A method for isolation and culture of lymphocytes from endoscopic biopsies

Journal of Immunological Methods, 54 (1982) 55~63 Elsevier Biomedical Press

55

A Method for Isolation and Culture of Lymphocytes from Endoscopic Biopsies H.A. Brown, J. Douglas, C.B. W i l l i a m s a n d J.A. W a l k e r - S m i t h Academic Department of Child Health, Queen Elizabeth Hospital for Chddren, London E2, and St. Bartholomew's Hospital, London ECI, U.K.

(Received 26 November 1981. accepted 10 March 1982)

Human small and large intestinal lamina propria lymphocytes have been successfully prepared from endoscopic biopsies by a combined enzymatic and mechanical method which gives higher yields of viable mucosal lymphocytes than previously reported, despite the small size of the biopsy samples. Viability of the cells was demonstrated by dye exclusion and they could be satisfactorily maintained in short-term culture. Phytohaemagglutinin-P (PHA-P) transformation characteristics of intestinal lymphoid cells and those of peripheral blood were studied in 20 patients with Crohn's disease and 10 control subjects. Peripheral blood lymphocytes were separated according to this technique, no decrease in viability being observed when compared to a standard FicolI-Hypaque gradient technique. Endoscopically abnormal (EA) and endoscopically normal (EN) Crohn's tissue showed significantly different responses to PItA-P ( P <0.001 ), EA tissue lymphocytes giving lower blastogenic responses.

Key words: lamina propria lymphocytes -- ('rohn) disease btop.~y - . mucosal biopa3'

Introduction Within the past few years many reports have appeared on the isolation and characteristics of intestinal mucosal lymphocytes (Nind et al., 1973; Breucha et al., 1974; Rudzik and Bienenstock, 1974; Clancy, 1976; Bookman and Bull, 1979; Croften et al., 1978; Goodacre et al., 1979). A number of studies relating to cell-mediated immunity have been reported in patients with Crohn's disease. Several have recorded impaired cutaneous delayed hypersensitivity (Williams, 1965; Jones et al., 1969) but there have been conflicting reports concerning in vitro peripheral blood lymphocyte responsiveness to non-specific mitogens such as phytohaemagglutinin (PHA). Some report depressed in vitro response (Parent et al., 1971; Guillou et al., 1973; Sachar et al., 1973; Strickland et al., 1974), while others have shown no significant changes (Asquith et al., 1973; Bird and Britton 1974). Depressed responses have also been observed in mixed lymphocyte culture (Richens et al., 1974). The purpose of this paper is to assess whether a combination or modification of published techniques yields higher numbers of lymphocytes per mg of tissue and to 0022-1759/0000-0000/82/$02.75 ~.'~1982 Elsevier Biomedical Press

5~ report changes in the response to the mitogcn PIIA-P of mucosal lymphocytes isolated from endoscopically abnormal (t~A) ('rohn's, cndoscopically normal (I:,N) ('rohn's and control mucosa, as examples.

Methods and Materials

Sutyect.v Nineteen Crohn's patients were studied (mean age 16.6 years, range 6 months--30 .,,ears, 11 females and 8 males). The diagnosis of Crohn's colitis was established in 11. The disease involved both the colon and the ileum in 7. and was confined to the terminal ileum in one patient. Tissue was obtained from 2 of the patients at surgery, biopsy specimens being obtained from the rest at endoscopy. Twelve subjects not suffering from inflammatory bowel disease (IBD) served as controls (mean age 12.2 years, range 3 25 years, 5 females and 7 males), llistologically normal tissue sections were obtained from 10 of this group, after prior endoscopy for suspected IBD, diagnosis having been endoscopically and radiologically excluded. One patient had tissue obtained at surgery (Meckel's diverticulum of the ileum). |:,leven of the patient group and all the controls provided blood samples.

('ell isolation The mucosa and submucosa were dissected free of the muscle layer (surgical specimens) with a fine razor blade and dissecting microscope, washed thoroughly in i lanks" balanced salt solution (HBSS: Gibco Europe) to remove blood and debris and chopped into very fine fragments. The tissue was then weighed (wet weight). After dissection the tissue was washed in HBSS. supplemented with heat-inactivated pooled AB positive serum (Blood Transfusion ('entre, Brentwood. Essex l. gentamicin 50 p,g. ml and penicillin 100 U / m l (Flo'~v Laboratories. Scotland). It ~as incubated at room temperature for 15 rain, in IIBSS supplemented with 10c~" heat-inactivated foetal calf serum (F('S: Gibco Eur~pe) and containing I mM dithiothreitol (Sigma). This step successfully removed adheren! surface mucus. l'~pithelial cells were then removed b~, incubating the tissue, with stirring, at 370( ` for 90 rain in 10 ml of 0.75 mM EDTA in calcium and magnesium free HBSS, supplemented with 10% FCS. The tissue was left to soak overnight at 40( ` in HBSS containing collagenase 0.5 mg,/ml (Type 1: Sigma) and hyaluronidase I m g / m l (Type 1: Sigma). Antibiotics (100 U,/ml penicillin and 100 g g / m l gentamicin) and 20% FCS were also added. The enzyme/tissue suspension was incubated for 40 rain at 37°( ` in a water bath. to dissociate the lamina propria. Subsequently, the suspension was treated with 3 ml of 0.2% NaCI for 30 sec to lyse remaining erythrocytes, followed by the immediate addition of an equal volume of 1.6% NaCI to restore isotonicity. The lymphocytes were then counted in a Ncubauer chamber. The cells were washed 6 times in RPMI 1640 {Gibco Bio-('ult, Scotland) supplemented with 20% FCS and additional antibiotics as above. This inactivated an5 remaining ED'FA. The proportion of Ivmphocvtes were again assessed. The cell

57

suspension was then passed over a series of siliconizcd stainless steel scrcens of decreasing mesh size (18, 60, 100). The screens had previously been sterilized and then washed in phosphate-buffered saline (PBS; Oxoid), containing 50 p~g/ml gentamicin and 25% FCS. The addition of this step to the procedure significantly reduced the amount of contamination by dead cells and debris and increased the purification of viable lymphoid elements in the suspension. The next stage involved layering the cell suspension on an isokinetic gradient of Ficoll, the gradient being constructed exactly according to Bland et al. (1979), and the suspension spun at 450 r e v / m i n for 14 rain at 4°C. in an MSE Coolspin centrifuge. The 3 ml cell suspension placed on the gradient contained only platelets after centrifugation and was discarded. The next 3 × 5 ml fractions were retained and pooled. Cell viability and counts were carried out on the lymphocytes recovered in this pooled fraction. Recovered cells were washed twice in supplemented RPMI 1640 before inclusion in an in vitro transformation assay.

Viability and cell counting Viability of the cells was assessed by 0.4% trypan blue exclusion and the lymphocytes counted. A smear of the final cell suspension on slides was air dried. fixed in absolute methanol for 3 min and then stained with Leishman's stain. A differential cell count was performcd. Monocytes and macrophagcs were identified on Wright's stained preparations by their histological characteristics.

Isolation of peripheral blood lympho¢3,tes Peripheral blood samples were collected in heparinized tubes from 23 of the subjects and patients at the time of gut tissue collection. The blood was separated over Ficoll-Hypaque (B6yum, 1968), and the buffy coat layer of cells washed twice in HBSS. The cells were then included in the transformation assay. Experiments were also conducted in which the peripheral blood lymphocytes were separated according to the intestinal lymphocyte procedure, to negate any differences in mitogenic response due to separation methods used.

Thymidine assay Lymphocyte cell suspensions were adjusted to a concentration of 5 × 10 -~cells/ml in RPMI 1640 containing Hepes buffer. Phytohaemagglutinin-P (Difco Laboratories) was added to triplicate cultures of the cells in stoppered polypropylene tubes. The proliferative response of the cells was assessed by constructing a dose response curve, using PHA-P concentrations of 12, 6, 3, 1.50, 0.75, 0.37, 0.18 and 0.09 m g / m l . Control tubes without PHA-P were included. The tubes were incubated at 37°C for 72 h and I #Ci of tritiated thymidine (spec. act. 20 p,C i / m m o l ; Radiochemical Centre, Amersham) was added to each tube 18 h prior to termination. D N A was extracted and its activity estimated according to Dent (1971). The results were expressed as cpm per tube corrected for background and the results subjected to statistical analysis by the Student's t-test modified for small numbers. When sufficient mucosal lymphocytes were isolated, an E rosette assay was performed to ascertain the proportion of T-cells present,

58

Results

Cell yield and morphological characteristics The number of lamina propria lymphocytes recovered gives an indication of the efficiency of the method. After treatment, the final recovery of cells/rag of mucosal tissue, for the control, EN and EA Crohn's tissue groups was 2.6, 14.2 and 22.9 × 10 s respectively. More than 95% of the final cells recovered were judged viable by trypan bluc exclusion. Surgical specimens gave higher yields of lymphocytes per mg of mucosal tissue than did the biopsy specimens, at all stages of the isolation procedure. With the biopsy samples, higher yields of cells were obtained from Crohn's disease patients than from control tissue. EN Crohn's tissue gave lower yields than EA Crohn's tissue (see Tables I and II). The morphological characteristics of the intestinal cells obtained in the final cell suspension are shown in Table lIl. The majority of the cells were small lymphocytes. Typical plasma cells constituted less than 4% of the final cell suspension. Crohn's EA colonic cell populations showed an increase in the proportion of macrophagcs. while Crohn's EA ileal cell populations showed increases in both macrophages and eosinophils with a decreased percentage of lymphocytes. Proliferative response to P H A - P The optimal dose for blastogenesis with the isolated intestinal lymphocyte cell populations was 750 ~ g / m l and for the peripheral cell population was 190 p.g,/ml.

TABLE

I

M E A N F I N A L Y I E L D O F L Y M P H O C Y T E S , , mg O F I N T E S T I N A l . M U C O S A F i s s u e group

M e a n wet weight of b i o p s y s a m p l e (mg) `~

Control n:lOC n-71 E n d o s c o p i c a l l y n o r m a l tissue ( E N ) " n=10C n=-0 I

Mean ~ recovcr~, ,..

(,,t,

I~

C

I

1.5 ~1.0 1.7)

79

76

_

2.6 (2.1- 2.9) 2.1( 1.0 -- 3. I )

E n d o s c o p i c a l l y a b n o r m a l tissue (F.A) " n=:14 C n:8 I " t, c '~ ¢

Mean final cell yield ( × 105/rag mucosal tissue)

14.2 ( 10.0 -24.1 ) 22.9 (8.6-39.0)

_

_

86

11.5 ~7.2--26.11

~,

M e a n weight of the surgical s a m p l e s (n = 2) in the EA tissue group ~ 1550 nag (1000-2050). C = colon. I =ileum. EN = e n d o s c o p i c a l l y n o r m a l tissue. EA ~- endoscopically a b n o r m a l tissue.

85

73

59 T A B L E !I M E A N CELL YIELD A N D RECOVERY T H R O U G H O U T THE ISOLATION P R O C E D U R E COLON

After enzyme treatment After passage over stainless steel screens After Ficoll gradient % Recovery

ILEUM

C" ( n = 10)

EN h ( n = 10)

EA c ( n = 14)

C (n-7)

EA (n :=8)

3.2 ( + 1.8) 2.8 (-'2.7) 2.6 ( -- 1.5) 79

16.5 ( - 6.2) 15.1 ( ± I1.0) 14.2 ( .+ 8.3) 86

26.9 ( - 8.0) 24.2 (-'. 8.6) 22.9 ( ~2.5) 85

1.9 ( - 1.0) 1.6 (~0.2) 1.5 ( ~0.3t 76

15.7 "- 1.4) 12.9 ~8.5) 11.5 ("- 1.41 73

~. 1 standard deviation of mean. " C = control tissue. h EN =endoscopically normal tissue. ~ EA = endoscopically abnormal tissue.

Fig. 1 shows response.

the results for the intestinal

The

peripheral

greater than the responses No

valid difference

either isolation significantly groups.

blood

increased

response

plotted

at the peak dose

was significantly

(P < 0.001)

of either small or large intestinal iymphocytes

in r e s p o n s e

procedure

cell p o p u l a t i o n s

lymphocyte

was observed

with peripheral

( F i g . 2). C e l l s i s o l a t e d

response

Cells from Crohn's

to the mitogen

from

blood

the control

compared

(Table

group

by

showed

with cells from

EA tissue gave a smaller response

IV).

separated

a

patient

(P < 0.001) than cells

T A B L E Ill D I F F E R E N T I A L C O U N T S OF T H E ISOLATED INTESTINAL CELLS COLON

No. of samples Mean cell yield ( × 105/mg tissue) Plasma cells Lymphocytes Macrophages Eosinophils Basophils or mast cells

ILEUM

Na

EN ~

EA "

N

EA

10 2.6 3+ 1 81 ~-3 8'- I 5 ~- 2 3= 1

10 14.2 4-'-2 82-'3 8÷ 1 4± 1 2~ 1

14 22.9 1 -- 1 81 + 4 12 ~-2 zl± 2 2 4_ 1

7 1.5 4:~ 3 84.--3 7--- 1 3 4_ I 2± I

8 11.5 2 -~ I 68 ~ 5 14 '-2 13 ~ 2 3 '- I

'- I standard deviation. " N =control tissue, Crohn's disease having been radiologically, histologically and endoscopically excluded. ~' EN -- endoscopically normal tissue. " EA -- endoscopically abnormal tissue.

6t)

C.P.M. x 10 -3 30

•

.

20

•

•

~

•

;.~

I

10

i°

I

control

EN

EA

I

I

control

COLON

EA

ILEUM

Fig. I. Proliferati'.c r e s p o n s e of intestinal l y m p h ~ : y t e s to P H A - F ' (750 p . g / m l ) . EN, cndoscopicall'~ n o r m a l tissue; t:.A. e n d o s c o p i c a l l y a b n o r m a l tissue; no F,N ileal tissue w a s available. T r i a n g l e s r e p r e s e n t surgical s p e c i m e n s a n d s q u a r e s r e p r e s e n t those s p e c i m e n s t a k e n f r o m p a t i e n t s reccb.'ing a z a t h i o p r i n e . Bars t h r o u g h e a c h p o i n t r e p r e s e n t the s t a n d a r d e r r o r of the m e a n .

T A B L E IV M F . A N IN V I ' F R O U P T A K E O F T R I ' I I A T E I ) T H Y M I I ) I N E Tissue group

Unstimulated cpm

( ' o n t r o l s ( n - 17) Endoscopically normal ~n-IO) Endoscopically abnormal (n - 221

BY I . Y M P H O ( ' Y T E S PHA~

cpm

I "

I'B ~'

I

PB

I 711 ' 164 ~

I 292 • 192

2 6 8 9 6 " 4,52

64023'

12274

379"

63

745 • 89

2 1 8 0 0 " 6,53

57 73~

6 ~45

501 '

69

745 " 89

13(184 ' 427

57 738

6 845

" l -- intestine ~ PB- peripheral blood. • S t a n d a r d e r r o r of the m e a n .

"I'ABt, E V SPON'IANEOUS

E-ROSETTE FORMATION

W I T H S t l E E P ERY'I H R O ( ' Y ' I ES

Tissue g r o u p ('ontrols E n d o s c o p i c a l l y n o r m a l C r o h n ' s tissue E n d o s c o p i c a l l y a b n o r m a l C r o h n ' s tissue

% Rosettes (n - 17) ( n - 10) (n 2 2 ~

45(40-48) 6 ~ 5 6 69) 72(67-751

61 70

60

T C.P.M. x 10 3

?

T |

50

T

4o

30

r 20 10

~.

0 0

1:2

1:4

1:8

1:16

1:32

1:64

1:128

PHA-P dilution

Fig. 2. Comparison of the isolation of peripheral blood lyrnphocytcs by both the Ficoll-Hypaque gradient technique and the described intestinal cell procedure. • • , Ficoll-Ilypaque gradient: © O. intestinal cell procedure. Bars through each point represent one standard deviation around the mean.

from Crohn's EN tissue (Fig. 1). This is a valid difference since isolation procedure was shown not to affect cell response significantly (Fig. 2). The number of E-rosette forming cells is shown in Table V.

Discussion

A technique is described which combines features of previously published methods for isolation of viable lymphocytes from human small and large intestinal mucosal biopsies. The development of a method to give high yields is an essential preliminary step for evaluation of local cellular immune mechanisms in these organs. The combination of enzymatic and mechanical methods described gives high yields of mucosal lymphocytes, the mean final cell yields being 2,6, 14.2 and 22.9 × 10 -~ cells/mg tissue for control, EN and EA tissue groups respectively, despite the small size of the biopsies (mean size 2.5 mm × 2 mm). Cell yields of this magnitude have not been reported previously with such small samples. There was considerable variation in the range of cell recovery which may be explained in 3 ways. First, the extent of lymphoid cell infiltration may vary between mucosal samples. Second, the effectiveness of the enzymes in releasing cells from the lamina propria may vary for methodological reasons; and third, during preparation of suspensions of lymphoid cells from human mucosa there may be selective enrichment of certain cell types, yielding a final preparation unrepresentative of the lymphoid cell population in the

62 mucosa. Several features of published methods were found to affect cell yield considerably. Croften et al. (1978) found that leaving the enzyme/tissue suspension at 4°C overnight gave much better cell preparations and yields, presumably due to better penetration of the enzymes. There was little apparent loss of viabilit\, in lhc recovered cells. We found that incubation of the tissue for 40 rain in the enzymes gave much better results than incubation for 30 min. We also found that the action of I';DTA on the biopsy samples was not IO(Y} effective. Contamination of the cell suspensions with epithelial cells was quite apparent during initial experiments, even after many washes with EDTA. We thought that an enzyme with similar effects to ED'I'A on epithelial cells might help to reduce this contamination. Since hyaluronidase has been used by ('roften et al. (1978) in preference to ED'FA, this was the obvious choice. Combining EI)'FA and hyaluronidase completely removed all traces of epithelial cells. The Ficoll gradient of Bland et al. (1979) was used in the final stage of the isolation procedure. We noted that the longer the gradients had been made before use, the less effective was the separation of the lymphocytes. Use immediately after preparation avoided this. The greater cell yield obtained from Crohn's disease abnormal mucosae reflects the presence of the inflammatory cell infiltrate characteristic of the disease. ('hanges in the absolute numbers of cells, however, have to be distinguished from changes in proportions of different subpopulations. Thus, while the proportion of lymphocytes is reduced in Crohn's involved mucosae, the absolute number of lymphocytes per unit area is increased. The cell population obtained from Crohn's diseased tissue differed to that from control mucosa in the presence of cells with morphology ranging from medium sized lymphocytes to plasmablasts. Skinner and Whitehead (1974) using fixed tissue sections reported similar morphological data. l_,arge numbers of plasma cells can normally be se¢:n in fixed tissue sections, so it was surprising to see so few in the final cell suspensions. Rudzik and Bienenstock (1974) and Goodacre et al. (1979), however, obtained similar low yields of plasma cells. This may have been due to selective depletion during isolation. It also proved possible to maintain isolated lymphocytes in short-term culture, as demonstrated by our PtIA-P results. The difference observed between maximal dose response of intestinal and peripheral lymphocytes to PHA-P, may be due to cells isolated from intestinal tissue being less active or responsive, or alternatively being subject to regulation or suppressive influences persisting in the final cell suspensions. The difference in response to PHA-P between Crohn's EA and Crohn's EN cells suggests a local abnormality of T-cell function or dysfunction of a T-cell subpopulation in the pathogenesis of the disease. Fluctuation in subsets of T-cells may significantly influence immunological expression, especially if the decrease occurs within one subset (Dopp et al., 1980). However, since Crohn's EN cells respond significantly less than cells from controls, the results suggest a diffuse lesion of the gastrointestinal tract, in accord with Dunne et al. (1977), who described morphometric abnormalities and enzyme deficiency in histologically normal ('rohn's tissue. Another partial explanation may relate to the fact that lymphocytes were obtained from 3 of the patients in the study after surgical resection and that 3 of the patients had been having azathioprine treatment prior to endoscopy. Park et al.

63 (1971) h a v e s h o w n that d e p r e s s i o n of l y m p h o c y t e r e s p o n s e s to P H A - P s t i m u l a t i o n m a y o c c u r d u r i n g the first w e e k o r m o r e a f t e r s u r g e r y u n d e r g e n e r a l a n a e s t h e s i a . H o w e v e r , this d o e s not a c c o u n t for the s i m i l a r results with e n d o s c o p y s p e c i m e n s w h e r e the o n l y m e d i c a t i o n was p e t h i d i n e a n d d i a z e p a m . W e d e m o n s t r a t e d the a b i l i t y o f the T-cells to f o r m rosettes w i t h s h e e p c r y t h r o c y t e s ( E R ) as p r e v i o u s l y n o t e d by D o p p ct al. (1980) a n d B r e u c h a a n d R c i t h m u l l e r (1975), w h o u t i l i z e d l y m p h o c y t e s u s p e n s i o n s f r o m r e s e c t e d i l e u m a f f e c t e d by C r o h n ' s disease. F u r t h e r w o r k w i t h m o n o c l o n a l a n t i b o d i e s to T-cell subsets will be f a c i l i t a t e d by the use of this m e t h o d o f p r e p a r i n g high n u m b e r s of l y m p h o c y t e s f r o m m u c o s a l biopsies.

Acknowledgements W e are g r a t e f u l to M i s s V. W r i g h t a n d Mr. I. T o d d w h o p r o v i d e d us w i t h the surgical s p e c i m e n s . W e also t h a n k Miss P. R o b e r t s o n for t y p i n g o f the m a n u s c r i p t a n d Mr. P. H i n d o c h a for t e c h n i c a l assistance. T h e w o r k was s u p p o r t e d by a g r a n t f r o m the Q u e e n E l i z a b e t h H o s p i t a l for C h i l d r e n R e s e a r c h A p p e a l Trust.

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