preparation purification lymphocytes propria · lamina propria lymphocytes (see figure for flow...

Gut, 1981, 22, 481-488

Preparation and purification of lymphocytes from theepithelium and lamina propria ofmurine small intestineM D J DAVIES* AND D M V PARROTT

From the Department of Bacteriology and Immunology, Western Infirmary, Glasgow

SUMMARY Existing methods for the production of lymphocytes from the small intestine have provedunsatisfactory when applied to the mouse. We report here a new method for the production ofhighly pure suspensions of lymphoid cells from the epithelial layer and lamina propria of mousesmall intestine. The production and purification methods are described in detail. At least ten millionlymphocytes are obtainable from each small intestine from either the epithelium or lamina propriaand the cell suspensions are shown to be little contaminated by non-lymphoid cells. Preliminaryanalysis of the two cell types indicates that they belong either to two separate populations or to onepopulation in very different stages of differentiation. The use of purified lymphoid cells from theepithelium and lamina propria of the small intestine may enable examination of the generation ofcytotoxicity towards gut epithelial cells; this may be important in the development of inflammatorybowel diseases.

With increasing interest being shown in theimmunology of the gut, it has become necessaryto produce pure populations of lymphoid cellsfrom the intestine to examine their immuneeffector function. Potentially the small intestinecan yield lymphocytes both from the epitheliallayer, the so-called intraepithelial lymphocytes,and from the lamina propria. So far, the majorityof studies have been carried out on human smallor large bowel tissue where two methods of ex-traction have been tried: mechanicall-3 and en-zymic.3-5 Mechanical and enzymic extractiontechniques have also been tried with varyingsuccess on experimental animal intestinal tis-sue.A-8 In the vast majority of these studies,'mucosal' lymphocytes have been extracted withlittle regard to their origin from the epitheliallayer or lamina propria. In view of the obser-vation8s that cytotoxic T cells in the epitheliallayer appear to have a different cytotoxic poten-tial from those in the lamina propria, it is necess-ary to examine separately lymphoid cells fromthe two layers when assessing effector function.We report here an efficient, economical, and

reproducible technique for producing lymphoid*Address for correspondence: Dr M D J Davies, Department ofBacteriology and Immunology, Western Infirmary, Glasgow GIl1 6NTScotland.

Received for publication 18 December 1980.

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cells from mouse smalil intestine epithelium andlamina propria. The cells are obtainable in veryhigh purity and viability and preliminary obser-vations on some of the properties of the isolatedcells show that the two populations are different.

Methods

ANIMALSC57B1 10ScSn, DBA/2, CBA/Ca, and Balb/cmice were supplied and bred from departmentstocks. Age- and sex-matched mice were usedwithin any one experiment.

M E D I ARPMI 1640 X 10 concentrate (Gibco Biocult) wasbrought to isotonicity (308 mOsmol/kg) bydilution with 10 mM HEPES, 25 mM sodiumbicarbonate buffer, and corrected to pH 7-2 with5N sodium hydroxide. This medum was routinelysupplemented with 5% heat-inactivated newborncalf serum (NCS; Gibco), 2 mM L-glutamine,100 units/ml penicillin and 100 jig streptomycin(Gibco). The addition of 30 ug/ml gentamicin(Sigma) is recommended during the extractionprocedure if the cells are to be cultured.

Calcium and magnesium ion free Hanks' bal-anced salt solution (CMF) (from 10) was madeup as a X 10 concentrate and diluted to isoton-

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icity using the HEPES-bicarbonate buffer asabove. pH was corrected to 7.2.

CHEMI CALSChemicals and enzymes used in these experimentswere supplied from the following sources: ethyl-enediaminetetraacetic acid (EDTA BDH Analar);collagenase types C-2139 and C-6885, bovineserum albumin fraction V (Sigma); Worthingtoncolilagenase type CLSPA (Millipore UK); Percoll,Ficoll 400 (Pharmacia); Triosil 440 (Nyegaard;Vestric); all stains (Gurr; GD Searle).

ANTI-T CELL ANTISERUMRabbit anti-mouse T cell serum was preparedby Mr John Shields of this department by twointravenous injections of Balb/c and CBA thy-mocytes followed by a single subcutaneousinjection of 1 ml of a CBA-Balib/c brain homo-genate mixture in complete Freund's adjuvant.The rabbit was bled out two months later, theserum extracted, and heat inactivated for 45minutes at 56°C. The serum was absorbed twiceagainst homogenised mouse liver and onceagainst mouse bone marrow cells. This serum,at a 1: 40 dilution in a cytotoxic assay using 1: 5diluted guinea-pig serum as a source of comp-lement, killed 95% of thymocytes, 2% of bonemarrow cells, and 59% of peripheral lymph nodelymphocytes.

ENUMERATION OF T CELLSThe number of T cells was enumerated by treat-ing cell suspensions with the anti-mouse T cellserum, incubating for 45 minutes at 37°C, andthen adding guinea-pig serum as a source ofcomplement to a final dilution of 1: 10. Afterincubation for 45 minutes at 37'C, the numberof viable cells in the suspension was assessed.

STAINING METHODSStaining was performed on cell suspensions cyto-centrifuged on to slides. Methyl-green pyroninand Leishman staining followed standardmethods. For 18 hour Giemsa staining, slideswere fixed in methanol and stained overnight ina 1: 20 dilution of stock Giemsa in pH6.5 Soren-sen's phosphate buffer. Slides were differentiatedin 0.25% colophonium resin in methylated spiritand then mounted as usual. Astra blue staining:slides were fixed in Carnoy's fluid, dried and rehy-drated then stained for 30 minutes in 0.1% astrablue in 0.7N hydrochloric acid, pH0 3. Afterwashing in 0.7N HCI, slides were lightly counter-stained in safranin, washed, dehydrated, and

mounted. Acid a-naphthyl acetate esterase(ANAE) followed the method of Ranki, Totter-man, and Hayry'0 with counterstaining in 0-1%toluidine blue.

ROSETTING METHODSEnumeration of complement-receptor bearingcells (EAC') by the rosette method was per-formed using standard techniques with a suitabledifution of rabbit anti-sheep red cell haemolysin(Flow Labs.) and a 1: 10 dilution of autologousmouse serum as a source of complement.Rosettes were clarified by lightly staining with0-1% methylene blue.

MEASUREMENT OF CELL VIABILITYCell viability was assessed by the ability of viablecellfs to exclude 0.2% eosin in RPMI.

GLASSWAREAll glassware was thoroughly washed with deter-gent, treated with 1% silicone fluid (MS 1107)in ethyl acetate for 20 seconds, rinsed with dis-tilled water, and baked for one hour at 160°C.

PREPARATION OF CELL SUSPENSIONS1. Mesenteric lymph nodesNodes were removed and disrupted either byforcing through a fine stainless steel screen or byteasing apart with a scalpel blade. After filtrationthrough a small column of sterile glass wool(May & Baker) in a 10 ml syringe, cell sus-pensions were washed twice in medium beforeuse.

2. Lamina propria lymphocytes (see Figure forflow chart of method)

Stage 1 Small intestines were removed fromanimals and handled in batches of three. Theguts were thoroughly washed through with coldCMF using a 50 ml syringe fitted with a shortplastic cannula. This removed all food remainsand much mucus.Thorough washing of the gut is essential, not

only to remove food but also to clarify Peyer'spatches for their subsequent removal. Althougha flush through of 10 ml of CMF per gut issufficient to delineate Peyer's patches, the use of40-5O ml per gut results in less debris being en-countered in later stages. Mucus has not beenfound to be of any problem in the mouse and,although various mucolytic agents such as dithio-erythritol have been added during the initialincubations, they make no difference to the finalyield.

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Preparation andpurification oflymphocytesfrom the epithelium and laminapropria ofmurine small intestine 483

Stage 2 Stage 3

6 Stage 5 Stage 4

liagenase , JRPMI EDTA

tage 8

h Density steps

Fig. Flow diagram of preparation of lamina propria lymphocytes.

Stage 2 The cleaned small intestines were thenplaced on CMF-moistened paper towels and allPeyer's patches, blood vessels, fat, and mesenterywere completely removed.Removal of all macroscopic Peyer's patches

is essential, so that they do not contaminate thefinal preparations. Removal of all adherent fatfrom the intestine improves the final yield andremoval of blood vessels decreases the numberof red blood cells which need to be removed inthe purification stages.

Stage 3 The intestines were then opened longi-tudinally and cut laterally into small pieces(0-5-1 cm). These pieces were well washed inCMF in a 50 ml flask by aspiration of the super-natant and addition of clean CMF.

Separation of the gut into small pieces (about05 cm long) is advisable for maximal cell yieldand, again, good washing of the pieces in CMFis advantageous.

Stage 4 After addition of 25 ml 5 mM EDTAin CMF, the guts were incubated at room tem-perature with a magnetic stirrer at 250 rpm to

remove the epithelium. When the supernatantfluid became sufficiently clouded with cells, itwas removed and fresh EDTA/CMF added(usually every 15 minutes). After about 90 min-utes, no more cells were seen in the supernatantindicating that all the epithelium had beenremoved. This was confirmed by histologicalexamination of the remaining fragments.EDTA/CMF at 25°C has been found to be

optimal. Although it was suggested that 90 min-utes was sufficient to remove all the epithelium,it is necessary to carry on the EDTA treatmentuntil the supernatant is clear of cells. This cansometimes take as long as 120 minutes. The gutpieces can be left in EDTA/CMF for severalhours longer without obvious detriment asjudged by histological examination of the re-maining pieces. However, as no tests on thesubsequent functional capacity of cells havebeen carried out after long incubations inEDTA, it would seem advisable to process thesamples as quickly as possible.

Stage 5 The pieces were then washed withCMF and incubated for 20 minutes with stirring

Stage

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in 25 ml RPMI/NCS to inactivate any remain-ing EDTA.

Stage 6 The remaining fragments were thentransferred to flasks containing 15 ml RPMI/NCS containing collagenase. The amount ofcollagenase used varied for each type; typesC-2139 and C-6885 were used at between 10 unitsand 90 units/ml, and CLSPA at 10 units/ml.The flasks and contents were then incubated at37°C in an orbital incubator (Gallenkamp typeIH 460) for 90 minutes at 180 cycles/minute.For the last 15 minutes of this incubation, theflasks were transferred again to the magneticstirrer to facilitate final disruption of the gutpieces. Use of the orbital incubator alone resultsin poor yields; using the stirrer alone results inmany of the freed cells being killed, hencedecreasing the yield of viable cells after purifica-tion. As an alternative to the magnetic stirrer,the gut pieces can be put into a universal con-tainer and pumped slowly up and down througha 5ml syringe to effect total disruption. Thechoice of collagenase type depends on what thecells are subsequently required for. Type C-2139has been found to be the most efficient at 90U/ml for a maximum yield, although 1OU/mland the use of syringe disruption rather thanmagnetic stirrer produces adequate yields. Thistype of collagenase does have some trypsinactivity, which may affect the expression of sur-face components or cellular function. However,it does not affect cytotoxic T cell activity as

already reported.'1 Type C-6885 used at 10 U/ml produces lower yields of cells but has no

detectable effects on the cells which could beattributed to trypsin. Type CLSPA at 10 U/mlis the least efficient in terms of yield but, as

this enzyme is free from tryptic and proteaseactivity, i's use in some experiments might berequired. The use of type CLSPA at 90 U/mlis not necessary, nor, we consider, financiallywarranted.

Stagje 7 The resultant 'soufp' was then filteredthrough a glass wool column as previously de-scribed and washed twice with RPMI/NCS. Thisprovided a single cell suspension still significantlycontaminated with dead cells and debris whichneeded further purification as described below(stage 8).

3. Intraepithelial lymphocytesThe method used was adapted from a techniquedeveloped by Dr. Allan Mowat (personalcommunication, BSc project, 1975). The smallintestines were prepared initially in the same

way as described for lamina propria cells.After stage 3, the small gut pieces wereput into a 50 ml siliconised flask with 20 mlRPMI/2% NCS (more serum causes foaming)and incubated for 30 minutes at 37°C in a shak-ing water bath. The gut pieces were then trans-ferred to a universal container and shakenvigorously for 15 seconds. The supernatant fluidwas removed and replaced and the shaking pro-cess repeated. When the supernatant fluid wasclear, the gut pieces were incubated in a conicalflask for another 30 minutes at 37°C in theshaking water bath and the original procedurerepeated. All the supernatants were then pooled,centrifuged, resuspended, and filtered throughglass wool and then washed several times. Theresulting cell suspension contained lymphocytes,epithelial cells, and debris and required furtherpurification as described in stage 8 below. Com-ments for stages 1 to 3 above apply also in thiscase. It should be stressed that adequate washingof gut pieces is necessary before incubation at37°C to reduce the amount of debris. At theshaking stage, removal of the supernatant fluidand replacement with fresh medium should berepeated until the supernatant is relatively cell-free as this improves the cell yield.

Stage 8 Purification of intraepithelial and laminapropria cell suspensions: three different methodswere tried:

1. Ficoll-Triosil This method was repeated asdescribed by Davidson and Parish12 and usuallyprovided a reasonable purification of viablemono-nuclear cells, although the yield of cellswas often low. However, if the original prepara-tion was grossly contaminated, then purifica-tion was usually incomplete.

2. Bovine serum albumin (BSA-6) Discon-tinuous steps of 10%, 28%, and 35% w/v BSAwere used. Apart from the usual handling prob-lems of high concentrations of BSA and the factthat the crude material had to be purified beforeuse, pure mononuclear cell suspensions werefound at the 28% /35% BSA interface. Thetechnique suffered the same problems as withFicoll-Triosil, coupled with those due to the highviscosity of BSA solutions.

3. Percoll Stock working solutions of iso-tonic Percoll were made up according to Ulmerand Flad.13 Solutions of density 1-055 g/ml and1085 g/ml were made up from the 1.120 g/mlstock by dilution with RPMI only. Densities werechecked by refractometry at ?0°C with a standardcurve of refractive index (RI) against density.Density steps were made up at 40C in IOX 1.5 cm

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Preparation andpurification oflymphocytes from the epithelium and laminapropria ofmurine small intestine 485

siliconised glass tubes; 1 ml 1.085 g/ml Percoll(RI=13464) was overlaid with 2 ml 1055 g/mlPercoll (RI 13420) then 3 ml RPMI/NCS. Alter-natively, 2 ml 1055 g/ml and 4 ml 1055 g/mlPercoll in a siliconised universal container were

used. T'he cell suspension was resuspended ineither the 1'055 g/ml layer or the overlayingRPMI/NCS. After centrifugation at 600g forabout 20 minutes at 40C (MSE Mistral 2L, swing-out head), highly viable mononuclear cells couldbe recovered from the 1055/1 085 g/ml interface.Dead cells, debris, and epithelial cells were foundat the upper interface and red blood cells in thepellet. The yield of purified cells depends on thenumber of cells loaded on to the Percoll steps andthc contamination of the suspension by debris andepithelial cells. If this is the case, it is better in-itially to resuspend the cells in 1'055 g/ml Percollinstead of in RPMI/NCS. If care is taken through-out the preparation, the yield of viable cells fromthe second Percoll interface can be as high as90% of the number of viable cells put on at thebeginning. In the case of high contaminationof the original suspension, the resultant cellyield at the second interface is reduced, althoughthe purity does remain high. The viabilty of cellstaken from the second interface always exceeds90%.The use of Percoll to purify the cell suspen-

sions is found to be the simplest and mosteconomic method and results in the best yields.

Results

Methods for producing purified lymphoid cellsfrom the epithelial layer and lamina propria ofthe small intestine of the mouse have beendescribed. Although the two techniques aredescribed separately, the two cell suspensions canbe produced from the same group of intestines.If, after isolating the intraepithelial lymphocytes(IEL), suspensions of lamina propria lymphocytes(LPL) are required, the LPL separation tech-nique can be resumed at stage 4, the time for theEDTA incubation being reduced by half.

TIME FOR PREPARATION OF CELLS

Preparation time for intraepithelial and laminapropria lymphocytes from the time of killing theanimals to the end of final purification is threeto four hours and four to five hours respectively.

VARIATION OF CELL YIELD WITH

MOUSE STRAIN AND AGE

Lamina propria cells have been prepared fromC57B1, DBA/2, Balb/c, and CBA/Ca mice. Theredoes not appear to be any difference in the cellyield from any of these strains. Within C57BImice, cell preparations have been made fromanimals varying in age between 6 and 24 weeks.Although all the cell yields were within the rangequoted below, it has been found that mice of 8-12 weeks in age represent a compromise of gutsize and lack of fat deposits both of which affectthe final cell yield. The average cell yield was9 X 106 to 18 X 107 cells per mouse for intra-epithelial lymphocytes and 5X 106 to 16 X 107cells per mouse for lamina propria lymphocytes.

CHARACTERISATION OF PURIFIEDCELLSComposition of cell populationsOn the basis of Leishman staining, IEL and LPLsuspensions have been characterised as shown inTable 1. After purification on Percoll, both cellsuspensions contain at least 90% lymphocytes. Inthe IEL, the contaminating cells are mainlyepithelial while the LPL are slightly contaminatedwith macrophages and eosinophils. The twolymphocyte populations from the gut do howevervary in size. Table 2 shows the size classificationof IEL, LPL, and mesenteric lymph node lympho-

Table 2 Size characteristics of cell populations

Mouse and Lymphocyte size (% of total) No. of cellscell type Small Medium Large counted

C57BI MLN 85 4 9-2 5-4 500C57B1 IEL 86-5 8-0 5-5 2561C57BI LPL 49.7 39 0 11-3 600

Table I Leishman staining characteristics of lymphocytes isolated from small intestine

Mouse and % of total counted* Total no. ofcell type Lymphocytes Macrophages Granulocytes Others cells counted

C57BI IEL 94-2±1.8 1.1±0.5 t 4.7±1.7 2718C57Bl LPL 90.3±37 4.1±2.2 4 0±2.6 1.5±1.1 4500

Others: epithelial cells, mast cells, free nuclei.*Mean ±SD of three separate experiments.tLess than 1 cell in 500.



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486

cytes (MLN) based on their staining character-istics. MLN and IEL are very similar in size,while LPL suspensions contain a larger propor-tion of medium and large lymphocytes.

RNA synthesis of cell populationsTable 3 shows the number of RNA synthesisingcells, assessed by pyronin staining, found in MLN,IEL, and LPL suspensions. Nearly half thelymphocytes isolated from the lamina propria arepyronin positive with only 10% in MLN. IELcontain intermediate numbers of pyronin positivecells to MLN and LPL.

Surface markers on cell populationsData for Thy 1.2 and complement-receptorbearing cells are shown in Table 4. IEL and LPLboth have low numbers of complement-receptorbearing cells compared with MLN. The measure-ments on LPL were made on cells extracted withcollagenase type C-6885 which was not found toalter the levels of receptors found on MLN cellsso treated. MLN and TEL contain similarnumbers of T cells exnressing the Thv 1.2 marker,while LPL suspensions have significantly moreT cells.

Granule containing cellsAfter staining with Giemsa overnight or withAstra blue, some lymphocytes have been found tocontain cytonlasmic granules. These cells haveotherwise typical lymnhoid morpholotw and donot resemble mast cells. IEL suspensions havebeen found to contain 36.2+4.2% of cellscontaining such granules. However, no granulecontaining cells have been found in MLN or LPL

Table 3 Methyl-green pyronin stainingcharacteristics of cell populations

Mouse and Pyronin Total no. ofcell type positive cells(%) cells counted

C57RIMLN 10 1000C57B1 TEL 19.7 2000C57B1 LPL 42-4 4070

Table 4 Surface markers on cell populations

Mouse and EAC' positive* Thv 1.2 positive*cell tvpe (%) (%)

C57BIMLN 19.1±55 60.8+9.4C57B1 TEL 2.9±0-6 65-2±8.1C57BI LPL 6.6+2.1 799±6.7

*Mean±SD of four to six separate experiments. At least 2090 cellcounted in total.

Davies and Parrott

suspensions after the examination of at least10 000 cells.

Acid a-naphthyl acetate esterase (ANAE) stainingPunctate staining of cells with ANAE positivematerial is a marker for resting T cells in themouse.10 We have stained MLN, IEL, and LPLsuspensions many times with ANAE and havefound the results unreliable. As the number ofactivated T cells in the guts of normal animalsvaries, the ANAE staining is probably of littlevalue. However, initial data suggest that MLN,IEL, and LPL all contain around 40% ANAEpositive cells.

Discussion

Much of our knowledge of the gut mucosaleffector system has stemmed from experiments on

mesenteric lymph node and Peyer's patch lympho-cytes. However, to understand mucosal effectorfunctions clearly, we must examine lymphocytesfrom within the gut itself. The methods describedin this paper provide the means to examine suchfunctions. We have already used such methods to

show that cytotoxic T cell capacities in mouse

mesenteric lymph nodes, Peyer's patches, andlamina propria vary markedly."The majority of lymphocyte preparation tech-

niques from the gut, both in humans and inexperimental animals, produce suspensions of'mucosal lymphocytes' which are clearly a

mixture of IEL and LPL.1 2 5 In view of our

observationsa that cvtotoxic T cell activities inIEL and LPL vary, it is necessary that we shouldexamine the two populations separately. We haveseveral reasons for believing that we have isolatedtwo different populations. Firstly, the preparationtechnique for IEL does not disrupt the laminapropria as assessed by histological examination.Secondly, the EDTA treatment used in the LPLpreparation completely removes identifiable epi-thelium. Thirdly, it appears that lymphocytes con-

taining cytoplasmic granules are a marker for TELand such cells have never been found in our LPLpreparations. Fourthly, the cytotoxic T cellexperiments already mentioned lead us to believethat both TEL and LPL represent differentpopulations or a single population at verydifferent stages of differentiation.The preparation technique for IEL is

particularly amenable for the subsequent analysisof effector cell function, as it does not require the



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eparation andpurification oflymphocytesfrom the epithelium and laminapropria ofmurine small intestine 487

use of enzymes or dissociation media. We havetried preparing LPL by a mechanical techniqueto obviate the use of enzymes. After EDTAtreatment, the gut pieces were homogenised in aloose-fitting glass homogeniser. However, inagreement with the observations of Bland et al.,3we found cell clumping to be a major problemand thus cell yields were low. The addition ofindomethacin to the medium was found todecrease clumping but this agent is known toalter many cellular functions including cytotoxicactivity14 and its use should be avoided.To our knowledge, there has only been one

published study on mouse mucosal lymphocytes,that of Guy-Grand et al.'5 on IEL. Theirpreparation technique is less rigorous than ours,which explains the lower yields, and the scantdetails given of the purification technique do notallow much further comparison. Our experienceof the Ficoll-Triosil gradients as used by themhave not been favourable and cell suspensionsare often grossly contaminated. Their datasuggest that there are equal numbers of T cells inthe epithelium and lamina pronria amounting to80-90% of the total number of lymohocvtes; ourdata would suggest that there are slightly fewerT cells in the epithelial layer. We do confirm thedata on the number of granulated cells found inthe epithelium but not the size profile of TEL.The Drofile that Guy-Grand et al. give for thesize of TEL is similar to that which we have foundfor LPL, whereas we find that IEL are rathersmaller cells. Size classifications done on thehnsis of stained cell nrenrations. however. arerather subjective and thus it is difficult tocomnare exoeriments done bv different workers.

Neither LPL nor TEL contain sipnificantnumbers of cells bearing recentors for the C3hcomponent of comolement. This is sunnorted bythe observation that C3b recentor bearing I'-nositive B lymnhoblasts from mesenteric lvmnhnodes do not home to the gut in siRnificantnumbers."' The data shown in Table 4, along withthe observation of Guv-Grand et al. that thereare very few surface Tg bearing cells in IEL,would suggest that there are some 20% of cellsin IEL without normal surface markers-'nullcells'.The preparation of human gut mucosal lympho-

cytes has been utilised to measure cell-mediatedreactions ecneciallv in natients with bowel disease.The data on the cytotoxic capacity of gut lympho-cytes from such patients are very variable3 17 andprobably a consequence of the assortment ofpreparation techniques used. Some of the

enzymes used can alter the cytotoxic potentialexpressed by cells,'8 while mechanical disruptionof mucosal tissue induces the release of prosta-glandins which can also alter cytotoxic expres-sion.'9 Using an animal model of gut lymphocytepreparation, we can examine the effects ofdifferent preparation techniques on cell functionand explore some of the properties of the cells ina system which is more open to experimentation.In particular, we can now examine the generationof cytotoxicity in LPL and IEL against gutepithelial cells.

It is a pleasure to acknowledge the experttechnical assistance of Mrs Sheila MacKenzie.This work was funded by the MRC, grant numberG977/623/S.

References

'Clancy R. Isolation and kinetic characteristics ofmucosal lymphocytes in Crohn's disease. Gastro-enterology 1976; 70:177-80.2Goodacre R, Davidson R, Singal D, Bienenstock J.Morphologic and functional characteristics ofhuman intestinal lymphoid cells isolated by amechanical technique. Gastroenterology 1979; 76:300-8.

3Bland PW, Richens ER, Britton DC, Lloyd JV.Isolation and purification of human large bowelmucosal lymphoid cells: effect of separation tech-nique on functional characteristics. Gut 1979; 20:1037-46.

4Bull DM, Bookman MA. Isolation and functionalcharacterisation of human intestinal mucosallympoid cells. J Clin Invest 1977; 59:966-74.

5Crofton RW, Cochran C. McClelland DBL. Pre-paration of lympoid cells from small specimens ofhuman gastro-intestinal mucosa. Gut 1978; 19:898-906.6Rudzik 0, Bienenstock J. Isolation and character-istics of gut mucosal lymphocytes. Lab Invest 1974;30:260-6.7Castro GA, Roy SA, Stockstill RD. Trichinellaspiralis: peroxidase activity in isolated cells from therat intestine. Exp Parasitol 1974; 36:307-15.8Arnaud-Battandier F, Bundy BM, O'Neill M,Bienenstock J, Nelson DL. Cytotoxic activities ofgut mucosal lymphoid cells in guinea pigs. JImmunol 1978; 121:1059-65.

saDavies MDJ, Parrott D.MV. Cytotoxic T cells insmall intestine epithelial, lamina propria, and lunglymphocytes. Immunology 1981 (in press).9Hanks JH, Wallace RE. Relation of oxygen andtemperature in the preservation of tissue by re-frigeration. Proc Soc Exp Biol Med 1949; 71:196-200.

'0Ranki A, T6tterman TH, Hiyry P. Identificationof mouse T and B lymphocytes from cytocentrifugedcell smears. Clin Exp Immunol 1976; 26:632-40.



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"Davies MDJ, Parrott DMV. The early appearanceof specific cytotoxic T cells in murine gut mucosa.Clin Exp Immunol 1980; 42:273-9.

"Davidson WF, Parish CR. A procedure for remov-ing red cells and dead cells from lymphoid cellsuspensions. J Immunol Methods 1975; 7:291-300.

"Ulmer AJ, Flad H-D. Discontinuous densitygradient separation of human mononuclear leuco-cytes using Percoll as gradient medium. J ImmunolMethods 1979; 30:1-10.

t4Droller MJ, Perlmann P, Schneider MU. Enhance-ment of natural and antibody-dependent lymphocytecytatoxicity by drugs which inhibit prostaglandinproduction by tumour target cells. Cell Immunol1978; 39:154-64.

t5Guy-Grand D, Griscelli C, Vassalli P. The mouse

gut T lymphocyte, a novel type of T cell. J ExpMed 1978; 148:1661-77.

16McWilliams M, Phillips-Quagliata JM, Lamm ME.Characteristics of mesenteric lymph node cellshoming to gut-associated lymphoid tissue insyngeneic mice. J Immunol 1975; 115:54-8.

'7Clancy R, Pucci A. Absence of K cells in humangut mucosa. Gut 1978; 19:273-6.

lsKedar E, Ortiz De Landazuri M, Fahey JL.Enzymatic enhancement of cell-mediated cyto-toxicity and antibody-dependent cell cytotoxicity.J Immunol 1974; 112:26-36.

'9Droller MJ, Schneider MU, Perlmann P. A possiblerole of prostaglandins in the inhibition of naturaland antibody-dependent cell-mediated cytotoxicityagainst tumour cells. Cell Immunol 1978; 39:165-77.



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