastaken as significant.

Results

SEPARATION PROCESS AND CELL YIELDHistological sections of tissue fragments,acquired during the separation procedure,showed that after EDTA treatment allepithelial cells of the villi and most of theintraepithelial lymphocytes had been removed.After the four hour incubation with collagenasethe connective tissue of the villi had beendigested, but the lamina propria between thecrypts was still intact. The tissue of 5 day oldpigs and of germ free animals was alreadytotally dispersed after this incubation step. Inall other samples of adult pigs, the whole tissuewas digested at the end of the experimentsafter a further 12 hours of collagenase incuba-tion. The lymphocyte viability was greater than90% in the trypan blue exclusion test. Phasecontrast microscopy was an efficient method tocount the number of lymphoid cells in theresulting suspensions. The lymphocyte yieldwas expressed in lymphocytes per gram oftissue. One gram was equivalent to 5-2 (0 9)cm2 gut tissue in 14 month old pigs. Figure 1shows the lymphocyte yield of 5 day and of 14month old pigs. Most cells were isolated in thefirst and the third fractions (EDTA incubationand 12 hour collagenase treatment). Fewerlymphocytes were obtained from the ileal thanthe jejunal mucosa (Fig 1). In 45 day old germfree animals, the cell yield was comparablewith that of 5 day old normal pigs (jejunumfirst fraction: 3-8 (2-8)X106, second fraction:1-5 (2) X 106). Lymphocyte numbers isolated

from the tissues of the 9 month old pigs wereabout 50% lower than from the 14 month oldanimals.

CELL DIFFERENTIATIONThe cytospots contained debris and manydead epithelial cells and these were excluded-from the evaluation. While the percentagesof lymphocytes, granular lymphocytes,eosinophils, and mast cells were easily deter-mined, it was difficult to differentiate betweenlarge lymphocytes and immature plasma cells.Small lymphocytes and eosinophils predomi-nated in the jejunum and ileum of 5 day oldanimals (for example, second fraction ofileum: lymphocytes=36-9 (85)%/,eosinophils=54 1 (10-2)%) and of germ freepigs. In the jejunum of 14 month old pigs (Fig2), the first EDTA fraction contained manysmall or granular lymphocytes and a signifi-cant proportion of eosinophils (4 0 (2-3)%).In the second fraction after four hours ofcollagenase treatment, the percentage of gran-ular lymphocytes from the jejunum haddecreased but more eosinophils were found(12-9 (3.5)O/0). Further collagenase incuba-tion for 12 hours resulted in an increase in theplasma cells and mast cells (Fig 2). In theileum of 14 month old pigs the comparativenumber of small lymphocytes was two thirdsand that of plasma cells only one third of thepercentages seen in the jejunal samples. In theileum, however, about a threefold increase inthe percentage of eosinophils was found afterEDTA and collagenase incubation. Therewere no differences in the cellular composi-tions of the jejunal as well as the ileal samplesbetween tissues of the 9 and 14 month oldpigs. In all animal groups the percentage ofeosinophils was significantly higher in theileum. The absolute numbers of mast cellsand eosinophils were calculated based onthe lymphocyte counts and the percentageof lymphoid cells in the differential cellcounts. In the ileum of 14 month old pigs,the first fraction contained 3-2 (1 9)X 106eosinophils/g, and the fractions after colla-genase treatment up to 4-7X 106eosinophils/g.

M Large lymphocytes = Granular lymphocytesEl= Plasma cells 1MM EosinophilsM Small lymphocytes = Mast cellsa-

4hour 12 hourEDTA Collagenase

Figure 2: Mean percentage oflymphoid cells, eosinophils,and mast cells injejunal cell suspensions isolatedfrom themucosa of 14 month old pigs afterEDTA and twosubsequent collagenase incubations. The cell differentiationwas performed on Giemsa stained cytospots.

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Lymphoid and non-lymphoid cells in the epithelium and lamina propria of intestinal mucosa ofpigs

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Figure 3: Flow cytometric analysis of mucosal cell suspensions isolatedfrom thejejunum ofa 14 month old pig. (A) and (B) Dot plots showing the forward and side scatter values ofall isolated cells. (A) Intraepithelial lymphocytes (IEL): cells isolated by EDTA treatment;(B) lamina propria lymphocytes (LPL): cells isolated during the overnight 12 hourcollagenase incubation. The delineated populations show the isolated lymphocytes(lymphocyte gate), in the LPL panel some of the lymphocytes have a higherforward scattervalue, which shows that after collagenase incubation a population of larger cells is isolatedfrom the lamina propria. (C) and (D)Same cell population as in (B) after gating on

lymphocytes. The cells were stained using CD2+ and IgA+ antibodies andimmunofluorescence. The dot plots show the values of the forward scatter and of thefluorescence intensity. (C) LPL, CD2+ T cells: the window shows the cells positive for theT cell marker, many of the cells are small (lowerforward scatter values); (D) LPL, IgA+cells: the surface IgA+ cells in the window have higherforward scatter values pointing totheir large size. There is a population of cells with largerforward scatter values that were

not stained by T cell antibodies and were negative for surface immunoglobulin, which are

putative plasma cells.

FLOW CYTOMETRYBlood and mesenteric lymph node lymphocytesuspensions were incubated using the samemethods to exclude the possible effects of theseparation protocol on the surface antigenexpression of the mucosal lymphocytes. Theforward scatter values of these suspensionsbefore and after treatment were comparable.

Some differences were found in the lympho-cyte subsets studied, for example after incuba-tion the percentage of CD2+ cells was reducedby <5%, but about 100% less surface Ig+ (sIg+)cells were found. In all suspensions tested, theexpression values of the lymphocyte antigenswere not affected by the incubation procedure.The lymphoid cells in the tissue suspensions

were identified and gated using their character-istic forward and side scatter properties in theflow cytometer (Fig 3). As young and germfree animals had few lymphocytes/g tissue (Fig1), the gating was difficult, because the smallnumbers of lymphocytes were engulfed in a

large proportion of red blood cells, deadenterocytes, and small tissue fragments. Thus,the gated population contained a certainnumber of events that were not lymphoid cells.This resulted in small percentages of lympho-cyte subsets in young and germ free pigs. Thecomparative number of CD2+ lymphocyteswas low, especially in the ileum (Table I). Inboth adult groups (9 and 14 months) thegating was easier (Fig 3). In the third fractionof these pigs, a significant proportion oflymphoid cells was found with higher forwardscatter values, consistent with their larger cellsize (Fig 3), and immunofluorescent stainingshowed that some of these larger cells were

sIgt (Fig 3). In this fraction in 9 month oldpigs, however, between 5 and 25% of the cellsin the gate were not labelled by T or

macrophage antibodies and expressed no sIg+(Table I). As the forward scatter values ofthese unlabelled cells were higher than those ofthe CD2+ cells (Fig 3), it is unlikely that thesewere red blood cells in the gated population.

Figure 4 and Table I show the percentagesof lymphocyte subsets in jejunal cell suspen-

sions of 5 day and 14 month old animals. Thenumber of macrophages was always lower than5%, therefore this subset was not included inTable I. In 5 day old animals a larger propor-

tion of CD8+ lymphocytes was found in the

TABLE I Proportions of lymphocyte subsets in mucosal cell suspensions

_ejunum Ileum

Collagenase Collagenase

EDTA 4 hour 12 hour EDTA 4 hour 12 hour

5 days (%) CD2 59-9 (11-9) 45-8 (8-0) 36-8 (13-0) 46-1 (0-5)CD4 7-0 (2-4) 6-1 (1-6) 3-2 (1-8) 6-5 (3-4)CD8 20-2 (14-1) 6-4 (6 7) ND 5 9 (4 5) 12-9 (11-0) NDsIgA 2-8 (2-4) 2-6 (2-1) 1-7 (1-3) 2-2 (1 9)sIgM 2-5 (1-9) 2-0 (1 0) 3-4 (4-2) 2-6 (2 9)

Germ free (%/o) CD2 55-9 (6 7) 71-1 (20 5) 12-8 (10 9) 74-2 (11 0)CD4 20-7 (30 2) 8-8 (2-0) 3-1 (3 4) 11-2 (3 5)CD8 4-2 (4 8) 15-3 (12-1) ND 1-3 (0 5) 23-6 (10-4) NDsIgA 1-5 (0 7) 1-7 (2-1) 0-4 (0-1) 2-0 (1-8)sIgM 1-2 (1-3) 2-7 (1-9) 0-8 (0-1) 2-9 (0 9)

9 months (%) CD2 86-0 (6-5) 90 9 (10 0) 57-6 (20 0) 84-1 (7 7) 93-4 (5-9) 77-5 (8-1)CD4 7-2 (3 3) 33-4 (12-6) 14-0 (6 8) 3 9 (1 0) 30.7 (9 4) 15-9 (6 5)CD8 67-5 (10-4) 52-7 (13-7) 32-6 (24-7) 54-5 (10-1) 50 4 (9 6) 39-6 (10-8)sIgA 1-8 (0 9) 2-7 (2 6) 5 9 (3 6) 1 9 (1 0) 2-8 (1-4) 3-7 (1-5)sIgM 1-6 (1 1) 6-6 (9 5) 4-1 (3 4) 1-8 (1-4) 1 9 (1-2) 2-3 (1 3)

14 months (/) CD2 95-1 (4-0) 93-6 (2-4) 84-4 (12-8) 95-0 (1-2) 94-9 (2-3) 89-3 (4-4)CD4 5-6 (2-2) 47-5 (21-6) 26-5 (4-1) 11-5 (10-6) 51-5 (26-3) 37-0 (12-3)CD8 85-7 (8 8) 86-0 (16-5) 61-1 (29 7) 80-1 (10-4) 83-6 (17-1) 72-9 (23 7)sIgA 2-2 (1-4) 3-4 (2 0) 12-9 (5-13) 1 9 (0 6) 2-7 (1-5) 8-7 (3 2)sIgM 1-3 (0-7) 2-1 (1-3) 4-3 (2-4) 1-1 (0-8) 1-4 (1 0) 2-5 (1-8)

Percentages of lymphocyte subsets (mean (SD)) in the mucosal cell suspensions of the jejunum and ileum in 5 day old normal,45 day old germ free, and 9 and 14 month old normal pigs. The results were obtained by flow cytometric analysis of epithelial cellsuspensions isolated by EDTA incubation and of lamina propria suspensions separated by two subsequent collagenaseincubations. ND=In 5 day old and in germ free animals no 12 hour collagenase incubation was performed as the mucosa wascompletely digested after the four hour collagenase incubation.

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A

3CD2+ M CD4+ M IgA+OM1 CD8+ IgM+|

4 hour 12 hourEDTA Collagenase

4 hour 12 hour

EDTA Collagenase

Figure 4: Percentage of CD2+, CD4+, CD8+, sIgA+, andsIgM+ cells in mucosal cell suspensions isolated after EDTAand two collagenase incubations (four hours and 12 hours).Suspensions isolatedfrom the jejunum of (A) 5 day oldpiglets and (B) 14 month old pigs. * In 5 day old animalsthe tissue was already digested after the first collagenaseincubation and the second collagenase incubation was notperformed.

EDTA fraction than in the collagenasefraction. The sum of the comparative numberofCD4+ and CD8+ cells was less than the per-centage of CD2+ cells, providing evidence of a

CD2+CD4-CD8- cell population in youngpigs. Very few sIg+ cells were found (Fig 4A,Table I). In the ileal suspensions from thesame animals and in the suspensions from 45

day old germ free pigs, a comparableCD2+/sIg+ subset pattern was seen (Table I).In germ free pigs more CD4+ than CD8+ wereharvested after EDTA incubation (Table I),but immunohistological examinations showedthat almost all of these CD4+ cells werelocated in the lamina propria (results notincluded).

In jejunal and ileal samples of adult pigs (9and 14 months), the lymphocyte compositionof the three cell suspensions showed consider-able differences (Fig 4B, Table I). As expected,the suspensions after short and overnight colla-genase treatment (second and third fraction)contained more CD4+ cells than the EDTAfraction. In comparison with CD2+ cells, thehigh percentages of CD4+ and CD8+ cellsshow that in the gut samples of these adult pigsthere must be a proportion of CD4 CD8double positive cells, especially in the fractionafter short collagenase treatment (Fig 4B,Table I). In the first two suspensions, a smallnumber of sIg+ cells was detected, but in thethird fraction there was an increased percent-age of sIgA+ and sIgM+ cells (Table I). In 9month old animals there was a proportion ofnon-T/non-sIg+ cells in the cell fraction after12 hours of collagenase incubation, as the sumof CD2+ T cells, sIgA+ and sIgM+ cells wasless than 100% (Table I).

Table I shows the percentages of subsets inthe jejunal and ileal suspensions of all fouranimal groups. Although the comparativesubset distribution is important, in view of thedifferences in the sizes of each compartmentthe absolute numbers (for example, cell yieldper g of tissue) are equally important,especially for comparing the results of differentstudies. Table II shows the absolute numbersof lymphocytes in each subset per g of tissue inthe jejunum and ileum in all four experimentalgroups. Very few cells, mostly CD2+, werefound in the ileum of germ free pigs. In 9month old animals, a significant proportionof sIg+ cells was isolated after the EDTA

TABLE II Absolute numbers per gram of tissue in each subset in lymphocyte suspensions from the mucosa

J3ejunum Ileum

Collagenase Collagenase

EDTA 4 hour 12 hour EDTA 4 hour 12 hour

5 daysxlO6/g CD2 3-3 (2-1) 0-6 (0-4) 1 1 (1 0) 0-7 (0 3)CD4 0 4 (0 3) 0-1 (0-1) 0-1 (0 1) 0-1 (0-1)CD8 1-2 (0 9) 0 1 (0 1) ND 0-2 (0-3) 0-2 (0-3) NDsIgA 0-2 (02) <0-1 0-1 (0-1) 01 (01)sIgM 0-2 (0 2) <0 1 0-1 (0-2) 0-1 (0 1)

Germ freeXl06/g CD2 2-0 (1-2) 1-4 (2-0) 0-2 (0 1) 0-1 (0 1)CD4 0 4 (0 4) 0-2 (0 2) <0 1 <0 1CD8 0-2 (0 2) 0-2 (0 2) ND <0 1 <0 1 NDsIgA <01 <0 1 <0 1 <0 1sIgM 0-1 (0-1) 0 1 (0 1) <0 1 <0 1

9 monthsxl06/g CD2 15-8 (6 3) 3-3 (2-8) 7-1 (6-1) 10-4 (4 0) 3-9 (0 6) 5-6 (2-1)CD4 1-3 (0 6) 1 0 (0-5) 1-7 (1 1) 0 4 (0 2) 1-3 (0 7) 1-2 (0 6)CD8 12-1 (3 9) 2-1 (2 0) 3-8 (3 6) 6-7 (2-6) 2-1 (0-5) 2-8 (1-2)sIgA 0 3 (0 2) 0 1 (0 2) 0 9 (0 8) 0-3 (0 2) 0 1 (0 1) 0-3 (0 2)sIgM 03 (02) 0-2 (03) 07 (08) 0-2 (02) 01 (01) 0-2 (01)

14 monthsX 106/g CD2 25-7 (10-7) 7-4 (3 7) 29-7 (9 8) 17-7 (7 5) 4-9 (2 9) 13-3 (6 4)CD4 1-3 (0 6) 2-9 (1-2) 8-6 (3 9) 1-6 (1-6) 1-9 (1-2) 4-5 (3-1)CD8 23-5 (8 2) 6-1 (2 9) 21-9 (14-1) 14-8 (6 5) 4-3 (2 8) 11-7 (7 8)sIgA 0 7 (0 7) 0-3 (0-3) 4-8 (2-7) 0-3 (0-1) 0-1 (0-1) 1-2 (0-6)sIgM 0-4 (0-3) 0-2 (0-1) 1-6 (1-2) 0-2 (0 1) 0-1 (0 1) 0 3 (0 2)

Absolute yield of lymphocyte subsets/g gut tissue (mean (SD)) in the same experiments shown in Table I. The results werecalculated based on the lymphocyte cell yield determined in phase contrast microscopy and the percentages of lymphocyte subsetsgiven in Table I (ND, see Table I).

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Lymphoid and non-lymphoid cells in the epithelium and lamina propria of intestinal mucosa ofpigs

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EDTA CollagenaseFigure 5: Newly formed cells in cell suspensions obtainedafter the EDTA and 12 hour collagenase treatment steps of9 month old pig tissues. The animals were given a singledose ofBrdU and the intestines were sampled 24 hourslater. Using an immunocytochemical double stain on

cytospots, the relative numbers of CD2+, CD8+, IgA+,and IgM+ cells that had incorporated BrdU weredetermined.

incubation, but in both adult animal groupsthe largest proportion of sIgA+ and sIgM+cells was found after the 12 hour collagenaseincubation.

IN VIVO PROLIFERATION STUDYIn the first cell fraction, only the proliferationof T and CD8+ cells was analysed becausethere were too few Ig+ cells in this compart-ment (Fig 5). In this first jejunal and ileal cellfraction equal numbers of T cells had incor-porated BrdU. In the third cell fraction, theproportion of proliferating lymphocytes was

lower (T cells= 1 1 (0O5)%). Small numbers ofIg+ cells were BrdU+ (IgA+=1 2 (06)%;IgM+=08 (05)%).

DiscussionThis study was performed to analyse thekinetics of separation of intraepithelial andlamina propria lymphocytes and to study theinfluence of age and microbial stimuli on thedevelopment of these cells during postnatallife. In all experiments, the mucosa was com-

pletely digested and the resulting cell suspen-sions were not purified. The separationprotocol had no important effects on the cellswith respect to the parameters studied.Important findings concerning the techniquewere made that are often not taken intoaccount in mucosal cell isolation. Firstly,within the cell suspensions isolated with theepithelium significant numbers of eosinophilsand sIg+ cells were present, in addition tomany granular lymphocytes and the expectedlarge proportion of CD8+ cells. Under normalcircumstances, eosinophils and Ig+ cells do notoccur in the intestinal epithelium, as confirmedby histological examination. Their presence inthe first cell fraction is clear evidence thatthese suspensions also contain cells from thelamina propria, perhaps immediately under theepithelium. Secondly, after collagenaseincubation, granular lymphocytes were seen,which are normally only found in the epithe-lium of the mucosa.25 26 Their occurrence afterthe first collagenase incubation is evidence of a

contamination from the epithelial cell pool.Morphological examination of the cell suspen-sions using Giemsa stained cytospots wasessential for these findings; the results couldnot have been obtained by flow cytometryalone. Thus, it is important in evaluating pub-lished data to take into consideration howintraepithelial or lamina propria cell suspen-sions have been isolated and purified andwhether the compositions of the cell popula-tions have been checked before analysis usingsensitive techniques.19 27 Thirdly, it was foundadvantageous to split the collagenase incuba-tion into two sequential steps. Regardingthe total cell composition (lymphoid/non-lymphoid cells) and the lymphocyte subsetcomposition, the suspensions after a short col-lagenase treatment (four hours) were morecomparable with intraepithelial cell fractions.Only after an overnight collagenase incubation(12 hours) were all cell populations in thelamina propria harvested (CD4+ lymphocytes,sIg+ cells, plasma cells, and mast cells). Thisreflects the histological structure of themucosa. During the first collagenase incuba-tion, the villus lamina propria was preferen-tially dissociated, as seen in histologicalsections of the tissue fragments. In immuno-histological examinations, however, the Ig+cells were mainly found in the crypts of themucosa.428 In agreement with this distribu-tion, very few sIg+ cells were found after thefirst collagenase incubation. The complete dis-sociation of the crypt areas was necessary toisolate most Ig+ cells. Based on these findings,an incomplete digestion of the lamina propriawill selectively reduce the proportion of sIg+cells in lamina propria suspensions.One further aspect was that in young

animals, the bulk of cells was already obtainedafter two hour EDTA incubation, even with-out collagenase treatment, because there is lessconnective tissue. In mice, a comparable quickseparation has been seen.'2 Therefore, it ismuch more difficult to obtain pure intraepithe-lial lymphocyte suspensions in young animalsor in smaller animals like rodents. Thesefindings are also relevant for separation experi-ments in inflamed tissue. The questionremains, however, whether the incubation pro-tocol should be adapted to the tissue underexamination or whether a standard protocolshould always be used. Harvesting theseparated cells at short intervals was an advan-tage, because it prevented cell loss resultingfrom cell death in the incubation flask andmade it possible to analyse the cell suspensionsin detail. If the tissue localisation of a specificcell subpopulation is unclear, an immunohisto-logical examination can help to find out if thecells in the suspensions originate from theepithelium or the lamina propria.As the number of intraepithelial or lamina

propria lymphocytes is low in gut samples ofyoung or germ free animals,4 29 the highproportion of epithelial cells and small tissuefragments may disturb the flow cytometricanalysis of the cell suspensions. This results inlow percentages of lymphocyte subsets (TableI). Although not performed in this study, the

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Rothkotter, Kirchhoff, Pabst

proportion of lymphocytes in the gatedsuspension can be determined using an anti-CD45 monoclonal antibody. Another difficultywas the occurrence of a population of cells notstained by T or anti-Ig antibodies. These non-T/non-sIg+ cells had higher forward scattervalues than the CD2+ cells (Fig 3) and it can bediscounted that they were red blood cells.Plasma cells have little if any immunoglobulinon their surface,30 so they may not be stainedwhen anti-Ig antibodies are used in immunoflu-orescence. The non-T/non-sIg+ cells with largeforward scatter values in the third suspensionsin adult animals (Fig 3) are probably plasmacells. The discrepancy between the percentageof sIg+ cells (flow cytometric analysis, Fig 3, 4B,and Table I) and the large numbers of plasmacells (Giemsa cytospots, Fig 2) supports thishypothesis. So far there is no explanation whythe 9 month old animals had more non-T/non-sIg+ cells than the 14 month old pigs.Further experiments should include techniquesto detect intracytoplasmic and surface immuno-globulin to analyse all intestinal lymphoid cellsof the B cell family.

In humans, no data are available on cellseparation in very young children. In youngpigs the cell yield was only 10% of thatobtained in older animals. In germ free pigletsof 45 days of age, cell yield and subset patternswere comparable with those in 5 day oldnormal animals. Together with earlier data,4 29this is evidence that live microbial antigens arethe main stimuli leading to a postnatal increasein the T lymphocyte compartments in the gutwall. The occurrence of a CD2+CD4-CD8-lymphocyte subset in the flow cytometricanalysis of suspensions obtained from younganimals is confirmation of a comparablefinding in immunohistology.4 It cannot beexcluded that these cells are ry/8 T cell receptorpositive cells, as in pigs there is evidence for aCD2+ -y/8 T cell subset.31 Lymphocytesdouble positive for CD4 and CD8 have beenseen not only in the lamina propria of pigs butalso among the intraepithelial lymphocytes inrats.32 The finding of the CD4+CD8+lymphocyte subset in the lamina propria ofadult animals showed that these lymphocytesare activated to a high extent, becauseCD4+CD8+ lymphocytes were found afteractivation of pig lymphocytes.33The relative proportions of mast cells to

lymphoid cells isolated from the mucosa in thethird fraction are comparable with resultsobtained by histological examination usingspecific detection methods for mucosal mastcells.34 The isolation technique is an easymethod to determine the population of mastcells and other non-lymphoid immune cells inall mucosal cell suspensions. Large numbers ofnon-lymphoid cells can be isolated for func-tional tests in vitro. In relative numbers, thesmall intestine of 5 day old pigs contains manymore non-lymphoid immune cells thanlymphocytes. In particular, eosinophils are thefirst cells extravasating into the intestinalmucosa and performing non-specific immuneresponses to the antigens appearing in the gutafter birth.

The turnover of the intraepithelial lympho-cytes is not yet clear. It has been reported thatthese cells proliferate in vivo16 18 19 and invitro.8 In contrast, in a study using the mono-clonal antibody Ki-67, which stains proliferat-ing cells, Halstensen et al 17 could not detectpositively stained cells in the normal humanintestinal epithelium. In our experiments manymore intraepithelial T cells were seen in thecell cycle during the 24 hour observationperiod than T or Ig+ cells in the laminapropria. Intradermal lymphocytes have a com-parable production rate with those in theintestinal epithelium, but the number ofBrdU+ cells in peripheral lymph nodes was 10-fold lower.35 It is a matter of speculationwhether all labelled cells were produced in theepithelium or lamina propria of the small intes-tine or whether they migrated into these tissuesites within the 24 hour period after the BrdUinjection, or a mixture of both.The total cell yield seen in adult pigs

(7X107/g) was within the range reported forhuman mucosa.10 13 36 It was difficult to com-pare these data as few studies stated whetherthe whole tissue had been digested and howmany cells had been lost during densitygradient centrifugation.13 In this study theabsolute cell yield was expressed per gram ofgut tissue, that is the weight of the mucosa andthe muscle layers. It is suggested that the cellyield per gram of stripped mucosa is possibly abetter basis for comparison as changes in theweight of the musculature would not theninfluence the results. The absolute cell yieldper gram of tissue can be used as a basis toestimate the size of the mucosal lymphocytepools. In this study, the similar cell numbers inadult animals after the EDTA and collagenasetreatments show that the intraepithelial andlamina propria cell pools are comparable inthese pigs. Based on these results, it can beextrapolated that the small intestines of 14month old pigs weighing about 50 kg contain atotal of 7 X 109 intraepithelial and laminapropria lymphocytes per metre (weight of 1 cmof small intestine= 1 g; total lymphocyte yieldper g=about 7-0X107/g; 10OX7-0X 107 lym-phocytes=7>X 109 cells/m). This is low in com-parison with the absolute number of IOX109plasma cells per metre reported in the humansmall intestine.37 However, the order of the cellpool size is comparable. It has been calculatedthat a 30 kg pig has a total of 321 x 1O9 lym-phocytes.38 As the small intestine of 14 monthold animals is 7 to 10 metres in length, morethan 10% of the total lymphocytes of the pigsbody are distributed in the epithelium andlamina propria of the small intestinal mucosa.

In conclusion, the results show the impor-tance of a careful separation protocol, modifiedaccording to the normal or diseased state of thetissue examined. A combination of lymphocytecounts, calculation of the cell yield/g, morpho-logical cell differentiation, and lymphocytesubset analysis is essential to acquire reliabledata about lymphoid and non-lymphoid cellsin the compartments of the mucosa. Thesestudies, especially those in young piglets, areimportant for clinical research. They can serve

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Lymphoid and non-lymphoid cells in the epithelium and lamina propria of intestinal mucosa ofpigs 1589

as a model for the examination of evolvingmucosal cell populations in postnatal develop-ment when the body is first facing microbialand nutritional antigens in the gut lumen.The study was supported by the DeutscheForschungsgemeinschaft, SFB 280 'Gastrointestinal Barrier',Project C 1. The authors thank Professor Dr Amtsberg of theDepartment of Microbiology, Veterinary School of Hanover forproviding the intestines of the germ free animals and Dr FKlobasa of the Institute for Animal Breeding and Behaviour(FAL), Mariensee, Neustadt for providing some of the pigs.Monoclonal antibodies were kindly supplied by Drs A Bianchi(Lelystad, The Netherlands), R M Binns (Cambridge, UK),and A Saalmuller (Tiibingen, Germany). The skilful technicalassistance of S Fa,Bbender, A Herden, and S Schlecht, thehelp of Dr N N Barman, and the correction of English andsecretarial help by S Fryk are gratefully acknowledged.These data were presented in part at the 7th International

Congress of Mucosal Immunology, August 1992, Prague.

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2 Harvey J, Jones DB. Human mucosal T-lymphocyte andmacrophage subpopulations in normal and inflamedintestine. Clin Exp Allergy 1991; 21: 549-60.

3 Perkkio M, Savilahti E. Time of appearance ofimmunoglobulin-containing cells in the mucosa of theneonatal intestine. Pediatr Res 1980; 14: 953-5.

4 Rothkotter HJ, Ulbrich H, Pabst R. The postnatal develop-ment of gut lamina propria lymphocytes: number,proliferation, and T and B cell subsets in conventionaland germ-free pigs. Pediatr Res 199 1; 29: 237-42.

5 Spencer J, Isaacson PG, Walker-Smith JA, MacDonald T-T.Heterogeneity in intraepithelial lymphocyte subpopula-tions in fetal and postnatal human small intestine. JPediatr Gastroenterol Nutr 1989; 9: 173-7.

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12 Van der Heijden PJ, Stok W. Improved procedure for theisolation of functionally active lymphoid cells from themurine intestine. Jf Immunol Methods 1987; 103: 161-7.

13 Selby WS, Janossy G, Bofill M, Jewell DP. Intestinal lym-phocyte subpopulations in inflammatory bowel disease:an analysis by immunohistological and cell isolationtechniques. Gut 1984; 25: 32-40.

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15 Shulman RJ, Henning SJ, Nichols BL. The miniature pig asan animal model for the study of intestinal enzymedevelopment. Pediatr Res 1988; 23: 311-5.

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CD4+ oJl3 cells in the lamina propria but proliferation(Ki-67) of os1, and -y/8 cells in the epithelium. Eur JImmnunol 1993; 23: 505-10.

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19 Yamamoto M, Fujihashi K, Beagley KW, McGhee JR,Kiyono H. Cytokine synthesis by intestinal intraepithe-lial lymphocytes: both y/8 T cell receptor-positive andox/3 T cell receptor-positive T cells in the GI phase ofcell cycle produce IFN-y and IL-5.JlItnnunol 1993; 150:106-14.

20 Dziaba KA, Lambrecht G, Petzoldt K. Intestinal and serumantibody response in gnotobiotic piglets to oral immuniza-tion with Escherichia coli. Co7np Immnunzol Microbiol InfectDis 1985; 8: 267-72.

21 James SP, Graeff AS. Spontaneous and lymphokine-induced cytotoxic activity of monkey intestinal mucosallymphocytes. Cell Iminunol 1985; 93: 387-97.

22 Lunney JK, Pescovitz MD. Differentiation antigens ofswine lymphoid tissues. In: Trmka Z, Miyasaka M, eds.Differentiation antigens in /ymnphohemopoietic tissues. NewYork: Dekker, 1988: 421-51.

23 Lunney JK. Characterization of swine leukocyte differentia-tion antigens. Immnunol Today 1993; 14: 147-8.

24 Westermann J, Ronneberg S, Fritz FJ, Pabst R.Proliferation of lymphocyte subsets in the adult rat: acomparison of different lymphoid organs. Eur J Itn?nunol/1989; 19: 1087-93.

25 Marsh MN, Leigh RJ, Loft DE, Gamer GV, Gordon DB.Studies of intestinal lymphoid tissue X-observations ongranular epithelial lymphocytes (gEL) in normal anddiseased human jejunum. Virchows Arch [A] 1988; 412:365-70.

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27 Guy-Grand D, Vanden Broecke C, Briottet C, Malassis-Seris M, Selz F, Vassalli P. Different expression of therecombination activity gene RAG-1 in various popula-tions of thymocytes, peripheral T cells and gut thymus-independent intraepithelial lymphocytes suggests twopathways ofT cell receptor rearrangement. EurJ7 Inmnunol1992; 22: 505-10.

28 Baklien K, Brandtzaeg P. Immunohistochemical character-ization of local immunoglobulin formation in Crohn'sdisease of the ileum. Scand J Gastroenterol 1976; 11:447-57.

29 Guy-Grand D, Griscelli C, Vassalli P. The mouse gut Tlymphocyte, a novel type ofT cell. Y Exp Med 1978; 148:1661-77.

30 Brown SL, Morrison SL. Regulation of the productionof secretory and membrane immunoglobulin duringlymphocyte development. Clin I?nmunol Ilntnunopathol1989; 50: 155-70.

31 Reddehase MJ, Saalmuller A, Hirt W. -y/8 T-lymphocytesubsets in swine. Curr Top Microbiol Iln"nunol 1991; 173:113-7.

32 Takimoto H, Nakamura T, Takeuchi M, Sumi Y, TanakaT, Nomoto K, et al. Age-associated increase in number ofCD4+ CD8+ intestinal intraepithelial lymphocytes in rats.Eur_7 Immunol 1992; 22: 159-64.

33 Saalmuller A, Reddehase MJ, Buhring HJ, Jonjic S,Koszinowski UH. Simultaneous expression of CD4 andCD8 antigens by a substantial proportion of restingporcine T lymphocytes. Eur J Iinmunol 1987; 17:1297-301.

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