JOURNAL OF PATHOLOGY, VOL.
164: 15 1-1 58 (I 99 I )
ADHESION OF NORMAL AND NEOPLASTIC LYMPHOID CELLS TO FIBROBLASTS A. S. JACK AND K. J. CHAPMAN
Department ojPathology, University of Leeds, Leeds LS2 9JT, West Yorkshire, U.K. Received 27 July 1990 Accepted11 January 1991
SUMMARY Adhesion between lymphocytes and other cells iscritical to many processes in the normal immune system. Alteration in the expression of cell adhesion molecules may be important in determining the behaviour of malignant lymphomas. In this study, the adhesion of normal lymphocytes to fibroblasts is compared with the adhesion of the T-cell lymphomas 56 and Hut78 ICRF. 56 was significantly more adherent to fibroblasts than either Hut78 or PBL despite the fact that 56 expresses almost no LFA-1. Anti-LFA-1 had little effect on the basal adhesion of Hut78 ICRF or PBL. Addition of anti-CD2 caused enhanced adhesion of 56 and PBL but not Hut78 ICRF, which expresses little of this molecule. This enhancement was abrogated by anti-LFA-l . Anti-CD45 also caused enhanced adhesion of PBL. This was largely due to LFA-1-mediated homotypic cell adhesion. The tumour cell lines display no such homotypic adhesion and the small enhancement of fibroblast adhesion was much less affected by the anti-LFA-1 antibody. These results show the complex interactions which occur between adhesion molecules and that differences in patterns of expression between normal and neoplastic cells could be a major determinant of tumour cell behaviour. KEY
WORDS-Malignant
lymphoma, fibroblastic adhesion
INTRODUCTION It is now recognized that cellular adhesion is of major importance in the normal functioning of the immune system. Antigen presentation’.* and lymphocyte-mediated cytotoxicity3 are examples of adhesion-dependent processes. The adhesion molecules which mediate these processes belong to the integrin and immunoglobulin supergene families. Of central importance is LFA-1 (CDI la/CD18) adhesion to ICAM-I (CD54).46 Other important molecules involved in lymphocyte adhesion include CDlIbjCD18, CDl lc/CD18, and the VLA group of integrin~.’.~ Of particular interest is the role of CD2 as an adhesion molecule which is a direct link between the processes of lymphocyte activation and adhesion.’-’’ Interaction with endothelia and lymphocyte homing are mediated by the LECCAM group of adhesion molecules and possibly Addressee for correspondence: D r A. S. Jack, Department of Pathology, University of Leeds, Leeds LS2 9JT, West Yorkshire, U.K.
0022-3417~91/050151-08 $05.00 Q 1991 by John Wiley & Sons, Ltd.
by CD44.12.’3These are carbohydrate-binding molecules which are structurally distinct from each other and from the immunoglobulin supergene family and integrin family members. CDI Ia/CD18 has also been found to have an ancillary role in the adhesion of lymphocytes to endothe1i~m.l~ The altered expression of adhesion molecules which occurs in some lymphoma^'^ may be important in determining the behaviour of these tumours. Some studies have examined the question of lymphoma endothelial interaction and although it is still unclear how this affects malignant behaviour. A property which characterizes all malignant lymphomas is the displacement and destruction of normal cellular elements in lymph nodes and other tissues. An explanation of this phenomenon could lie in differential adhesion to connective tissue cells and matrix proteins. To begin to investigate this possibility, an in vitro assay has been devised to look for differences in both the extent and the mechanisms of adhesion of lymphoma cells and normal lymphocytes to connective tissue cells.
152
A. S. JACK AND K . J . CHAPMAN
MATERIALS AND METHOD
Cell culture Human embryonic fibroblasts were obtained from Flow Laboratories Ltd. They were maintained in RPMI 1640 (Gibco), containing 10 per cent heatinactivated fetal calf serum, 2 m of ~ glutamine, penicillin and streptomycin, and buffered to pH 7.2 with 3 N morpholinosulphonic acid. cells were passed with magnesium- and calcium-free medium containing 0.5 per cent trypsin and 0.02 per cent EDTA. Peripheral blood lymphocytes (PBLs) were prepared from volunteer donor blood by density centrifugation on Lymphoprep (Nygaard, U.K., Ltd.) and resuspended to 2 x lo7 per ml in RPMI 1640 containing 5 per cent fetal calf serum. The T-cell lymphoma cell lines 56 and Hut78 ICRF were a gift from Professor A. W. Boylston. Both lines were maintained in suspension culture. Adhesion a s s q
The fibroblasts were plated at 5 x 104cellsper well on a 24-well plate (1.75 cm2 growth surface) 48 h prior to utilization. Wells were greater than 70 per cent confluence on the day of use. Cells were not used at confluence because the subsequent rigorous washing of the confluent monolayer led to extensive detachment of fibroblasts and hence underestimation of adhesion. Visual examination of the cultures showed that this was not a problem at around 70 per cent. To compensate for variation in cell density five replicate wells were used per test. Lymphocytes (PBLs. tumour cells) were labelled with medium containing 2.7 MBq/ml of "Cr as (Na,CrO,) or a specific activity of 1.35-2.7 x 10' MBq/g. Cells were maintained in this medium for 1 h at 37°C in a flat-bottomed flask. This removed plastic adherent cells from the peripheral blood preparation. Non-adherent cells were washed in serum, then placed in RPMI 1640 containing 5 per cent fetal calf serum. Cells were then incubated with anti-CD2, anti-LFA- I , anti-CD45, or combinations oftheseantibodies. Detailsoftheantibodies used are stated below. In each case, the cells were incubated for 40 min at 37°C before further washing and resuspending to 2 x lo6 cells per ml. One ml of the suspension containing labelled cells was added to wells containing fibroblasts and the wells were incubated for 40 min at 37°C. After incubation the cells were washed seven times by adding PBS to one side of the well and removing it from the opposite
side. Adherent cells were then lysed in Triton X-100 and the gamma emission of the supernatant was measured. Five replicates were performed for each assay. The specific labelling intensity of the cells was determined from control samples and the proportion of added cells which adhered was then calculated. Additional experiments were performed in which the fibroblast monolayer was pretreated with anti-ICAM-1 and the assay was then performed as above. To validate the assay, a number of preliminary studies were performed. To exclude the possibility that antibody-coated cells were attaching to fibroblasts through Fc receptors, the assay was performed in the presence of varying concentrations of human AB serum in order to saturate any Fc receptors present on the surface of the fibroblasts. The presence of serum in concentrations of up to 20 per cent did not affect the results. The final washing protocol was determined by experiment. Seven washes were found to be required to completely remove plastic adherent lymphoid tumour cells. Performing the assay at 4"C, room temperature, or 37°C had minimal effect on adhesion. For this reason it was decided to perform the study at 37°C. Lymphoid cells coated with the antibodies used in the study were maintained at the temperature for the assay period and studied by indirect immunofluorescence. No significant internalization of antibody was observed. Statistical comparisons were performed using the Student's r-test. Imm unoplieno type All three cell types were labelled with CD2 (Dako Ltd., clone MT910), CD3 (Dako Ltd., clone T34B5), CD45 (Dako Ltd., mixture ofclones 2B11 and PD7/26), CD45RO (Dako Ltd., clone UCHLl), CD25 (Dako Ltd., clone ACT-l), CDI la (Serotec Ltd., clone 25.3. I), and anti-ICAM-1 (Serotect Ltd.) using an indirect immunofluorescence technique and antibodies at previously defined optimal dilutions. These antibodies were also used as described in the adhesion assays. (In some cases, an alternative anti-CD1 l a was used: Serolab clone SPV-L7.) These were measured and counted using an EPICS5 flow cytometer (Coulter Ltd.). Ten thousand cells were counted for each assay. An anti-melanoma antibody which does not react with lymphoid cells served as a negative control. The phenotype of adherent peripheral blood lymphocytes was determined using fibroblasts on
ADHESION OF NORMAL AND NEOPLASTIC LYMPHOCYTES
coverslips rather than on 24-well plates. At the end of the assay period, the coverslip was fixed and stained by the indirect immunoperoxidase technique. The numbers of adherent cells labelled with antibodies to CD3, CD22, CD4, and CD8 were counted. Scanning electron rnicroscopjl Fibroblasts were prepared as above and plated o n acetate coverslips. Adhesion assays were then performed as above, but using unlabelled cells. After washing, the preparations were fixed in 3 per cent buffered glutaraldehyde, dehydrated for 5 min each in graded acetone (30-90 per cent), then for 15 min in two changes of 100 per cent acetone. The specimen was then critical point-dried and gold-coated. The specimens were examined and photographed in a JEOL 1200 electron microscope in the scanning mode. Table I-Lymphocyte
immunophenotypes
Hut78 ICRF Antibody
PBL (Yo)
CD3 -CD2 -CDI l a CD25 CD45 CD45R UCHLl CD4 CD8
81 82 90 2.5 94 51 38 nd nd
56 (Yo)
("/.I ~
81 16 2 0 60 5 10
< 10 < 10
81 0 90 0 85 0 88 60 0
RESULTS Cell phenotype
The expression of a variety of lymphocyte markers by three cell types is shown in Table I. Two points should be noted. First, very few 56 cells constitutively express LFA-I, the few cells which did show a very low intensity of expression (Fig. I). Second, Hut78 ICRF showed a very low level of expression of CD2. In other respects, Hut78 ICRF resembled a peripheral T-cell lymphoma, whereas 56 was more difficult to classify, but probably represents an earfier state of T-cell maturation. Using
153
the same method, 45 per cent of a suspension of fibroblasts was positive with anti-ICAM-I. The phenotype of adherent peripheral lymphocytes was determined by the indirect immunoperoxidase technique. It was found that 89 per cent of adherent cells were T cells; 1 1 per cent were B cells with no macrophages. There was a slight enrichment of T cells with respect to peripheral blood. The CD4:CD8 ratio was 2.4:l. Using a similar method, ICAM- I was found to be expressed on the fibroblast monolayers. Almost all the cells appeared positive. Adhesion assay The data reported here are from representative experiments and represent a mean of five replicate wells with a standard deviation of less than 10 per cent of the mean in each case. In all of these studies, 56 was about twice as adherent to fibroblasts as PBLs or Hut78 ICRF (P < 0.001). This is illustrated in Fig. 2. The addition of anti-LFA-l to cells had no significant effect on Hut78 ICRF or PBLs. As expected, there was no effect on 56, which did not express this molecule on the cell surface. When anti-CD2 was added, there was a significant enhancement of adhesion by the CDZexpressing cells, PBLs (P
164: 15 1-1 58 (I 99 I )
ADHESION OF NORMAL AND NEOPLASTIC LYMPHOID CELLS TO FIBROBLASTS A. S. JACK AND K. J. CHAPMAN
Department ojPathology, University of Leeds, Leeds LS2 9JT, West Yorkshire, U.K. Received 27 July 1990 Accepted11 January 1991
SUMMARY Adhesion between lymphocytes and other cells iscritical to many processes in the normal immune system. Alteration in the expression of cell adhesion molecules may be important in determining the behaviour of malignant lymphomas. In this study, the adhesion of normal lymphocytes to fibroblasts is compared with the adhesion of the T-cell lymphomas 56 and Hut78 ICRF. 56 was significantly more adherent to fibroblasts than either Hut78 or PBL despite the fact that 56 expresses almost no LFA-1. Anti-LFA-1 had little effect on the basal adhesion of Hut78 ICRF or PBL. Addition of anti-CD2 caused enhanced adhesion of 56 and PBL but not Hut78 ICRF, which expresses little of this molecule. This enhancement was abrogated by anti-LFA-l . Anti-CD45 also caused enhanced adhesion of PBL. This was largely due to LFA-1-mediated homotypic cell adhesion. The tumour cell lines display no such homotypic adhesion and the small enhancement of fibroblast adhesion was much less affected by the anti-LFA-1 antibody. These results show the complex interactions which occur between adhesion molecules and that differences in patterns of expression between normal and neoplastic cells could be a major determinant of tumour cell behaviour. KEY
WORDS-Malignant
lymphoma, fibroblastic adhesion
INTRODUCTION It is now recognized that cellular adhesion is of major importance in the normal functioning of the immune system. Antigen presentation’.* and lymphocyte-mediated cytotoxicity3 are examples of adhesion-dependent processes. The adhesion molecules which mediate these processes belong to the integrin and immunoglobulin supergene families. Of central importance is LFA-1 (CDI la/CD18) adhesion to ICAM-I (CD54).46 Other important molecules involved in lymphocyte adhesion include CDlIbjCD18, CDl lc/CD18, and the VLA group of integrin~.’.~ Of particular interest is the role of CD2 as an adhesion molecule which is a direct link between the processes of lymphocyte activation and adhesion.’-’’ Interaction with endothelia and lymphocyte homing are mediated by the LECCAM group of adhesion molecules and possibly Addressee for correspondence: D r A. S. Jack, Department of Pathology, University of Leeds, Leeds LS2 9JT, West Yorkshire, U.K.
0022-3417~91/050151-08 $05.00 Q 1991 by John Wiley & Sons, Ltd.
by CD44.12.’3These are carbohydrate-binding molecules which are structurally distinct from each other and from the immunoglobulin supergene family and integrin family members. CDI Ia/CD18 has also been found to have an ancillary role in the adhesion of lymphocytes to endothe1i~m.l~ The altered expression of adhesion molecules which occurs in some lymphoma^'^ may be important in determining the behaviour of these tumours. Some studies have examined the question of lymphoma endothelial interaction and although it is still unclear how this affects malignant behaviour. A property which characterizes all malignant lymphomas is the displacement and destruction of normal cellular elements in lymph nodes and other tissues. An explanation of this phenomenon could lie in differential adhesion to connective tissue cells and matrix proteins. To begin to investigate this possibility, an in vitro assay has been devised to look for differences in both the extent and the mechanisms of adhesion of lymphoma cells and normal lymphocytes to connective tissue cells.
152
A. S. JACK AND K . J . CHAPMAN
MATERIALS AND METHOD
Cell culture Human embryonic fibroblasts were obtained from Flow Laboratories Ltd. They were maintained in RPMI 1640 (Gibco), containing 10 per cent heatinactivated fetal calf serum, 2 m of ~ glutamine, penicillin and streptomycin, and buffered to pH 7.2 with 3 N morpholinosulphonic acid. cells were passed with magnesium- and calcium-free medium containing 0.5 per cent trypsin and 0.02 per cent EDTA. Peripheral blood lymphocytes (PBLs) were prepared from volunteer donor blood by density centrifugation on Lymphoprep (Nygaard, U.K., Ltd.) and resuspended to 2 x lo7 per ml in RPMI 1640 containing 5 per cent fetal calf serum. The T-cell lymphoma cell lines 56 and Hut78 ICRF were a gift from Professor A. W. Boylston. Both lines were maintained in suspension culture. Adhesion a s s q
The fibroblasts were plated at 5 x 104cellsper well on a 24-well plate (1.75 cm2 growth surface) 48 h prior to utilization. Wells were greater than 70 per cent confluence on the day of use. Cells were not used at confluence because the subsequent rigorous washing of the confluent monolayer led to extensive detachment of fibroblasts and hence underestimation of adhesion. Visual examination of the cultures showed that this was not a problem at around 70 per cent. To compensate for variation in cell density five replicate wells were used per test. Lymphocytes (PBLs. tumour cells) were labelled with medium containing 2.7 MBq/ml of "Cr as (Na,CrO,) or a specific activity of 1.35-2.7 x 10' MBq/g. Cells were maintained in this medium for 1 h at 37°C in a flat-bottomed flask. This removed plastic adherent cells from the peripheral blood preparation. Non-adherent cells were washed in serum, then placed in RPMI 1640 containing 5 per cent fetal calf serum. Cells were then incubated with anti-CD2, anti-LFA- I , anti-CD45, or combinations oftheseantibodies. Detailsoftheantibodies used are stated below. In each case, the cells were incubated for 40 min at 37°C before further washing and resuspending to 2 x lo6 cells per ml. One ml of the suspension containing labelled cells was added to wells containing fibroblasts and the wells were incubated for 40 min at 37°C. After incubation the cells were washed seven times by adding PBS to one side of the well and removing it from the opposite
side. Adherent cells were then lysed in Triton X-100 and the gamma emission of the supernatant was measured. Five replicates were performed for each assay. The specific labelling intensity of the cells was determined from control samples and the proportion of added cells which adhered was then calculated. Additional experiments were performed in which the fibroblast monolayer was pretreated with anti-ICAM-1 and the assay was then performed as above. To validate the assay, a number of preliminary studies were performed. To exclude the possibility that antibody-coated cells were attaching to fibroblasts through Fc receptors, the assay was performed in the presence of varying concentrations of human AB serum in order to saturate any Fc receptors present on the surface of the fibroblasts. The presence of serum in concentrations of up to 20 per cent did not affect the results. The final washing protocol was determined by experiment. Seven washes were found to be required to completely remove plastic adherent lymphoid tumour cells. Performing the assay at 4"C, room temperature, or 37°C had minimal effect on adhesion. For this reason it was decided to perform the study at 37°C. Lymphoid cells coated with the antibodies used in the study were maintained at the temperature for the assay period and studied by indirect immunofluorescence. No significant internalization of antibody was observed. Statistical comparisons were performed using the Student's r-test. Imm unoplieno type All three cell types were labelled with CD2 (Dako Ltd., clone MT910), CD3 (Dako Ltd., clone T34B5), CD45 (Dako Ltd., mixture ofclones 2B11 and PD7/26), CD45RO (Dako Ltd., clone UCHLl), CD25 (Dako Ltd., clone ACT-l), CDI la (Serotec Ltd., clone 25.3. I), and anti-ICAM-1 (Serotect Ltd.) using an indirect immunofluorescence technique and antibodies at previously defined optimal dilutions. These antibodies were also used as described in the adhesion assays. (In some cases, an alternative anti-CD1 l a was used: Serolab clone SPV-L7.) These were measured and counted using an EPICS5 flow cytometer (Coulter Ltd.). Ten thousand cells were counted for each assay. An anti-melanoma antibody which does not react with lymphoid cells served as a negative control. The phenotype of adherent peripheral blood lymphocytes was determined using fibroblasts on
ADHESION OF NORMAL AND NEOPLASTIC LYMPHOCYTES
coverslips rather than on 24-well plates. At the end of the assay period, the coverslip was fixed and stained by the indirect immunoperoxidase technique. The numbers of adherent cells labelled with antibodies to CD3, CD22, CD4, and CD8 were counted. Scanning electron rnicroscopjl Fibroblasts were prepared as above and plated o n acetate coverslips. Adhesion assays were then performed as above, but using unlabelled cells. After washing, the preparations were fixed in 3 per cent buffered glutaraldehyde, dehydrated for 5 min each in graded acetone (30-90 per cent), then for 15 min in two changes of 100 per cent acetone. The specimen was then critical point-dried and gold-coated. The specimens were examined and photographed in a JEOL 1200 electron microscope in the scanning mode. Table I-Lymphocyte
immunophenotypes
Hut78 ICRF Antibody
PBL (Yo)
CD3 -CD2 -CDI l a CD25 CD45 CD45R UCHLl CD4 CD8
81 82 90 2.5 94 51 38 nd nd
56 (Yo)
("/.I ~
81 16 2 0 60 5 10
< 10 < 10
81 0 90 0 85 0 88 60 0
RESULTS Cell phenotype
The expression of a variety of lymphocyte markers by three cell types is shown in Table I. Two points should be noted. First, very few 56 cells constitutively express LFA-I, the few cells which did show a very low intensity of expression (Fig. I). Second, Hut78 ICRF showed a very low level of expression of CD2. In other respects, Hut78 ICRF resembled a peripheral T-cell lymphoma, whereas 56 was more difficult to classify, but probably represents an earfier state of T-cell maturation. Using
153
the same method, 45 per cent of a suspension of fibroblasts was positive with anti-ICAM-I. The phenotype of adherent peripheral lymphocytes was determined by the indirect immunoperoxidase technique. It was found that 89 per cent of adherent cells were T cells; 1 1 per cent were B cells with no macrophages. There was a slight enrichment of T cells with respect to peripheral blood. The CD4:CD8 ratio was 2.4:l. Using a similar method, ICAM- I was found to be expressed on the fibroblast monolayers. Almost all the cells appeared positive. Adhesion assay The data reported here are from representative experiments and represent a mean of five replicate wells with a standard deviation of less than 10 per cent of the mean in each case. In all of these studies, 56 was about twice as adherent to fibroblasts as PBLs or Hut78 ICRF (P < 0.001). This is illustrated in Fig. 2. The addition of anti-LFA-l to cells had no significant effect on Hut78 ICRF or PBLs. As expected, there was no effect on 56, which did not express this molecule on the cell surface. When anti-CD2 was added, there was a significant enhancement of adhesion by the CDZexpressing cells, PBLs (P
