Autoimmunity, 1992, Vol. 13, pp. 233-241 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
LFA-1 AND ICAM-1 MOLECULE EXPRESSION IN JEJUNAL MUCOSA FROM CHILDREN WITH AUTOIMMUNE ENTEROPATHY SUSAN HILL*, PETER J. MILLA*, ANNA CIAMPOLILLO, GIORGIO NAPOLITANO, GIAN F R A N C 0 BOTTAZZO and RITA MIRAKIAN *Department of Child Health, Institute of Child Health, London WCI IEH, and Department of Immunology, The Royal London Hospital Medical College, London E l 2AD
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(Received November 21,1991; in final form May 27.1992) The expression of adhesion molecules by cells of the small intestinal mucosa was compared in gut biopsies from children with autoimmune small intestinal enteropathy and normal controls and related to HLA-DR expression by the same tissue. Jejunal biopsies were stained by IFL with monoclonal antibodies to LFA-I (TS1/22 and CDlla/25.3.I) and ICAM-1 (RRI/I and 84H10) molecules. LFA-I and ICAM-I positive cells were observed in the lamina propria in all cases and the counts were increased in autoimmune enteropathy compared with controls. In addition, in 4 of 7 cases of autoimmune enteropathy crypt enterocytes were positives for ICAM-I when stained with RRI/I and 3 of the 4 were also positive for LFA-I when stained with both LFA-I reagents. We speculate on the role of adhesion molecule expression in autoimmune enteropathy.
KEY WORDS: Autoimmune enteropathy, ICAM-I, LFA-I, HLA-DR, adhesion molecule.
LFA- 1 binds to its counter receptors, the Intercellular Adhesion Molecules (ICAM-1 and 2)15-'*which are physiologically expressed on endothelial and APCs but generally not on resting T cells or on epithelial ceIIs'6,'y. The LFA-l/ICAM-1 interaction is an early event in the process of T cell activation and plays a critical role in effector T cell activation'".". Cytokines, such as IFNy alone and/or in combination with IL-1 and T N F a can induce/enhance ICAM-1 expression in v i t u ~ ' ~ -in~ the ' , same way as HLA Class I1 molecule induction has been already demonstrated22 (reviewed in Ref. 23). Cytokine production has been recently confirmed in vivo near sites of active mononuclear infiltration in tissues affected by autoimmune diseases and by epithelial structures
INTRODUCTION Inappropriate expression of HLA Class I1 products has been detected in crypt enterocytes from children affected by autoimmune enteropathy',2.Similar observations have been made previously in the intestinal epithelium derived from patients with inflammatory bowel disease (IBD) (reviewed in Refs 3 and 4), coeliac disease5 and in gland epithelium from patients with classical autoimmune disorders (reviewed in Ref. 6). These findings have constituted an important step in understanding target cell autorecognition and destruction by T activated lymphocytes in autoimmune disorders. Epithelial cells which express de novo HLA Class I1 molecules become capable of directly operating as antigen presenting cells (APC) to autologous T helper/inducer CD4+ve lymphocyte subsets without macrophage participation (reviewed in Refs 6, 7 and 8). However, additional prerequisites for effective antigen presentation include the expression on the cell surface of "accessory" molecules which enhance cell contact and adhesion9-". One of these molecules is the Lymphocyte Function Associated Antigen (LFA- l ) , a heterodimer, expressed on the surface of lymphocytes, monocytes and some APCs'"'' (reviewed in Ref. 14).
The aim of this study was to investigate the possible upregulation of adhesion molecules on cells of the intestinal mucosa affected by autoimmune enteropathy and colitis2*. It was also important to compare this expression with the aberrant expression of Class I1 molecules on enterocytes, and with the expression of activation molecules on morphologically and histologically normal jejunal mucosa. PATIENTS
Correspondence to: Dr R. Mirakian, Department of Immunology, The Royal London Hospital Medical College, 56-76 Ashfield Street, London El 2AD.
Two groups of patients were selected: 7 children with autoimmune enteropathy, and a control group of 5
233
234
S. HILL ET AL
Table 1 Autoimmune enteropathy: clinical and morphological features. PIS
A'v
Ses
AutoAbs
Jejitnal biopsy
Treatment
I
2 Yr
M
+
None
2
8 Yr
F
1/64
3
1 Yr
M
11128
4
l m
M
I18
5
3 yr
F
1/64
6
S yr
M
11128
I
15 m
M
I18
Mild PVA LP: normal infiltrate PVA LP: heavy infiltrate Severe PVA LP: heavy infiltrate PVA LP: moderate infiltrate Very mild PVA LP: mild infiltrate Severe PVA LP: moderate infiltrate PVA LP: moderate infiltrate
Azathioprine Prednisolone ALathioprine None Prednisolone Azathioprine Prednisolone Prednisolone
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* P V A : partial v~llou?~ atrophy: LP: i a r n i n ~proprra. yr.;. years' m. rnonrhs.
children with histologically normal small intestinal mucosa. The 7 children with autoimmune enteropathy were aged from 9 months to 8 years (median 2 years). Five were males and 2 females. They had all presented with chronic diarrhoea and failure to thrive, had had circulating enterocyte antibodies (EcAbs). on at least two occasions and an enteropathy of variable severity (Table 1). Five of the 7 were on treatment with prednisolone when studied. The 5 control children were aged from 1 to 10 years (median 4 years). Two were male and 3 female. They all underwent small intestinal biopsy for diagnostic purposes and had histologically normal mucosa. Two had presented with persistent diarrhoea and failure to thrive (in one a diagnosis of cystic fibrosis was made and in the other congenital chloridorrhoea), 1 with abdominal distension (in whom aerophagy was diagnosed) and 2 with loose stool (in whom no intestinal abnormality was detected). No Abs were observed in the control sera.
METHODS Informed parental consent was obtained before small intestinal biopsies were taken from the duodenojejunal flexure with a double port Watson Paediatric capsule. One of the two specimens was fixed in formalin and sections were stained with haematoxylineosin for histological analysis. The other sample was snap frozen and stored at -70°C until 4 ,U cryostat sections were cut, fixed for 5 min in cold acetone and stained using an indirect immunofluorescence technique (IFL). The list of monoclonal antibodies (MoAbs) used is reported in Table 2. In particular, 2 separate batches of affinity purified ascites fluid of TS1/22, TS1/18 and RRI/l (T. Springer) were tested.
Table 2
Mouse monoclonal antibodies used in IFL studies
C h e
Specijicity
Class
Source
Mid 3 RFDR W6132 PI IH4 UCHTI CD19 TS 1/22 TSI/18 RRI/I 25.3 84H10
HLA-DR HLA-DR HLA-ABC Mouse thyroglobulin Pan T B cells LFA-I achain LFA- ID chain ICAM- 1 LFA- 1 a chain ICAM- 1
IgG I IgM IgG2a IgG I IgG 1 IgG I IgG 1 IgG 1 IgG 1 IgGl IgG 1
P. Lydyard G. Janossy J . Bodmer P. Lydyard P.C.L. Beverly lmmunotech T. Springer T. Springer T. Springer Immunotech Immunotech
The specificity of LFAl (clone 25.3.1) (Immunotech) was similar to TS1/2212. In order to assess the working dilution of each antibody batch, all reagents were tested on lymph node sections before screening the gut biopsies. The 84H 10 MoAb (Immunotech) was used at the final dilution of 1/50 whereas LFA-I (Immunotech) was always applied at 1/20 and both batches of TS1/22 (T. Springer) were used at 1/40 dilution. The first batch of TS1/18 (T. Springer) was used at 1/100 whereas the second batch optimal dilution was 1/25. The first batch of R R l / l (T. Springer) was satisfactory at 1/50 whereas the working dilution of the second batch was 1/10. UCHTI and CD19 were used neat and RFDR working dilution was 1/2. Both the specific and the unrelated MoAbs were applied on the sectiomfor 30 min followed by 25 min incubation with FITC conjugated rabbit anti mouse IgG (Dako). The incubation period was stopped by a 15 min washing step with phosphate buffered saline (PBS). Omission of specific antibodies and the use of P11 MoAb were used as controls of non specific staining. The staining was repeated on at least 2 sequential sections at 3 different levels of each frozen block.
235
ADHESION MOLECULES IN GUT MUCOSA
Table 3 LFA-I and ICAM-I cell counts in the lamina propria in control and pathological specimens: their relationship with HLA-DR. Control specimens
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LFA- I +ve cells
10+2.9
LFA- 1+vel DR+ve cells
5.1+3.3%
ICAM- I+ve cells
6.6+2.9
ICAM- I+ve/ DR+ve cells
6.3+3.7%
Pathological specimens 14.2+3.1 8.2+4.1% 13+4.9 4.2+3.2%
The sections were then mounted with glycerol mounting medium and the results were read blindly by 2 independent observers. To compare the number of positive intraepithelial (IEL) and lamina propria (LP) cells in diseased and control biopsies at least 4 different fields for each section were screened under 1 mm grid graticule of 21 mm diameter (Graticule LTD). The final cell count of each preparation was obtained calculating the average of the 4 readings for each preparation. To document whether adhesion molecule positive mononuclear cells were co-expressing immune activation markers, such as HLADR products, double IFL was performed. This consisted of a 30 min incubation with either LFA- 1 a or ICAM- 1 (Immunotech) MoAbs followed by the application of 30 min incubation with TRITC conjugated goat anti mouse 1gG (Southern). The third layer consisted of a 30 min incubation
with RFDR IgM followed by a FITC conjugated goat anti mouse IgM (Southern). Each incubation step was followed by 15 min washing in PBS. This sequence was intentionally maintained throughout the study although the same results were obtained when HLADR MoAb was applied in the first step of the double IFL. MoAb to mouse thyroglobulin was used as negative control in all experiments and the specific MoAb for each incubation step was excluded from at least one control section in each experiment. Not less than 200 HLADR+ve cells were counted.
RESULTS Morphological studies
In all 7 cases of autoimmune enteropathy the jejunal biopsy showed partial villous atrophy of various degrees ranging from very mild ( 1 case) to severe ( I case). This was associated in 6 samples with LP mononuclear cell infiltration of varying intensity whereas in case No. 1 no abnormal cell infiltration was clearly detected. No histological abnormality was found in any of the jejunal biopsies of control children. Immunolngicul studies Adhesion molecule expression in the LP cells
LFA-1.
Mononuclear cells both within the intraepi-
Figure 1 Cryostat section of jejunal biopsy from a child affected by autoimmune enteropathy stained by IFL with TS1/22. Numerous LFA-1 +ve cells in the infiltrated lamina propria ( X 250). See Color Plate at back of issue.
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S . HILL ET AL
Figure 2 Double IFL on jejunal biopsy from a child affected by autoimmune entcropathy. (a) Section incubated with MoAb to HLADR (RFDR) and stained with FITC rabbit antinlouse IgM. IFL staining on scattered mononuclear cells in the lamina propria and also on thc crypt epithelium; (b) same section incubated with MoAb to LFA-I (TS1/22) and contrastained with TRITC rabbit I@. Lymphocyte-like antimouse appearance of lamina propria +ve cells. No clear staining in the crypt epithelium. Most HLADR +ve cells in the lamina propria do not co-express LFA-1 products (~400)(see arrows). See Color Plate at back of issue.
thelial and the LP compartments reacted with LFA-I ( a and /? chains) in normal and diseases gut biopsies with all reagents used. By comparing LFA-I staining with that of adjacent sections stained for pan T and B cells, the majority of LFA-I positive cells showed a lymphocyte morphology although there were also a proportion with a macrophage/monocyte-like appearance. The number of LFA-I LP positive cells in autoimmune enteropathy specimens was moderately increased in comparison with normal control specimens (14.2k3.1 versus 10.3k2.9) (meankSD) (Table 3). This observation was more evident within areas of
mononuclear cell infiltration although at times a precise estimate of the cell counts in these locations was not posible because of the intimate contact among cells (Figure 1). In the IEL compartment no appreciable variation in the LFA-counts were observed in the two groups studied. By double IFL technique it was shown that in both control and diseased tissues most LFA-I positive cells were not expressing the HLADR activation marker (Figure 2a, b) but the co-expression of HLADR and LFA-I products was restricted to a proportion of cells (5.1 %k3.3%). A moderate but not significant increase
ADHESION MOLECULES IN GUT MUCOSA
231
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(a)
Figure 3 Cryostat section of jejunal biopsy from a control sample (hhowing 2 intestinal villi): (a) compared with that from a child affcctcd by autoimmune enteropathy (showing mucosal crypts), (h) stained by IFL with R R I / I MoAb. Moderate increase in ICAM- I +ve cells in the lamina propria in the affected sample (b) in comparison with the control (a). Both villus and crypt epithelium are negative ( ~ 2 5 0 )See . Color Plate at back of issue.
in the numbers of LFA-1 positive cells co-expressing the HLADR surface marker was observed in the LP cells of biopsies from patients with autoimmune enteropathy in comparison with control specimens (8.2%+ 4.1 %) (Table 3).
I C A M - I . ICAM-1 reacting cells were generally larger in size, showed a dendritic, endothelial or macrophage-like morphology and were not very frequent in the normal control mucosa (6.6k2.9). Again the number of LP reacting cells were generally increased in samples affected by autoimmune enteropathy compared with control biopsies but this increase was not statistically significant ( 13f4.9)
(Table 3). ICAM-1 positive staining was particularly intense in the autoimmune specimens within vascular endothelial cells and within the infiltrated areas (Figure 3a, b). No ICAM-1 positive cells were seen in the IEL compartment. These results were clearly reproducible with both the commercial and non commercial antibody preparations. Most of the ICAM-1 positive cells did not show the HLADR marker (Figure 4a b). In the autoimmune samples a slight increase in the number of cells co-expressing HLADR and ICAM-1 molecules was demonstrated in comparison with normal biopsies (6.3%f3.7% versus 4:2%+3.2%) (Table
238
S. HILL ET AL
3). This finding was clearer within the infiltrated areas although in these locations the counts could not always be very accurate because of the very tight contacts among cells. Adhesion molecule expression on epithelial cells
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L F A - l a and P chains were not detected in the epithelium of the villi and crypts in all 5 controls with either the commercial or non commercial MoAb preparations. LFA- 1 a products were detectable within the crypt epithelium in 3/7 patients with both TS1/22 and 25.3 (Figure 5). TS1/18 MoAb reacted with LFA-1P
Figure 4 Double IFL on jejunal biopsy lrom a child affected by autoiinmune enieropathy. (a) Section stained with RFDR MoAb followed by FITC-rabbit antimouse IgM: IFL reaction on many lymphornononuclear cells in the lamina propria and on the gut crypt epithelium. (h) Same section contrastained with RRI/I MoAb followed by TRITC rabbit anti-mouse IgG: Lamina propria +ve cells, crypt epithelium -vc. The majority of HLA DR +ve cells d o not co-express ICAM1 products ( ~ 2 5 0 ) .
molecules in the epithelium of only 1/7 gut biopsies from children with autoimune enteropathy. No ICAM-1 expression was detected in either crypt or villus enterocytes of the control biopsies, though isolated goblet cells were positive in 2/5 specimens from controls. In contrast, goblet cells and crypt enterocytes from 4/7 patients with autoimmune enteropathy were positive for ICAM-I. However these observations were only demonstrated with the RRl/I preparation, not with 84H 10. Villus epithelial cells of both patients and control biopsies invariably did not express either LFA-I or ICAM- 1. These results are summarized in Table 4. In
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ADHESION MOLECULES IN GUT MUCOSA
239
Figure 5 IFL staining with TS1/22 of a cryostat section from a jejunal biopsy from a child affected by autoimmune enteropathy. Both crypt epithelial and lamina propria cells show a positive reaction ( ~ 2 5 0 )See . Color Plate at back of issue.
Table 4 Relationship between the histological site and the MoAb expression in pathological specimens.
expression of class 11 molecules could be demonstrated in the crypt epithelium of all patients with autoimmune enteropathy’.
TS1/22 a 25.3
TS1118
RRIII
84H10
IEL cells*
-9
--f
-
-
LP cells**
t
t
1‘
t
DISCUSSION
Crypt enterocytes 317
I/7
417
-
Villuq enterocytes-
-
-
-
The present investigation has shown an increase of LFA-I and ICAM-1 cell counts in the L P of gut biopsies of children with autoimmune enteropathy in comparison with control samples whereas LFA-1 and ICAM- 1 molecule expression in the IEL compartment did not differ in the- two groups. No expression of LFA-I and a very low level of ICAM-1 was demonstrated in the epithelium from control intestinal mucosa samples, thus confirming previous
* h t r d e p l l h d l d l lymphocyte5
**ldmlnd
proprld
all the preparations non specific staining was not seen either with the use of the unrelated MoAb or with the incubation of the conjugate alone.
observation^^^. HLA products HLA Class I molecules Both intestinal enterocytes and LP mononuclear cells strongly expressed HLA Class I molecules with a similar pattern and intensity in patients and controls. HLA Class I1 molecules These products were expressed in the mature enterocytes of villi and in the L P mononuclear cells in both pathological and control specimens. An increased number of HLA/DR positive LP cells were detected in the gut of patients with autoimmune enteropathy. No reactivity was found in the crypt epithelium for normal controls whereas aberrant
Conversely, it was surprising to demonstrate an upregulation of ICAM-I products in the crypt epithelium of a proportion of jejunal biopsies from children with autoimmune enteropathy using one specific MoAb reagent but not the other. A possible explanation could be on the recent demonstration that RR 1/1 84H 10 MoAbs recognize spatially separate epitopes on the same ICAM-I molecule”. Thus, we speculate that if during chronic inflammatory processes ICAM- 1 molecules displayed a spatial configuration on the cell surface different from that usually observed under physiological situations, distinct ICAM-1 epitopes also would be displayed and would become accessible targets to the binding of distinct MoAbs. As different determinants of the ICAM1 products appear to play a differential functional role
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240
S. HILL ET A L
in immunological phenomena2’, the preferential expression of certain ICAM- 1 epitopes might confer to the positive cells a more effective antigen presenting ability. The role of accessory molecules in enhancing antigen presentation has been emphasized in transfection experiments. A mouse fibroblastoid cell line transfected with DR cDNA could efficiently present antigens to specific DR restricted T cell clones or to alloreactive T cells only when the levels of DR expression were high. With more modest levels of HLA Class I1 products, antigen presentation could be performed’” only after supertransfection with the ICAM-I accessory molecule genome. Thus, a clear evidence for a synergistic action between Class I1 and ICAM-I has been provided in this way. However, a definite demonstration of a co-expression of these activation molecules within chronically affected epithelial in vivo has not been confirmed either in the present condition or in the more classical autoimmune diseases such as the thyroid autoimmune disease”,” (reviewed in Ref. 33). This is obviously surprising as both HLADR and ICAM-I products are expected to be under the control of similar sets of cytokines. What is certain is that the in vitro direct effect of cytokines such as I F N y a n d T N F a on the up-regulation of DR and ICAM-I products is a clear cut phenomenon whereas the in vivo circuits and interrelations of various cytokine network constitute a much more intricate cascade which is still far from being understood. Detailed in siru hybridization studies performed in different stages of the autoimmune process and standardized MoAb to cytokines are already providing preliminary information on the in vitro cytokine role in these clinical condition^^^. In the present study, a co-expression of accessory molecules and HLA Class I1 products was selectively restricted to a subpopulation of L P cells but not to IEL cells. It was not certain, with the available reagents, whether crypts in a proportion of cases shared the same property. These findings would indicate that in autoimmune intestinal disease the levels of HLA Class I1 expression on LP cells ought to be in general adequate to allow the generation and perpetuation of a full mechanism for (auto)-antigen presentation leading to enterocyte destruction”. It can be speculated that in the eventuality that the crypt epithelium also was able to coexpress HLADR and adhesion products, autoantigen presentation and epithelial cell destruction would follow the interaction between the cells and the underlying CD4 positive lymphocytes in the L P with which the epithelium appears to be in close contact”. However, the ability of the crypt epithelium to present autoantigens to CD4 subsets still needs a full demonstration. The present findings that only a small proportion of LP cells co-express adhesion and HLADR Class I1 molecules parallel those obtained in classical
inflammatory bowel diseasesz7. Differently from our results, in those pathological conditions, the actual counts of LFA-I/ICAM-I positive cells in the LP were significantly increased in comparison to control specimens, thus confirming an increased cell adhesiveness, tighter T cell interactions and a mouse active local immune response in the LP of those pathological disorders27. It has been shown that viruses” and parasites (reviewed in Ref. 11) use ICAM-1 as their receptors. However, the lack of adhesion molecule expression on villus epithelium, both in physiological and pathological conditions appears not to substantiate the role of common infectious agents in this clinical entity. Conversely, this finding might reflect a protective role of the gut epithelium against potentially hazardous sensitizations to repeatedly ingested oral antigens. In conclusion, the present demonstration of a moderate up-regulation of adhesion products on LP cells from gut specimens of children with autoimmune enteropathy reflects an accessory rather than a primary role of these molecules in the pathogenesis of this conditions.
Acknowledgements We are grateful to Prof. T. Springer (Boston, USA) for the generous gift of TS1/22, TS1/18 and RRI/l MoAbs. We are indebted to Immunotech (Marseille) for the donation of CDI 1a/25.3.1, 84HI0 (CD 54) and CD19 and to Dr J. Bodmer (ICRF, London) for W6/32. We are grateful to Dr P. Lydyard for Mid 3 and PI I MoABs, Prof. P. Beverley for supplying us with UCHTI and Prof. G. Janossy for providing us with RFDR reagent. Mrs Teresa Caseseca, supported by CICRA, has provided invaluable technical assistance. Mrs Virpi Smith has helped us with the histology. Dr S. Hill was supported by an Action for Research into Crippling Diseases of Childhood Fellowship.
References I . Mirakian R, Richardson A, Milla PJ, Unsworth J, WalkerSmith J , Savage M, Bottazzo GF. Idiopathic protracted diarrhoea of infancy: an autoimmune variant. Brit Med J 1986; 293: 1 132-1 I36 2. Mirakian R, Hill S, Richardson A, Milla PJ, Walker-Smith J. Bottazzo GF. HLA product expression and lymphocyte subpopulations in jejunum biopsies of children with idiopathic protracted diarrhoea and enterocyte auloantibodies. J A u t ~ i n i niunity 1988; 1: 263-271 3. Brandtzaep P, Halstensen TS, Kett K, Krajci P, Kvale D, Rognum TO, Scott H, Sollid LM. Immunology and immunopathology of human gut mucosa: humoral immunity and intraepithclial lymphocytes. Gosfrornler-ology 1989; 97: 1562-1584 4. Mayer L, Eisenhardt D, Salomon P, Bauer W, PIOUS R , Piccinini L. Expression of Class 11 molecules on intestinal epithelial cells in humans. Gastroenterology 1991; 100: 3-12 5 . Ciclitira PJ, Nelufer JM, Ellis HJ, Evans DJ. The effect of gluten on HLADR in the small intestinal epithelium of patients with coeliac disease. CIiu Exp Immunol 1986; 63: 101-104 6. Bottazzo GF, Todd I , Mirakian R , Belfiore A, Pujol-Borrell R.
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ADHESION MOLECULES IN GUT MUCOSA Organ-specific autoimmunity: a 1986 overview. Immunol Rev 1986; 94: 137-169 7. Mayer L. Shlian R. Evidence for function of Ia molecules on gut epithelial cells in man. J Exp Med 1987; 166: 1471-1482 8. Grubek-Lobenstein B, Londei M, Greenall C, Pirich K, Kassal H, Waldhausl W, Feldmann M. Pathogenic relevance of HLA Class I1 expressing thyroid follicular cells in non toxic goitre and in Graves disease. J Clin Invest 1988; 81: 1608-1612 9. Shaw S, Lucc GEG. The lymphocyte function associated antigen (LFA)- 1 and CD2/LFA3 pathways of antigen-independent human T cell adhesion. J Inimunology 1987; 139: 1037-1045 10. Makgoba MW, Sanders ME, Shaw S. The CD2-LFA3 and LFA-l/ICAM- 1 pathways: relevance to T cell recognition. Innnunology Today 1989; 10: 4 1 7 4 2 2 11. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 4 2 5 4 3 4 12. Sanchez-Madid F, Krensky AM, Ware CF, Robbins E, Strominger JL, Burdkoff JJ, Springer TA. Three distinct antigen associated with human T lymphocyte cytolysis, LFAI, LFA2 and LFA3. PNAS 1982; 79: 7489-7493 13. Rothlein R, Springer TA. The requirement for lymphocyte function associated antigen I in homotypic leukocyte adhesions stimulated by phorbol-ester. .I Exp Med 1982; 163: 1132-1 140 14. Springer TA, Dustin ML, Kishimoto TK, Marlin SD. The lymphocyte function associated LFA-I, CD2 and LFA3 molecules: cell adhesion receptors of the immune system. Ann Rev Immunu/ 1987; 5: 223-252 15. Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular molecule (ICAM-I) distinct from LFA-I. J . Immuno/ 1986; 137: 1270-1274 16. Marlin SD, Springer TA. Purified intercellular adhesion molecule (ICAM-1) is a ligand for lymphocyte function-associated antigens (LFA-I). Cell 1987; 51: 813-819 17. Makgoba MW, Sanders ME, Luce GAG, Dustin ML, Springer TA. Clark EA, Mannoni P, Shaw S. ICAM-1, a ligand for LFAI dependent adhesion of B, T and myeloid cells. Nature 1988; 331: 86-88 18. Staunton DE, Dustin ML, Springer TA. Functional cloning of ICAM-2, a cell adhesion ligand for LFA-I homologous to ICAM-I. Nature 1989; 339: 61-64 19. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL- I and IFN-y. Tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-I). J lmmunolo,qy 1986; 137: 245-254 20. Campbell IL, Cutri A, Wilkinson D, Boyd AW, Harrison LC. Intercellular adhesion molecule I is induced on isolated endocrine islet cells by cytokines but not by reovirus infection. PNAS 1989; 86: 42824286 21. Pujol-Borrell R, Vives M, Badenas J, Foz M, Buscema M, Soldevila G, Bottazzo GF. Modulation of intercellular adhesion molecule-I (ICAM-I) in human islet cells. Diaheres 1989; 38 (suppl. 22): 733A 22. Weetman AP, Wolkman DJ, Bumann KD, Gerrard TL, Fauci AS. The in vitro regulation of human thyrocytes HLADR antigen expression. J Clin Endocrinol Metah 1985; 61: 8 17-824
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23. Pujol-Borrel R, Todd I. Inappropriate Class 11 expression in autoimmunity. Is it the primary event? In Bottazzo GF, Donaich D (eds), Clinics in Immunology and Allergology. BailliL.re’s. 1987; 1-29 24. Grubeck-Loebenstein B, Buchan G, Chantry H, Kassl H, Londei M, Pirich K, Barrett K, Turner M, Waldhausl W, Feldmann M. Analysis of intrathyroid cytokine production in thyroid autoimmune disease: Thyroid follicular cells produce interleukin-la and interleukin 6. Clin E.wp Immunol 1989; 77: 324-330 25. Zheng R, Abney E, Chu Q, Field M, Grubeck-Loebenstein B, Maini RN, Feldmann M. Detection of IL-6 and IL-I production in human thyroid epithelial cells by non-radioactive in siru hybridization and immunohistochemical methods. Clin Exp Immunol I99 1; 83: 3 14-3 I9 26. Hamilton F, Black M, Farquharson MA, Stewart C, Foulis AK. Spatial correlation between thyroid epithelial cells expressing Class I1 MHC molecules and interferon- y containing lymphocytes in human thyroid autoimmune diseases. Clin Exp Immuno/ 1991; 83: 64-68 27. Malizia C, Calabrese A, Cottone M, Raimondo M, Ludwik K, Trejdosiewicz R, Smart CJ, Oliva L, Pagliaro L. Expression of leukocyte adhesion molecules by mucosal mononuclear phagocytes in inflammatory bowel disease. Gastroenternlngy 199 I ; 100: 150-159 28. Hill S, Milla PJ, Bottazzo GF, Mirakian R. Autoimmune enteropathy and colitis: is there a generalized autoimmune gut disorder? Gut 199 I ; 32: 3 6 4 2 29. Maio M, Tessitori G, Pinto A, Temponi M, Colombatti A, Ferrone S. Differential role of distinct determinants of intercellular adhesion molecule- I phenomena. J Immunmology 1989; 143: 181-188 30. Altimann DM, Hogg N, Trowsdale J , Wilkinson D. Co-transfection of ICAM-I and HLADR reconstitutes human antigenpresenting cell function in mouse L cells. Nature 1989; 338: 512-516 31. Bagnasco M, Caretto A, Olive D, Pedini GW, Canonica GW, Betterle C. Expression of ICAM- I on thyroid epithelial cells in Hashimoto’s thyroiditis and not in Graves’ disease or capillary carcinoma. Clin. exp. Immunol. 199 1 ; 83: 309-3 13 32. Ciampolillo A, Napolitano G, Mirakian R, Miyasaki A, Giorgino R, Bottazzo GF. Intercellular adhesion molecule (ICAM-I) in Graves’ disease: in vivo versus in vitro contrasting results. J Autoimmunity (in press) 33. Bottazzo GF, Drexhage H. Recent advances in thyroid autoimmunity. Current Opinion in Immunology (in press) 34. Zheng ROH, Abney ER, Chev CQ, Field M, Maini RN, Lamb J, Feldman M. Detection of in vivo production of T N F a by human thyroid epithelial cells. Immunology 1992; 75: 4 5 6 4 6 2 35. Russell GJ, Hermatz PR. MHC Class I1 expression on enterocytes: to present or not to present. Gastroenterology 1991; 199: 274-276 36. Hashimoto Y, Komer T. Close relationship between the cells of the immune system and the epithelial cells in the rat small intestine. Cell Tissue Res 1988; 254: 4 1 4 7 37. Staunton DE, Merfurri VJ, Rothleim R, Barton R, Marlin SD, Springer TA. Cell adhesion molecule ICAM-1 is the major surface receptor for Rhinovirus. Cell 1989; 56: 849-853
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
LFA-1 AND ICAM-1 MOLECULE EXPRESSION IN JEJUNAL MUCOSA FROM CHILDREN WITH AUTOIMMUNE ENTEROPATHY SUSAN HILL*, PETER J. MILLA*, ANNA CIAMPOLILLO, GIORGIO NAPOLITANO, GIAN F R A N C 0 BOTTAZZO and RITA MIRAKIAN *Department of Child Health, Institute of Child Health, London WCI IEH, and Department of Immunology, The Royal London Hospital Medical College, London E l 2AD
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(Received November 21,1991; in final form May 27.1992) The expression of adhesion molecules by cells of the small intestinal mucosa was compared in gut biopsies from children with autoimmune small intestinal enteropathy and normal controls and related to HLA-DR expression by the same tissue. Jejunal biopsies were stained by IFL with monoclonal antibodies to LFA-I (TS1/22 and CDlla/25.3.I) and ICAM-1 (RRI/I and 84H10) molecules. LFA-I and ICAM-I positive cells were observed in the lamina propria in all cases and the counts were increased in autoimmune enteropathy compared with controls. In addition, in 4 of 7 cases of autoimmune enteropathy crypt enterocytes were positives for ICAM-I when stained with RRI/I and 3 of the 4 were also positive for LFA-I when stained with both LFA-I reagents. We speculate on the role of adhesion molecule expression in autoimmune enteropathy.
KEY WORDS: Autoimmune enteropathy, ICAM-I, LFA-I, HLA-DR, adhesion molecule.
LFA- 1 binds to its counter receptors, the Intercellular Adhesion Molecules (ICAM-1 and 2)15-'*which are physiologically expressed on endothelial and APCs but generally not on resting T cells or on epithelial ceIIs'6,'y. The LFA-l/ICAM-1 interaction is an early event in the process of T cell activation and plays a critical role in effector T cell activation'".". Cytokines, such as IFNy alone and/or in combination with IL-1 and T N F a can induce/enhance ICAM-1 expression in v i t u ~ ' ~ -in~ the ' , same way as HLA Class I1 molecule induction has been already demonstrated22 (reviewed in Ref. 23). Cytokine production has been recently confirmed in vivo near sites of active mononuclear infiltration in tissues affected by autoimmune diseases and by epithelial structures
INTRODUCTION Inappropriate expression of HLA Class I1 products has been detected in crypt enterocytes from children affected by autoimmune enteropathy',2.Similar observations have been made previously in the intestinal epithelium derived from patients with inflammatory bowel disease (IBD) (reviewed in Refs 3 and 4), coeliac disease5 and in gland epithelium from patients with classical autoimmune disorders (reviewed in Ref. 6). These findings have constituted an important step in understanding target cell autorecognition and destruction by T activated lymphocytes in autoimmune disorders. Epithelial cells which express de novo HLA Class I1 molecules become capable of directly operating as antigen presenting cells (APC) to autologous T helper/inducer CD4+ve lymphocyte subsets without macrophage participation (reviewed in Refs 6, 7 and 8). However, additional prerequisites for effective antigen presentation include the expression on the cell surface of "accessory" molecules which enhance cell contact and adhesion9-". One of these molecules is the Lymphocyte Function Associated Antigen (LFA- l ) , a heterodimer, expressed on the surface of lymphocytes, monocytes and some APCs'"'' (reviewed in Ref. 14).
The aim of this study was to investigate the possible upregulation of adhesion molecules on cells of the intestinal mucosa affected by autoimmune enteropathy and colitis2*. It was also important to compare this expression with the aberrant expression of Class I1 molecules on enterocytes, and with the expression of activation molecules on morphologically and histologically normal jejunal mucosa. PATIENTS
Correspondence to: Dr R. Mirakian, Department of Immunology, The Royal London Hospital Medical College, 56-76 Ashfield Street, London El 2AD.
Two groups of patients were selected: 7 children with autoimmune enteropathy, and a control group of 5
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S. HILL ET AL
Table 1 Autoimmune enteropathy: clinical and morphological features. PIS
A'v
Ses
AutoAbs
Jejitnal biopsy
Treatment
I
2 Yr
M
+
None
2
8 Yr
F
1/64
3
1 Yr
M
11128
4
l m
M
I18
5
3 yr
F
1/64
6
S yr
M
11128
I
15 m
M
I18
Mild PVA LP: normal infiltrate PVA LP: heavy infiltrate Severe PVA LP: heavy infiltrate PVA LP: moderate infiltrate Very mild PVA LP: mild infiltrate Severe PVA LP: moderate infiltrate PVA LP: moderate infiltrate
Azathioprine Prednisolone ALathioprine None Prednisolone Azathioprine Prednisolone Prednisolone
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* P V A : partial v~llou?~ atrophy: LP: i a r n i n ~proprra. yr.;. years' m. rnonrhs.
children with histologically normal small intestinal mucosa. The 7 children with autoimmune enteropathy were aged from 9 months to 8 years (median 2 years). Five were males and 2 females. They had all presented with chronic diarrhoea and failure to thrive, had had circulating enterocyte antibodies (EcAbs). on at least two occasions and an enteropathy of variable severity (Table 1). Five of the 7 were on treatment with prednisolone when studied. The 5 control children were aged from 1 to 10 years (median 4 years). Two were male and 3 female. They all underwent small intestinal biopsy for diagnostic purposes and had histologically normal mucosa. Two had presented with persistent diarrhoea and failure to thrive (in one a diagnosis of cystic fibrosis was made and in the other congenital chloridorrhoea), 1 with abdominal distension (in whom aerophagy was diagnosed) and 2 with loose stool (in whom no intestinal abnormality was detected). No Abs were observed in the control sera.
METHODS Informed parental consent was obtained before small intestinal biopsies were taken from the duodenojejunal flexure with a double port Watson Paediatric capsule. One of the two specimens was fixed in formalin and sections were stained with haematoxylineosin for histological analysis. The other sample was snap frozen and stored at -70°C until 4 ,U cryostat sections were cut, fixed for 5 min in cold acetone and stained using an indirect immunofluorescence technique (IFL). The list of monoclonal antibodies (MoAbs) used is reported in Table 2. In particular, 2 separate batches of affinity purified ascites fluid of TS1/22, TS1/18 and RRI/l (T. Springer) were tested.
Table 2
Mouse monoclonal antibodies used in IFL studies
C h e
Specijicity
Class
Source
Mid 3 RFDR W6132 PI IH4 UCHTI CD19 TS 1/22 TSI/18 RRI/I 25.3 84H10
HLA-DR HLA-DR HLA-ABC Mouse thyroglobulin Pan T B cells LFA-I achain LFA- ID chain ICAM- 1 LFA- 1 a chain ICAM- 1
IgG I IgM IgG2a IgG I IgG 1 IgG I IgG 1 IgG 1 IgG 1 IgGl IgG 1
P. Lydyard G. Janossy J . Bodmer P. Lydyard P.C.L. Beverly lmmunotech T. Springer T. Springer T. Springer Immunotech Immunotech
The specificity of LFAl (clone 25.3.1) (Immunotech) was similar to TS1/2212. In order to assess the working dilution of each antibody batch, all reagents were tested on lymph node sections before screening the gut biopsies. The 84H 10 MoAb (Immunotech) was used at the final dilution of 1/50 whereas LFA-I (Immunotech) was always applied at 1/20 and both batches of TS1/22 (T. Springer) were used at 1/40 dilution. The first batch of TS1/18 (T. Springer) was used at 1/100 whereas the second batch optimal dilution was 1/25. The first batch of R R l / l (T. Springer) was satisfactory at 1/50 whereas the working dilution of the second batch was 1/10. UCHTI and CD19 were used neat and RFDR working dilution was 1/2. Both the specific and the unrelated MoAbs were applied on the sectiomfor 30 min followed by 25 min incubation with FITC conjugated rabbit anti mouse IgG (Dako). The incubation period was stopped by a 15 min washing step with phosphate buffered saline (PBS). Omission of specific antibodies and the use of P11 MoAb were used as controls of non specific staining. The staining was repeated on at least 2 sequential sections at 3 different levels of each frozen block.
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ADHESION MOLECULES IN GUT MUCOSA
Table 3 LFA-I and ICAM-I cell counts in the lamina propria in control and pathological specimens: their relationship with HLA-DR. Control specimens
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LFA- I +ve cells
10+2.9
LFA- 1+vel DR+ve cells
5.1+3.3%
ICAM- I+ve cells
6.6+2.9
ICAM- I+ve/ DR+ve cells
6.3+3.7%
Pathological specimens 14.2+3.1 8.2+4.1% 13+4.9 4.2+3.2%
The sections were then mounted with glycerol mounting medium and the results were read blindly by 2 independent observers. To compare the number of positive intraepithelial (IEL) and lamina propria (LP) cells in diseased and control biopsies at least 4 different fields for each section were screened under 1 mm grid graticule of 21 mm diameter (Graticule LTD). The final cell count of each preparation was obtained calculating the average of the 4 readings for each preparation. To document whether adhesion molecule positive mononuclear cells were co-expressing immune activation markers, such as HLADR products, double IFL was performed. This consisted of a 30 min incubation with either LFA- 1 a or ICAM- 1 (Immunotech) MoAbs followed by the application of 30 min incubation with TRITC conjugated goat anti mouse 1gG (Southern). The third layer consisted of a 30 min incubation
with RFDR IgM followed by a FITC conjugated goat anti mouse IgM (Southern). Each incubation step was followed by 15 min washing in PBS. This sequence was intentionally maintained throughout the study although the same results were obtained when HLADR MoAb was applied in the first step of the double IFL. MoAb to mouse thyroglobulin was used as negative control in all experiments and the specific MoAb for each incubation step was excluded from at least one control section in each experiment. Not less than 200 HLADR+ve cells were counted.
RESULTS Morphological studies
In all 7 cases of autoimmune enteropathy the jejunal biopsy showed partial villous atrophy of various degrees ranging from very mild ( 1 case) to severe ( I case). This was associated in 6 samples with LP mononuclear cell infiltration of varying intensity whereas in case No. 1 no abnormal cell infiltration was clearly detected. No histological abnormality was found in any of the jejunal biopsies of control children. Immunolngicul studies Adhesion molecule expression in the LP cells
LFA-1.
Mononuclear cells both within the intraepi-
Figure 1 Cryostat section of jejunal biopsy from a child affected by autoimmune enteropathy stained by IFL with TS1/22. Numerous LFA-1 +ve cells in the infiltrated lamina propria ( X 250). See Color Plate at back of issue.
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Figure 2 Double IFL on jejunal biopsy from a child affected by autoimmune entcropathy. (a) Section incubated with MoAb to HLADR (RFDR) and stained with FITC rabbit antinlouse IgM. IFL staining on scattered mononuclear cells in the lamina propria and also on thc crypt epithelium; (b) same section incubated with MoAb to LFA-I (TS1/22) and contrastained with TRITC rabbit I@. Lymphocyte-like antimouse appearance of lamina propria +ve cells. No clear staining in the crypt epithelium. Most HLADR +ve cells in the lamina propria do not co-express LFA-1 products (~400)(see arrows). See Color Plate at back of issue.
thelial and the LP compartments reacted with LFA-I ( a and /? chains) in normal and diseases gut biopsies with all reagents used. By comparing LFA-I staining with that of adjacent sections stained for pan T and B cells, the majority of LFA-I positive cells showed a lymphocyte morphology although there were also a proportion with a macrophage/monocyte-like appearance. The number of LFA-I LP positive cells in autoimmune enteropathy specimens was moderately increased in comparison with normal control specimens (14.2k3.1 versus 10.3k2.9) (meankSD) (Table 3). This observation was more evident within areas of
mononuclear cell infiltration although at times a precise estimate of the cell counts in these locations was not posible because of the intimate contact among cells (Figure 1). In the IEL compartment no appreciable variation in the LFA-counts were observed in the two groups studied. By double IFL technique it was shown that in both control and diseased tissues most LFA-I positive cells were not expressing the HLADR activation marker (Figure 2a, b) but the co-expression of HLADR and LFA-I products was restricted to a proportion of cells (5.1 %k3.3%). A moderate but not significant increase
ADHESION MOLECULES IN GUT MUCOSA
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(a)
Figure 3 Cryostat section of jejunal biopsy from a control sample (hhowing 2 intestinal villi): (a) compared with that from a child affcctcd by autoimmune enteropathy (showing mucosal crypts), (h) stained by IFL with R R I / I MoAb. Moderate increase in ICAM- I +ve cells in the lamina propria in the affected sample (b) in comparison with the control (a). Both villus and crypt epithelium are negative ( ~ 2 5 0 )See . Color Plate at back of issue.
in the numbers of LFA-1 positive cells co-expressing the HLADR surface marker was observed in the LP cells of biopsies from patients with autoimmune enteropathy in comparison with control specimens (8.2%+ 4.1 %) (Table 3).
I C A M - I . ICAM-1 reacting cells were generally larger in size, showed a dendritic, endothelial or macrophage-like morphology and were not very frequent in the normal control mucosa (6.6k2.9). Again the number of LP reacting cells were generally increased in samples affected by autoimmune enteropathy compared with control biopsies but this increase was not statistically significant ( 13f4.9)
(Table 3). ICAM-1 positive staining was particularly intense in the autoimmune specimens within vascular endothelial cells and within the infiltrated areas (Figure 3a, b). No ICAM-1 positive cells were seen in the IEL compartment. These results were clearly reproducible with both the commercial and non commercial antibody preparations. Most of the ICAM-1 positive cells did not show the HLADR marker (Figure 4a b). In the autoimmune samples a slight increase in the number of cells co-expressing HLADR and ICAM-1 molecules was demonstrated in comparison with normal biopsies (6.3%f3.7% versus 4:2%+3.2%) (Table
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S. HILL ET AL
3). This finding was clearer within the infiltrated areas although in these locations the counts could not always be very accurate because of the very tight contacts among cells. Adhesion molecule expression on epithelial cells
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L F A - l a and P chains were not detected in the epithelium of the villi and crypts in all 5 controls with either the commercial or non commercial MoAb preparations. LFA- 1 a products were detectable within the crypt epithelium in 3/7 patients with both TS1/22 and 25.3 (Figure 5). TS1/18 MoAb reacted with LFA-1P
Figure 4 Double IFL on jejunal biopsy lrom a child affected by autoiinmune enieropathy. (a) Section stained with RFDR MoAb followed by FITC-rabbit antimouse IgM: IFL reaction on many lymphornononuclear cells in the lamina propria and on the gut crypt epithelium. (h) Same section contrastained with RRI/I MoAb followed by TRITC rabbit anti-mouse IgG: Lamina propria +ve cells, crypt epithelium -vc. The majority of HLA DR +ve cells d o not co-express ICAM1 products ( ~ 2 5 0 ) .
molecules in the epithelium of only 1/7 gut biopsies from children with autoimune enteropathy. No ICAM-1 expression was detected in either crypt or villus enterocytes of the control biopsies, though isolated goblet cells were positive in 2/5 specimens from controls. In contrast, goblet cells and crypt enterocytes from 4/7 patients with autoimmune enteropathy were positive for ICAM-I. However these observations were only demonstrated with the RRl/I preparation, not with 84H 10. Villus epithelial cells of both patients and control biopsies invariably did not express either LFA-I or ICAM- 1. These results are summarized in Table 4. In
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ADHESION MOLECULES IN GUT MUCOSA
239
Figure 5 IFL staining with TS1/22 of a cryostat section from a jejunal biopsy from a child affected by autoimmune enteropathy. Both crypt epithelial and lamina propria cells show a positive reaction ( ~ 2 5 0 )See . Color Plate at back of issue.
Table 4 Relationship between the histological site and the MoAb expression in pathological specimens.
expression of class 11 molecules could be demonstrated in the crypt epithelium of all patients with autoimmune enteropathy’.
TS1/22 a 25.3
TS1118
RRIII
84H10
IEL cells*
-9
--f
-
-
LP cells**
t
t
1‘
t
DISCUSSION
Crypt enterocytes 317
I/7
417
-
Villuq enterocytes-
-
-
-
The present investigation has shown an increase of LFA-I and ICAM-1 cell counts in the L P of gut biopsies of children with autoimmune enteropathy in comparison with control samples whereas LFA-1 and ICAM- 1 molecule expression in the IEL compartment did not differ in the- two groups. No expression of LFA-I and a very low level of ICAM-1 was demonstrated in the epithelium from control intestinal mucosa samples, thus confirming previous
* h t r d e p l l h d l d l lymphocyte5
**ldmlnd
proprld
all the preparations non specific staining was not seen either with the use of the unrelated MoAb or with the incubation of the conjugate alone.
observation^^^. HLA products HLA Class I molecules Both intestinal enterocytes and LP mononuclear cells strongly expressed HLA Class I molecules with a similar pattern and intensity in patients and controls. HLA Class I1 molecules These products were expressed in the mature enterocytes of villi and in the L P mononuclear cells in both pathological and control specimens. An increased number of HLA/DR positive LP cells were detected in the gut of patients with autoimmune enteropathy. No reactivity was found in the crypt epithelium for normal controls whereas aberrant
Conversely, it was surprising to demonstrate an upregulation of ICAM-I products in the crypt epithelium of a proportion of jejunal biopsies from children with autoimmune enteropathy using one specific MoAb reagent but not the other. A possible explanation could be on the recent demonstration that RR 1/1 84H 10 MoAbs recognize spatially separate epitopes on the same ICAM-I molecule”. Thus, we speculate that if during chronic inflammatory processes ICAM- 1 molecules displayed a spatial configuration on the cell surface different from that usually observed under physiological situations, distinct ICAM-1 epitopes also would be displayed and would become accessible targets to the binding of distinct MoAbs. As different determinants of the ICAM1 products appear to play a differential functional role
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S. HILL ET A L
in immunological phenomena2’, the preferential expression of certain ICAM- 1 epitopes might confer to the positive cells a more effective antigen presenting ability. The role of accessory molecules in enhancing antigen presentation has been emphasized in transfection experiments. A mouse fibroblastoid cell line transfected with DR cDNA could efficiently present antigens to specific DR restricted T cell clones or to alloreactive T cells only when the levels of DR expression were high. With more modest levels of HLA Class I1 products, antigen presentation could be performed’” only after supertransfection with the ICAM-I accessory molecule genome. Thus, a clear evidence for a synergistic action between Class I1 and ICAM-I has been provided in this way. However, a definite demonstration of a co-expression of these activation molecules within chronically affected epithelial in vivo has not been confirmed either in the present condition or in the more classical autoimmune diseases such as the thyroid autoimmune disease”,” (reviewed in Ref. 33). This is obviously surprising as both HLADR and ICAM-I products are expected to be under the control of similar sets of cytokines. What is certain is that the in vitro direct effect of cytokines such as I F N y a n d T N F a on the up-regulation of DR and ICAM-I products is a clear cut phenomenon whereas the in vivo circuits and interrelations of various cytokine network constitute a much more intricate cascade which is still far from being understood. Detailed in siru hybridization studies performed in different stages of the autoimmune process and standardized MoAb to cytokines are already providing preliminary information on the in vitro cytokine role in these clinical condition^^^. In the present study, a co-expression of accessory molecules and HLA Class I1 products was selectively restricted to a subpopulation of L P cells but not to IEL cells. It was not certain, with the available reagents, whether crypts in a proportion of cases shared the same property. These findings would indicate that in autoimmune intestinal disease the levels of HLA Class I1 expression on LP cells ought to be in general adequate to allow the generation and perpetuation of a full mechanism for (auto)-antigen presentation leading to enterocyte destruction”. It can be speculated that in the eventuality that the crypt epithelium also was able to coexpress HLADR and adhesion products, autoantigen presentation and epithelial cell destruction would follow the interaction between the cells and the underlying CD4 positive lymphocytes in the L P with which the epithelium appears to be in close contact”. However, the ability of the crypt epithelium to present autoantigens to CD4 subsets still needs a full demonstration. The present findings that only a small proportion of LP cells co-express adhesion and HLADR Class I1 molecules parallel those obtained in classical
inflammatory bowel diseasesz7. Differently from our results, in those pathological conditions, the actual counts of LFA-I/ICAM-I positive cells in the LP were significantly increased in comparison to control specimens, thus confirming an increased cell adhesiveness, tighter T cell interactions and a mouse active local immune response in the LP of those pathological disorders27. It has been shown that viruses” and parasites (reviewed in Ref. 11) use ICAM-1 as their receptors. However, the lack of adhesion molecule expression on villus epithelium, both in physiological and pathological conditions appears not to substantiate the role of common infectious agents in this clinical entity. Conversely, this finding might reflect a protective role of the gut epithelium against potentially hazardous sensitizations to repeatedly ingested oral antigens. In conclusion, the present demonstration of a moderate up-regulation of adhesion products on LP cells from gut specimens of children with autoimmune enteropathy reflects an accessory rather than a primary role of these molecules in the pathogenesis of this conditions.
Acknowledgements We are grateful to Prof. T. Springer (Boston, USA) for the generous gift of TS1/22, TS1/18 and RRI/l MoAbs. We are indebted to Immunotech (Marseille) for the donation of CDI 1a/25.3.1, 84HI0 (CD 54) and CD19 and to Dr J. Bodmer (ICRF, London) for W6/32. We are grateful to Dr P. Lydyard for Mid 3 and PI I MoABs, Prof. P. Beverley for supplying us with UCHTI and Prof. G. Janossy for providing us with RFDR reagent. Mrs Teresa Caseseca, supported by CICRA, has provided invaluable technical assistance. Mrs Virpi Smith has helped us with the histology. Dr S. Hill was supported by an Action for Research into Crippling Diseases of Childhood Fellowship.
References I . Mirakian R, Richardson A, Milla PJ, Unsworth J, WalkerSmith J , Savage M, Bottazzo GF. Idiopathic protracted diarrhoea of infancy: an autoimmune variant. Brit Med J 1986; 293: 1 132-1 I36 2. Mirakian R, Hill S, Richardson A, Milla PJ, Walker-Smith J. Bottazzo GF. HLA product expression and lymphocyte subpopulations in jejunum biopsies of children with idiopathic protracted diarrhoea and enterocyte auloantibodies. J A u t ~ i n i niunity 1988; 1: 263-271 3. Brandtzaep P, Halstensen TS, Kett K, Krajci P, Kvale D, Rognum TO, Scott H, Sollid LM. Immunology and immunopathology of human gut mucosa: humoral immunity and intraepithclial lymphocytes. Gosfrornler-ology 1989; 97: 1562-1584 4. Mayer L, Eisenhardt D, Salomon P, Bauer W, PIOUS R , Piccinini L. Expression of Class 11 molecules on intestinal epithelial cells in humans. Gastroenterology 1991; 100: 3-12 5 . Ciclitira PJ, Nelufer JM, Ellis HJ, Evans DJ. The effect of gluten on HLADR in the small intestinal epithelium of patients with coeliac disease. CIiu Exp Immunol 1986; 63: 101-104 6. Bottazzo GF, Todd I , Mirakian R , Belfiore A, Pujol-Borrell R.
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ADHESION MOLECULES IN GUT MUCOSA Organ-specific autoimmunity: a 1986 overview. Immunol Rev 1986; 94: 137-169 7. Mayer L. Shlian R. Evidence for function of Ia molecules on gut epithelial cells in man. J Exp Med 1987; 166: 1471-1482 8. Grubek-Lobenstein B, Londei M, Greenall C, Pirich K, Kassal H, Waldhausl W, Feldmann M. Pathogenic relevance of HLA Class I1 expressing thyroid follicular cells in non toxic goitre and in Graves disease. J Clin Invest 1988; 81: 1608-1612 9. Shaw S, Lucc GEG. The lymphocyte function associated antigen (LFA)- 1 and CD2/LFA3 pathways of antigen-independent human T cell adhesion. J Inimunology 1987; 139: 1037-1045 10. Makgoba MW, Sanders ME, Shaw S. The CD2-LFA3 and LFA-l/ICAM- 1 pathways: relevance to T cell recognition. Innnunology Today 1989; 10: 4 1 7 4 2 2 11. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 4 2 5 4 3 4 12. Sanchez-Madid F, Krensky AM, Ware CF, Robbins E, Strominger JL, Burdkoff JJ, Springer TA. Three distinct antigen associated with human T lymphocyte cytolysis, LFAI, LFA2 and LFA3. PNAS 1982; 79: 7489-7493 13. Rothlein R, Springer TA. The requirement for lymphocyte function associated antigen I in homotypic leukocyte adhesions stimulated by phorbol-ester. .I Exp Med 1982; 163: 1132-1 140 14. Springer TA, Dustin ML, Kishimoto TK, Marlin SD. The lymphocyte function associated LFA-I, CD2 and LFA3 molecules: cell adhesion receptors of the immune system. Ann Rev Immunu/ 1987; 5: 223-252 15. Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular molecule (ICAM-I) distinct from LFA-I. J . Immuno/ 1986; 137: 1270-1274 16. Marlin SD, Springer TA. Purified intercellular adhesion molecule (ICAM-1) is a ligand for lymphocyte function-associated antigens (LFA-I). Cell 1987; 51: 813-819 17. Makgoba MW, Sanders ME, Luce GAG, Dustin ML, Springer TA. Clark EA, Mannoni P, Shaw S. ICAM-1, a ligand for LFAI dependent adhesion of B, T and myeloid cells. Nature 1988; 331: 86-88 18. Staunton DE, Dustin ML, Springer TA. Functional cloning of ICAM-2, a cell adhesion ligand for LFA-I homologous to ICAM-I. Nature 1989; 339: 61-64 19. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL- I and IFN-y. Tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-I). J lmmunolo,qy 1986; 137: 245-254 20. Campbell IL, Cutri A, Wilkinson D, Boyd AW, Harrison LC. Intercellular adhesion molecule I is induced on isolated endocrine islet cells by cytokines but not by reovirus infection. PNAS 1989; 86: 42824286 21. Pujol-Borrell R, Vives M, Badenas J, Foz M, Buscema M, Soldevila G, Bottazzo GF. Modulation of intercellular adhesion molecule-I (ICAM-I) in human islet cells. Diaheres 1989; 38 (suppl. 22): 733A 22. Weetman AP, Wolkman DJ, Bumann KD, Gerrard TL, Fauci AS. The in vitro regulation of human thyrocytes HLADR antigen expression. J Clin Endocrinol Metah 1985; 61: 8 17-824
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23. Pujol-Borrel R, Todd I. Inappropriate Class 11 expression in autoimmunity. Is it the primary event? In Bottazzo GF, Donaich D (eds), Clinics in Immunology and Allergology. BailliL.re’s. 1987; 1-29 24. Grubeck-Loebenstein B, Buchan G, Chantry H, Kassl H, Londei M, Pirich K, Barrett K, Turner M, Waldhausl W, Feldmann M. Analysis of intrathyroid cytokine production in thyroid autoimmune disease: Thyroid follicular cells produce interleukin-la and interleukin 6. Clin E.wp Immunol 1989; 77: 324-330 25. Zheng R, Abney E, Chu Q, Field M, Grubeck-Loebenstein B, Maini RN, Feldmann M. Detection of IL-6 and IL-I production in human thyroid epithelial cells by non-radioactive in siru hybridization and immunohistochemical methods. Clin Exp Immunol I99 1; 83: 3 14-3 I9 26. Hamilton F, Black M, Farquharson MA, Stewart C, Foulis AK. Spatial correlation between thyroid epithelial cells expressing Class I1 MHC molecules and interferon- y containing lymphocytes in human thyroid autoimmune diseases. Clin Exp Immuno/ 1991; 83: 64-68 27. Malizia C, Calabrese A, Cottone M, Raimondo M, Ludwik K, Trejdosiewicz R, Smart CJ, Oliva L, Pagliaro L. Expression of leukocyte adhesion molecules by mucosal mononuclear phagocytes in inflammatory bowel disease. Gastroenternlngy 199 I ; 100: 150-159 28. Hill S, Milla PJ, Bottazzo GF, Mirakian R. Autoimmune enteropathy and colitis: is there a generalized autoimmune gut disorder? Gut 199 I ; 32: 3 6 4 2 29. Maio M, Tessitori G, Pinto A, Temponi M, Colombatti A, Ferrone S. Differential role of distinct determinants of intercellular adhesion molecule- I phenomena. J Immunmology 1989; 143: 181-188 30. Altimann DM, Hogg N, Trowsdale J , Wilkinson D. Co-transfection of ICAM-I and HLADR reconstitutes human antigenpresenting cell function in mouse L cells. Nature 1989; 338: 512-516 31. Bagnasco M, Caretto A, Olive D, Pedini GW, Canonica GW, Betterle C. Expression of ICAM- I on thyroid epithelial cells in Hashimoto’s thyroiditis and not in Graves’ disease or capillary carcinoma. Clin. exp. Immunol. 199 1 ; 83: 309-3 13 32. Ciampolillo A, Napolitano G, Mirakian R, Miyasaki A, Giorgino R, Bottazzo GF. Intercellular adhesion molecule (ICAM-I) in Graves’ disease: in vivo versus in vitro contrasting results. J Autoimmunity (in press) 33. Bottazzo GF, Drexhage H. Recent advances in thyroid autoimmunity. Current Opinion in Immunology (in press) 34. Zheng ROH, Abney ER, Chev CQ, Field M, Maini RN, Lamb J, Feldman M. Detection of in vivo production of T N F a by human thyroid epithelial cells. Immunology 1992; 75: 4 5 6 4 6 2 35. Russell GJ, Hermatz PR. MHC Class I1 expression on enterocytes: to present or not to present. Gastroenterology 1991; 199: 274-276 36. Hashimoto Y, Komer T. Close relationship between the cells of the immune system and the epithelial cells in the rat small intestine. Cell Tissue Res 1988; 254: 4 1 4 7 37. Staunton DE, Merfurri VJ, Rothleim R, Barton R, Marlin SD, Springer TA. Cell adhesion molecule ICAM-1 is the major surface receptor for Rhinovirus. Cell 1989; 56: 849-853
