Int. J. Cancer: 20, 881-886 (1977)
EBV SPECIFIC SECRETORY IgA IN SALIVA OF NPC PATIENTS. PRESENCE OF SECRETORY PIECE IN EPITHELIAL MALIGNANT CELLS C. DESGRANGES I, J. Y. LI and G . DE-THB Unit of Biological Carcinogenesis, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyons; and Laboratoire d'Histologie et d'Embryologie, UER MPdicale Lyon Sud- Ouest, 69600 Ouflins, France
Saliva samples from 59 patients with nasopharyngeal carcinoma (NPC) and from 20 normal individuals were studied to determine the nature and origin of the EBV-specific IgA in N P C saliva. About 50% of N P C saliva samples contained secretory IgA specific for EBV. The corresponding tumor IgA(a) was found in plasma cells surrounding the epithelial tumor cells and the secretory piece at the surface of epithelial cells. A slightly higher proportion of NPC saliva samples containing IgA was found in patients from Tunis than in samples from Hong Kong. Attention is drawn to the clinical value of the salivary IgA in diagnosis and monitoring of treatment of NPC.
Jijoye cells stained with fluorescein isothiocyanate conjugated anti-human IgA specific for alpha chains (FITC anti-a) and an anti-human secretory piece of IgA (FITC-anti-SP) both from Dakopatts (Copenhagen, Denmark). The specificity of the IgA(u) conjugate was tested on smears of myeloma cells and for IgA(SP) with positive sera for IgG, IgA(u) and IgM. Gel filtration analysis of EBV IgA antibodies from saliva samples
One-ml samples of IgA(u) and SP-positive saliva The presence of EBV-specific IgA in serum and (HK 976, Tu 21, Tu 19) were applied to columns saliva from nasopharyngeal carcinoma (NPC) of 6B Sepharose in 0.01 M Tris HCI buffer PH 7.2. patients was recently described (Henle and Henle, Fractions (3.5 ml) were collected, analyzed for 1976; Desgranges et al., 1977). As the lympho- absorbance at 280 nm, grouped by three, lyophilized, epithelium in the oro-pharynx is a site where IgA and resuspended in 0.5 ml distilled water. Each is synthesized (Tomasi et a/., 1965; Strober et a/., fraction was tested by immunofluorescence on 1970; Brandtzaeg and Baklien, 1976), an obvious fixed Jijoye cells for anti-EBV IgA(u), IgA(SP), question is whether the EBV-specific IgA in NPC IgG and IgM. patients could have a local origin. The present study was therefore done to determine the nature Specificity of anti-EBV IgA isolated from nasoof the 11s secretory IgA, or of 7s(a) chains, and to pharyngeal tumors search in the oro-pharynx for cells producing EBVThe specificity of anti-EBV IgA was determined specific IgA(a) as well as the secretory piece (SP). for five NPC tumors as follows: the tumors were carefully ground with sand in PBS; then, after centrifugation at 400 g for 15 min, the supernatant MATERIAL AND METHODS was tested for anti-EBV/IgA(u) and SP on Jijoye Saliva smears. As controls, extracts from inflamed adenoids Most of the saliva samples used for the present or tonsils, normal nasopharyngeal mucosa and study were described earlier (Desgranges et al., other head and neck tumors were used. 1977). A total of 79 saliva samples were studied: 30 from untreated and 8 from cobalt-treated Tunisian Immunohistopathological studies NPC patients (collected by Dr. Ellouz, Institut After fixation for 1-3 days in Bouin-Holland Salah Azaiz, Tunis); 21 from untreated Chinese NPC patients from Hong Kong (collected by fluid, without acetic acid, and with the addition Dr. J. H. C. Ho, Queen Elizabeth Hospital, Hong of 5 % saturated sublimate, the NPC biopsies were Kong); and 10 from normal individuals in both carefully washed in tap water, dehydrated and embedded in paraffin. Four-,um thick sections Tunis and Hong Kong. were put on glass slides with 0.5% aqueous gelatin Detection of EBV-specific IgA (u) and SP in saliva and treated with the various antisera, overnight at 4"C in a moist chamber. The sections were samples 1
The presence of IgA reactivity to EBVIVCA in saliva was detected using acetone-fixed smears of
Received: August 4, 1977.
882
DESGRANGES ET AL.
then rinsed in phosphate-buffered saline solution (PBS) at PH 7.4, washed for 1 h with three changes of PBS, and counterstained with Evans’ blue (1/10,000) for 10min. The sections were mounted in buffered glycerol and inspected with a fluorescence microscope. Photographs were taken using a Kodak high-speed Ektachrome film. Control experiments included treatment of tumor sections with normal rabbit serum and with nonconjugated specific antisera to block the staining by the fluorescein-conjugated sera. To facilitate localization of the different cells in the tumors, consecutive sections were stained either for immunofluorescence (IF) or for pathological examination by haemalum-erythrosine. Antisera
T o localize IgA(a) chain a fluorescein-conjugated antiserum (F/1092, Dakopatts, Copenhagen) and a horseradish peroxidase-labelled antiserum to human IgA(a) (Institut Pasteur, Paris) were used at a 1/40 dilution. To localize SP we first used a fluorescein-conjugated antiserum to SP (FIOSP, Dakopatts, Copenhagen) and, later, a non-conjugated antiserum a horseradish to SP followed by a FITC-or peroxidase-labelled sheep anti-rabbit y-globulin (Institut Pasteur, Paris). In the latter case the reaction product was revealed with 3-3’ diaminobenzidine (DAB) according to the method of Graham and Karnovsky (I 966). In attempts to localize cells producing EBVspecific immunoglobulins in tumors, we adsorbed concentrated P3HR.l virus (10 times concentrated) directly on Bouin-fixed sections. The sections were then washed very carefully for 1 h in PBS to remove non-specifically adsorbed virus particles. The remaining virus particles were probably bound to antiVCA antibodies in the sections and could be detected by treatment with a rabbit anti-VCA ( I /1280) antiserum obtained by immunization of a rabbit with purified and concentrated EBV, followed by staining with a FITC-conjugated goat antiserum to rabbit 7s y-globulin (Hyland, Costa Mesa, Calif.). After microscopic localization of the cells containing anti-EBV immunoglobulins, the FITC rabbit antiserum was dissociated from these positive cells by overnight treatment with a glycine buffer according to Nakane (1968). Finally, a second FITC-conjugated serum specific for IgA (a) was used for staining of IgA-producing cells in the same section. The previously positive cells, if positive also after this second step, were considered positive for EBV-specific IgA(a). The many staining steps were possible sources of artefacts. However, repeated experiments with similar results and the use of controls including non-
immunized rabbit serum, or omission of virus adsorption in the staining procedure, made us confident that the results were correct. EBV nuclear antigen ( E B N A ) in tumor sections The anti-complement immunofluorescence test (ACIF) as described by Reedman and Klein (1973) was carried out on frozen sect,ions of tumors (6 prn thick) after fixation for 2 min in acetone-methanol. Two EBNA-positive reference sera were used: RKOOl (INSERM reference) and Tu 119 (a Tunisian NPC) and one EBNA-negative reference RK003 (INSERM reference). RESULTS
Secretory nature of IgA in NPC saliva as seen by immunofluorescence
In a series of 21 saliva samples from non-treated NPC patients from Hong Kong 9 were positive for EBV/IgA(a) (43%) and 7 were positive for SP (34%) (Table I). Out of 29 NPC saliva samples from Tunisia, 18 were positive for EBV/IgA(a) (62%) and 15 were positive for SP (51%). But when the Tunisian saliva samples were divided into two groups (Table TI), according to the patients’ age (under or over 20 years), 20% were anti-EBV IgA(a) positive in the first group against 84% in the second, and 20% and 68% respectively for the secretory piece. TABLE I ANTI-EBV/IgA ( a ) A N D (SP) IN NPC SALIVA SAMPLES AS DETECTED BY IMMUNOFLUORESCENCE STAINING OF JIJOYE CELL SMEARS Hong Kong
EBV/IgA (a)-
Tunis
9/21
(43 %)
18/29
(02 %)
7/21
(34%)
15/29
(51 %)
positive EBV/IgA (SP)-
positive
TABLE I1 ANTI-EBV/IsA (a)A N D SP IN TUNISIAN NPC SALIVA SAMPLES DETECTED BY IMMUNOFLUORESCENCE Age
EBV/IgA (a)-
positive
EBV/IgA (SP)-
positive
221 years G20years >21 years G20years
% positive
16/19 2/10 13/19 2/10
(84%) (20%) (68%) (20%)
SECRETORY IGA
IN
NPC SALIVA AND TUMORS
ANTI
883
S P / I F lvca)
FIGURE 1
Gel filtration analysis on 6B Sepharose of an NPC case saliva sample (HK976). ANTI I R A D ( / I F l V C a )
ANTI I g G / I F l v c a i 16
8 4
Isolation of I g A ( a ) and SP from NPC saliva by chromatography
Figure 1 gives the profile obtained by separation of 1 ml of unconcentrated saliva (positive for IgA(a) and SP) (HK976) on Sepharose 6B. After lyophilization of the different fractions, their antiEBV IgA(u), IgA(SP), IgG and IgM reactivities were tested by indirect immunofluorescence on Jijoye cells. Using IgG as a 7s marker 7s IgA(a) was found in fractions 43 to 54, while 11s IgA (SP) was detected in fractions 37 to 40. No EBV/IgM activity was found in any fraction. EB V-specificIgA in tumor extracts The tumor extracts obtained as described above were put on Jijoye cells and tested for anti-EBVIgA(a) and anti-EBV-IgA(SP). Tumor extracts from patients positive for anti-EBV-IgA in the saliva were also EBV-IgA positive. The amount of anti-EBV-IgA(SP) in tumors was regularly lower than that of anti-EBV-IgA(u). Figure 2 shows IgA ( u ) in a Bouin-fixed Tunisian NPC (Tu601). Note that the positive cells are plasma cells surrounding epithelial tumor cells. Figure 3 shows another tumor (Tu40) having
FrGURE2
IgA (a) in plasma cells surrounding epithelial tumor cells (Tu601) (staining with fluorescein). x 300.
884
DESGRANGES ET AL.
plasma cells positive for IgA(a) (staining with peroxidase). The localization of the secretory piece in tumor tissue is given in Figures 4 and 5 . Figure 4 shows the presence of SP in the epithelium and the lumen of the glandular acini and ductules (staining with peroxidase), while Figure 5 shows epithelial tumor cells surface-positive for SP in I F ; the same cells were positive for EBNA in the ACIF test. In Figure 6, plasma cells previously stained positive for anti-EBV immunoglobulins (see “ Material
FIGURE 3 IgA ( a ) in plasma cells surrounding epithelial tumor cells (Tu40) (staining with peroxidase). x 160.
FIGURE 5 IgA (SP) on the surface of epithelial tumor cells (Tu603) (staining with fluorescein). x 300.
and Methods”) in a second staining step positive also for lgA(a), and these cells are accordingly considered as positive for anti-EBV/IgA(a). DISCUSSION
FIGURE 4 TgA (SP) in the glandular acini of an NPC biopsy (Tu602) (staining with peroxidase). x 320.
The marked increase of TgA in sera from NPC patients (Wara et al., 1975) is due to specific EBV antibodies (Henle and Henle, 1976). Recently, NPC throat-washings were also found to contain anti-EBV/IgA (Desgranges et at,, 1977; Ho et a/., 1977). We show here that these TgA molecules are mainly of a secretory nature (11s) and are produced in the nasopharyngeal tumors themselves.
SECRETORY IGA IN NPC SALIVA AND TUMORS
FIGURE 6
Anti-EBV/IgA (a) positive plasma celles (staining with fluorescein). x 470. IgA immunoglobulins of unknown specificity have been detected in the saliva of patients with oral cancer (Brown et a/., 1975), oropharyngeal and bronchopulmonary carcinomas (Mandel et al., 1973). In these cancers, as in NPC, the salivary IgA seems to be due to a local secretory response. The EBV specificity of these antibodies should be looked for, although in a previous study we did not detect EBV-specific IgA in 9 saliva samples from patients with head and neck tumors other than NPC. On the other hand, elevated titers of serum IgA were found in patients with cancers of epithelial secretory organs such as prostate (Ablin et al., 1972), uterus (Vasudevan et al., 1971) and lung (Waldman et a/., 1970). In these cancers
885
the elevation of serum IgA titers appears to result from a filtration of local secretory IgA into the circulating blood (Thompson and Asquith, 1970). In our hands no anti-EBVfIgA was found in throat washings of 10 Burkitt lymphoma patients, 20 infectious mononucleosis patients and 10 other conditions associated with EBV but only in NPC patients. This suggests an origin of EBV/IgA in the tumor itself, which reflects the involvement of both the epithelial cells and the lymphocytes and plasma cells in the tumor formation process. The differences observed between saliva samples obtained from Hong Kong and Tunis are not understood. In Hong Kong, Ng et al. (1977) obtained results similar to ours. The observation that young NPC patients in Tunisia lack IgA in the saliva probably reflects differences in the maturity of the local immune system of the Waldeyer ring (oropharyngeal synthesis of IgA in children is much lower than in adults, see L a m , 1976). The mechanism of secretion and transport of IgA in NPC may follow the model proposed by Tourville et a/. (1969) for normal mucosa. The IgA(u) is produced in plasma cells while SP is secreted by the epithelial cells lining the ducts of the salivary glands. In the biopsies studied here, SP was present on the surface of the epithelial tumor cells. The role of the EBV-specific IgA antibodies present in the tumor is not clear. They may inhibit the activity of sensitized T killer cells and therefore favor tumor growth. The diagnostic and prognostic use of IgA in NPC patients has been mentioned previously (Desgranges and deTh6, 1977). Their potential value in early detection of the tumor and in monitoring local relapse deserve further investigation. To this end, a follow-up study of NPC patients and a prospective epidemiological study on a cohort at high risk for NPC has been proposed. ACKNOWLEDGEMENTS
This work was supported by contract No. NO1 CP 43296 with the Virus Cancer Program of the National Cancer Institute, NIH, USA.
IgA SECRETOIRES SPECIFIQUES D E L’EBV DANS LA SALIVE D E MALADES NPC. PRESENCE D E PIECES SECRETOIRES SUR LES CELLULES TUMORALES EPITHELIALES La salive de 59 malades porteurs de cancer du rhinopharynx (NPC), originaires de Hong Kong et de Tunis, et de 20 t h o i n s , a BtB etudiee pour determiner la nature et I’origine des IgA spbcifiques de I’EBV, niises e n Bvidence r6cernrnent par Desgranges et coll. (1977). La moitie des salives de rnalades NPC contenait des IgA/EBV de nature s6cr6toire tandis que les turneurs correspondantes montraient la prbence d’lgA, dont certaines spbifiques de I’EBV dans les plasmocytes entouranr les travkes de cellules tumorales epitheliales, lesquelles possbdaient des pieces secretoires A leur surface. 11 est apparu que les salives NPC en provenance de Tunis contenaient plus souvent des IgA/EBV que celles originaires de Hong Kong. Ce point ainsi que la valeur diagnostique, pronostique et de surveillance du traiternent sont discutks.
886
DESGRANGES ET AL. REFERENCES
AHLIN,R., GORDEN,M. J., and SOANE,W. A., Level of immunoglobulin in the serum of patients with carcinoma of the prostate. Neoplasma, 19, 57-60 (1972). BRANDTZAEG, P., and BAKLIEN, K., Immunohistochemical studies of the formation and epithelial transport of immunoglobulins in normal and diseased human intestinal mucosa. Scand. J. Gasrroenterol., 11 (Suppl. 36), 1-45 (1976). BROWN,A. M., LALLY,E. T., FRANKEL, A., HARWICK, R.. DAVIS,L. W., and ROMINGER, C. J., The association of the IgA levels of serum and whole saliva with the progression of oral cancer. Cancer, 35, 1154-1162 (1975). DESGRANGES, C., and DE-THC, G., Presence of EpsteinBarr virus (EBV) specific IgA in saliva of nasopharyngeal carcinoma (NPC) patients: their activity, origin and possible clinical value. In; G. de-The and Y.Ito (ed.), Nasopharyngeal Carcinoma-Etiology and Control, International Agency for Research on Cancer (IARC Scientific Publications No. 18). Lyons (in press). DESGRANGES, C., DE-THB, G., Ho, J. H. C., and ELLOUZ,R., Neutralizing EBV-specific I g A in throat washings of nasopharyngeal carcinoma (NPC) patients. Int. 1. Cancer, 19, 627-633 (1 977). GRAHAM, R. C., and KARNOVSKY, M. J., The early stages of absorption of infected horse-radish peroxidase into the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Hisrochem. Cytochem., 14, 291-302 (1966). HENLE,G., and HENLE,W., Epstein-Barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma. Int. J. Cancer, 17, 1-7 (1976). Ho, H. C., NG, M. H., and KWAN,H. C., IgA antibodies to Epstein-Barr viral capsid antigens in saliva of nasopharyngeal carcinoma patients. Brit. J . Cancer, 35, 888-890 (1977). LAMM,M., Cellular aspects of immunoglobulin A. Advanr. Immunol., 22, 223-290 ( I 976). MANDEL, M. A., DVORAK, K., and DECOSSE, J. J., Salivary immunoglobulins in patients with oropharyngeal and bronchopulmonary carcinoma. Cancer, 31, 1408-1413 (1973).
NAKANE,P. K., Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J. Histochem. Cytochem., 16, 557-560 (1968). N c , M. H., The genetic and the antigenic basis for the IgA antibody responses t o infection with EBV. In: G. de-The and Y. Ito (ed.), Nasopharyngeal Carcinoma-Etiology and Control, International Agency for Research on Cancer (IARC Scientific Publications No. 18), Lyons (in press). REEDMAN, B. M., and KLEIN, G., Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J . Cancer, 11, 499-520 (1973). STROBER, W., BLAESE, R. M., and WALDMANN, T. A., The origin of salivary IgA. J. lab. clin. Med., 75, 856-862 (1970).
THOMPSON, R. A., and ASQUITH,P., Quantitation of exocrine IgA in human serum in health and disease. Clin. exp. Immunol., 7, 491-500 (1970). TOMASI, T. B., TAN,E. M., SOLOMON, A., and PENDERCAST,R. A., Characteristics of an immune system common t o certain external secretions. J . exp. Med., 121, 101-124 (1965). TOURVILLE, D. R., ADLER,R. H., BIENENSTOCK, J., and TOMASI, T. B., The human secretory immunoglobulin systey,: iinmunohistological localization of yA, secretory " piece , and lactoferrin in normal human tissues. J. exp. Mzd., 129,411-423 (1969). VASUDEVAN, D. M., BALAKRISHNAN, K., and TALWAR, G. P., Immunoglobulins in carcinoma of the cervix. Ind. J. med. Res., 59, 1653-1659 (1971). WALDMAN,R . H., MACK, J. P., STELLA,M. M., and ROWE,D. S., Secretory IgA in human serum. J . Immunol.. 105, 43-47 (1970). WARA, W. M., WARA, D. W., PHILLIPS,T. L., and AMMAN, A. J., Elevated IgA in carcinoma of the nasopharynx Cancer, 35, 1313-1315 (1975).
EBV SPECIFIC SECRETORY IgA IN SALIVA OF NPC PATIENTS. PRESENCE OF SECRETORY PIECE IN EPITHELIAL MALIGNANT CELLS C. DESGRANGES I, J. Y. LI and G . DE-THB Unit of Biological Carcinogenesis, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyons; and Laboratoire d'Histologie et d'Embryologie, UER MPdicale Lyon Sud- Ouest, 69600 Ouflins, France
Saliva samples from 59 patients with nasopharyngeal carcinoma (NPC) and from 20 normal individuals were studied to determine the nature and origin of the EBV-specific IgA in N P C saliva. About 50% of N P C saliva samples contained secretory IgA specific for EBV. The corresponding tumor IgA(a) was found in plasma cells surrounding the epithelial tumor cells and the secretory piece at the surface of epithelial cells. A slightly higher proportion of NPC saliva samples containing IgA was found in patients from Tunis than in samples from Hong Kong. Attention is drawn to the clinical value of the salivary IgA in diagnosis and monitoring of treatment of NPC.
Jijoye cells stained with fluorescein isothiocyanate conjugated anti-human IgA specific for alpha chains (FITC anti-a) and an anti-human secretory piece of IgA (FITC-anti-SP) both from Dakopatts (Copenhagen, Denmark). The specificity of the IgA(u) conjugate was tested on smears of myeloma cells and for IgA(SP) with positive sera for IgG, IgA(u) and IgM. Gel filtration analysis of EBV IgA antibodies from saliva samples
One-ml samples of IgA(u) and SP-positive saliva The presence of EBV-specific IgA in serum and (HK 976, Tu 21, Tu 19) were applied to columns saliva from nasopharyngeal carcinoma (NPC) of 6B Sepharose in 0.01 M Tris HCI buffer PH 7.2. patients was recently described (Henle and Henle, Fractions (3.5 ml) were collected, analyzed for 1976; Desgranges et al., 1977). As the lympho- absorbance at 280 nm, grouped by three, lyophilized, epithelium in the oro-pharynx is a site where IgA and resuspended in 0.5 ml distilled water. Each is synthesized (Tomasi et a/., 1965; Strober et a/., fraction was tested by immunofluorescence on 1970; Brandtzaeg and Baklien, 1976), an obvious fixed Jijoye cells for anti-EBV IgA(u), IgA(SP), question is whether the EBV-specific IgA in NPC IgG and IgM. patients could have a local origin. The present study was therefore done to determine the nature Specificity of anti-EBV IgA isolated from nasoof the 11s secretory IgA, or of 7s(a) chains, and to pharyngeal tumors search in the oro-pharynx for cells producing EBVThe specificity of anti-EBV IgA was determined specific IgA(a) as well as the secretory piece (SP). for five NPC tumors as follows: the tumors were carefully ground with sand in PBS; then, after centrifugation at 400 g for 15 min, the supernatant MATERIAL AND METHODS was tested for anti-EBV/IgA(u) and SP on Jijoye Saliva smears. As controls, extracts from inflamed adenoids Most of the saliva samples used for the present or tonsils, normal nasopharyngeal mucosa and study were described earlier (Desgranges et al., other head and neck tumors were used. 1977). A total of 79 saliva samples were studied: 30 from untreated and 8 from cobalt-treated Tunisian Immunohistopathological studies NPC patients (collected by Dr. Ellouz, Institut After fixation for 1-3 days in Bouin-Holland Salah Azaiz, Tunis); 21 from untreated Chinese NPC patients from Hong Kong (collected by fluid, without acetic acid, and with the addition Dr. J. H. C. Ho, Queen Elizabeth Hospital, Hong of 5 % saturated sublimate, the NPC biopsies were Kong); and 10 from normal individuals in both carefully washed in tap water, dehydrated and embedded in paraffin. Four-,um thick sections Tunis and Hong Kong. were put on glass slides with 0.5% aqueous gelatin Detection of EBV-specific IgA (u) and SP in saliva and treated with the various antisera, overnight at 4"C in a moist chamber. The sections were samples 1
The presence of IgA reactivity to EBVIVCA in saliva was detected using acetone-fixed smears of
Received: August 4, 1977.
882
DESGRANGES ET AL.
then rinsed in phosphate-buffered saline solution (PBS) at PH 7.4, washed for 1 h with three changes of PBS, and counterstained with Evans’ blue (1/10,000) for 10min. The sections were mounted in buffered glycerol and inspected with a fluorescence microscope. Photographs were taken using a Kodak high-speed Ektachrome film. Control experiments included treatment of tumor sections with normal rabbit serum and with nonconjugated specific antisera to block the staining by the fluorescein-conjugated sera. To facilitate localization of the different cells in the tumors, consecutive sections were stained either for immunofluorescence (IF) or for pathological examination by haemalum-erythrosine. Antisera
T o localize IgA(a) chain a fluorescein-conjugated antiserum (F/1092, Dakopatts, Copenhagen) and a horseradish peroxidase-labelled antiserum to human IgA(a) (Institut Pasteur, Paris) were used at a 1/40 dilution. To localize SP we first used a fluorescein-conjugated antiserum to SP (FIOSP, Dakopatts, Copenhagen) and, later, a non-conjugated antiserum a horseradish to SP followed by a FITC-or peroxidase-labelled sheep anti-rabbit y-globulin (Institut Pasteur, Paris). In the latter case the reaction product was revealed with 3-3’ diaminobenzidine (DAB) according to the method of Graham and Karnovsky (I 966). In attempts to localize cells producing EBVspecific immunoglobulins in tumors, we adsorbed concentrated P3HR.l virus (10 times concentrated) directly on Bouin-fixed sections. The sections were then washed very carefully for 1 h in PBS to remove non-specifically adsorbed virus particles. The remaining virus particles were probably bound to antiVCA antibodies in the sections and could be detected by treatment with a rabbit anti-VCA ( I /1280) antiserum obtained by immunization of a rabbit with purified and concentrated EBV, followed by staining with a FITC-conjugated goat antiserum to rabbit 7s y-globulin (Hyland, Costa Mesa, Calif.). After microscopic localization of the cells containing anti-EBV immunoglobulins, the FITC rabbit antiserum was dissociated from these positive cells by overnight treatment with a glycine buffer according to Nakane (1968). Finally, a second FITC-conjugated serum specific for IgA (a) was used for staining of IgA-producing cells in the same section. The previously positive cells, if positive also after this second step, were considered positive for EBV-specific IgA(a). The many staining steps were possible sources of artefacts. However, repeated experiments with similar results and the use of controls including non-
immunized rabbit serum, or omission of virus adsorption in the staining procedure, made us confident that the results were correct. EBV nuclear antigen ( E B N A ) in tumor sections The anti-complement immunofluorescence test (ACIF) as described by Reedman and Klein (1973) was carried out on frozen sect,ions of tumors (6 prn thick) after fixation for 2 min in acetone-methanol. Two EBNA-positive reference sera were used: RKOOl (INSERM reference) and Tu 119 (a Tunisian NPC) and one EBNA-negative reference RK003 (INSERM reference). RESULTS
Secretory nature of IgA in NPC saliva as seen by immunofluorescence
In a series of 21 saliva samples from non-treated NPC patients from Hong Kong 9 were positive for EBV/IgA(a) (43%) and 7 were positive for SP (34%) (Table I). Out of 29 NPC saliva samples from Tunisia, 18 were positive for EBV/IgA(a) (62%) and 15 were positive for SP (51%). But when the Tunisian saliva samples were divided into two groups (Table TI), according to the patients’ age (under or over 20 years), 20% were anti-EBV IgA(a) positive in the first group against 84% in the second, and 20% and 68% respectively for the secretory piece. TABLE I ANTI-EBV/IgA ( a ) A N D (SP) IN NPC SALIVA SAMPLES AS DETECTED BY IMMUNOFLUORESCENCE STAINING OF JIJOYE CELL SMEARS Hong Kong
EBV/IgA (a)-
Tunis
9/21
(43 %)
18/29
(02 %)
7/21
(34%)
15/29
(51 %)
positive EBV/IgA (SP)-
positive
TABLE I1 ANTI-EBV/IsA (a)A N D SP IN TUNISIAN NPC SALIVA SAMPLES DETECTED BY IMMUNOFLUORESCENCE Age
EBV/IgA (a)-
positive
EBV/IgA (SP)-
positive
221 years G20years >21 years G20years
% positive
16/19 2/10 13/19 2/10
(84%) (20%) (68%) (20%)
SECRETORY IGA
IN
NPC SALIVA AND TUMORS
ANTI
883
S P / I F lvca)
FIGURE 1
Gel filtration analysis on 6B Sepharose of an NPC case saliva sample (HK976). ANTI I R A D ( / I F l V C a )
ANTI I g G / I F l v c a i 16
8 4
Isolation of I g A ( a ) and SP from NPC saliva by chromatography
Figure 1 gives the profile obtained by separation of 1 ml of unconcentrated saliva (positive for IgA(a) and SP) (HK976) on Sepharose 6B. After lyophilization of the different fractions, their antiEBV IgA(u), IgA(SP), IgG and IgM reactivities were tested by indirect immunofluorescence on Jijoye cells. Using IgG as a 7s marker 7s IgA(a) was found in fractions 43 to 54, while 11s IgA (SP) was detected in fractions 37 to 40. No EBV/IgM activity was found in any fraction. EB V-specificIgA in tumor extracts The tumor extracts obtained as described above were put on Jijoye cells and tested for anti-EBVIgA(a) and anti-EBV-IgA(SP). Tumor extracts from patients positive for anti-EBV-IgA in the saliva were also EBV-IgA positive. The amount of anti-EBV-IgA(SP) in tumors was regularly lower than that of anti-EBV-IgA(u). Figure 2 shows IgA ( u ) in a Bouin-fixed Tunisian NPC (Tu601). Note that the positive cells are plasma cells surrounding epithelial tumor cells. Figure 3 shows another tumor (Tu40) having
FrGURE2
IgA (a) in plasma cells surrounding epithelial tumor cells (Tu601) (staining with fluorescein). x 300.
884
DESGRANGES ET AL.
plasma cells positive for IgA(a) (staining with peroxidase). The localization of the secretory piece in tumor tissue is given in Figures 4 and 5 . Figure 4 shows the presence of SP in the epithelium and the lumen of the glandular acini and ductules (staining with peroxidase), while Figure 5 shows epithelial tumor cells surface-positive for SP in I F ; the same cells were positive for EBNA in the ACIF test. In Figure 6, plasma cells previously stained positive for anti-EBV immunoglobulins (see “ Material
FIGURE 3 IgA ( a ) in plasma cells surrounding epithelial tumor cells (Tu40) (staining with peroxidase). x 160.
FIGURE 5 IgA (SP) on the surface of epithelial tumor cells (Tu603) (staining with fluorescein). x 300.
and Methods”) in a second staining step positive also for lgA(a), and these cells are accordingly considered as positive for anti-EBV/IgA(a). DISCUSSION
FIGURE 4 TgA (SP) in the glandular acini of an NPC biopsy (Tu602) (staining with peroxidase). x 320.
The marked increase of TgA in sera from NPC patients (Wara et al., 1975) is due to specific EBV antibodies (Henle and Henle, 1976). Recently, NPC throat-washings were also found to contain anti-EBV/IgA (Desgranges et at,, 1977; Ho et a/., 1977). We show here that these TgA molecules are mainly of a secretory nature (11s) and are produced in the nasopharyngeal tumors themselves.
SECRETORY IGA IN NPC SALIVA AND TUMORS
FIGURE 6
Anti-EBV/IgA (a) positive plasma celles (staining with fluorescein). x 470. IgA immunoglobulins of unknown specificity have been detected in the saliva of patients with oral cancer (Brown et a/., 1975), oropharyngeal and bronchopulmonary carcinomas (Mandel et al., 1973). In these cancers, as in NPC, the salivary IgA seems to be due to a local secretory response. The EBV specificity of these antibodies should be looked for, although in a previous study we did not detect EBV-specific IgA in 9 saliva samples from patients with head and neck tumors other than NPC. On the other hand, elevated titers of serum IgA were found in patients with cancers of epithelial secretory organs such as prostate (Ablin et al., 1972), uterus (Vasudevan et al., 1971) and lung (Waldman et a/., 1970). In these cancers
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the elevation of serum IgA titers appears to result from a filtration of local secretory IgA into the circulating blood (Thompson and Asquith, 1970). In our hands no anti-EBVfIgA was found in throat washings of 10 Burkitt lymphoma patients, 20 infectious mononucleosis patients and 10 other conditions associated with EBV but only in NPC patients. This suggests an origin of EBV/IgA in the tumor itself, which reflects the involvement of both the epithelial cells and the lymphocytes and plasma cells in the tumor formation process. The differences observed between saliva samples obtained from Hong Kong and Tunis are not understood. In Hong Kong, Ng et al. (1977) obtained results similar to ours. The observation that young NPC patients in Tunisia lack IgA in the saliva probably reflects differences in the maturity of the local immune system of the Waldeyer ring (oropharyngeal synthesis of IgA in children is much lower than in adults, see L a m , 1976). The mechanism of secretion and transport of IgA in NPC may follow the model proposed by Tourville et a/. (1969) for normal mucosa. The IgA(u) is produced in plasma cells while SP is secreted by the epithelial cells lining the ducts of the salivary glands. In the biopsies studied here, SP was present on the surface of the epithelial tumor cells. The role of the EBV-specific IgA antibodies present in the tumor is not clear. They may inhibit the activity of sensitized T killer cells and therefore favor tumor growth. The diagnostic and prognostic use of IgA in NPC patients has been mentioned previously (Desgranges and deTh6, 1977). Their potential value in early detection of the tumor and in monitoring local relapse deserve further investigation. To this end, a follow-up study of NPC patients and a prospective epidemiological study on a cohort at high risk for NPC has been proposed. ACKNOWLEDGEMENTS
This work was supported by contract No. NO1 CP 43296 with the Virus Cancer Program of the National Cancer Institute, NIH, USA.
IgA SECRETOIRES SPECIFIQUES D E L’EBV DANS LA SALIVE D E MALADES NPC. PRESENCE D E PIECES SECRETOIRES SUR LES CELLULES TUMORALES EPITHELIALES La salive de 59 malades porteurs de cancer du rhinopharynx (NPC), originaires de Hong Kong et de Tunis, et de 20 t h o i n s , a BtB etudiee pour determiner la nature et I’origine des IgA spbcifiques de I’EBV, niises e n Bvidence r6cernrnent par Desgranges et coll. (1977). La moitie des salives de rnalades NPC contenait des IgA/EBV de nature s6cr6toire tandis que les turneurs correspondantes montraient la prbence d’lgA, dont certaines spbifiques de I’EBV dans les plasmocytes entouranr les travkes de cellules tumorales epitheliales, lesquelles possbdaient des pieces secretoires A leur surface. 11 est apparu que les salives NPC en provenance de Tunis contenaient plus souvent des IgA/EBV que celles originaires de Hong Kong. Ce point ainsi que la valeur diagnostique, pronostique et de surveillance du traiternent sont discutks.
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