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In vitro induction of anti-intermediate filament antibody in lymphocyte cultures by Epstein-Barr virus
Immunology Letters, 5 (1982) 203-205
Elsevier Biomedical Press
IN VITRO INDUCTION OF ANTI-INTERMEDIATE FILAMENT ANTIBODY IN LYMPHOCYTE CULTURES BY E P S T E I N - B A R R V I R U S S. M. MORTAZAVI-MILANI*, H. E. STIERLE and E. J. HOLBOROW Bone and Joint Research Unit, The London Hospital Medical College, 2 5 - 2 9 Ash field Street, London E1 2AD, U.K.
(Received 4 August 1982) (Accepted 14 August 1982)
1. Summary Serum antibodies reactive with intermediate filaments of the cytoskeleton (anti-IF antibodies) are often present in infectious mononucleosis, some other viral diseases, and rheumatoid arthritis. The mechanism of their production is not known, but it is possible that the formation of this and other autoantibodies result from polyclonal activation of B-cells. Peripheral blood mononuclear cells from subjects with or without serum anti-IF antibody were therefore cultured in the presence or absence of Epstein-Barr virus (EBV). IgM anti-IF antibody was produced in both unfractionated and T-cell-depleted cultures, but not in the supernatants of the same cells cultured without added EBV.
2. Introduction Intermediate (7-11 nm) filaments are a widespread class of cytoskeletal structures in eukaryotic cells, distinct from both microf'llaments ( 5 - 6 nm) and microtubules ( 2 0 - 2 5 nm). Anti-IF antibodies are readily detected by indirect immunofluorescence, staining a fine network of filaments in the cytoplasm of cultured cells of different types [ 1]. They are present in infectious mononucleosis [2,3], some other acute viral infections [4], and in rheumatoid arthritis [5]. The mechanism of their production is unknown. We have separated blood mononuclear cells from * On sabbatical leave from the Department of Biology, University of Tehran, Tehxan, Iran.
the blood of subjects with or without anti-IF antibody, cultured them in the presence or absence of EBV and tested the supernatant culture fluids for anti-IF antibody.
3. Materials and methods Mononuclear cells separated from the blood of normal subjects and rheumatoid arthritis patients by gradient centrifugation on Lymphoprep (Nyegaard, Oslo) were divided into two lots for culture, one being exposed to EBV released into the fluid phase of transformed marmoset B95-8 cell cultures, the other being cultured without virus. T-Cell depletion was performed by removal of E-rosetting cells. Supernatants from transformed cell cultures were collected after 6 weeks, and tested after concentration (× 10-20). Supernatants of cultures without added EBV from the same subjects were collected at 4-day intervals for 14 days, pooled, concentrated as above and tested as controls. The culture supernatants were tested for the production of antibodies reactive with IF by indirect immunofluorescence on monolayers of cultured human foetal skin fibroblasts or HEp2 cells as substrates. Cells were seeded on to multispot slides after brief trypsinization of stock cultures and maintained in RPMI 1640 supplemented with 10% FCS and antibiotics until sub-confluency. After rinsing the slides in pre-warmed PBS (37°C) the cells were fixed in cold methanol (-20°C) for 10 min and thoroughly rinse d in PBS before sera or culture supernatants were applied. Positive culture fluids stained cytoplasmic arrays of filaments in both types of cell (Figs. 1 and 3) and in
0165-2478[82]0000-0000/$2.75 © 1982 Elsevier Biomedical Press
203
colchicine-treated fibroblasts perinuclear filamentous coils in typical intermediate filament pattern were seen (Fig. 2).
4. Results
Fig. 1.
Fig. 2.
Anti-IF antibody was present in supernatants from EBV-stimulated cultures of unfractionated lymphocytes (Table 1) from both rheumatoid patients and normals. Antibody was also present in T-depleted EBVstimulated lymphocyte culture supernatants. In two experiments anti-IF antibody was also found in the culture supernatant of umbilical cord blood lymphocytes exposed to EBV. Anti-IF antibody was in some cases found in EBV-stimulated cell cultures from subjects in whose sera anti-IF antibody was not detected (Table 2). Since it has been shown that in vitro EBV stimulation of rheumatoid or normal blood mononuclear cells induced IgM anti-IgG antibody (rheumatoid factor) [6], 4 culture fluids (2 rheumatoid and 2 normal) positive for anti-IF antibody were absorbed with heataggregated human IgG (63°C, 15 min, final concentration 10 mg/ml). This did not affect the positive staining for anti-IF antibody.
Table 1 Anti-IF antibody in culture supernatants Rheumatoids
Fig. 3. Figs. 1-3. Different staining patterns observed with culture fluids from EBV-stimulated cell cultures (×400). Human embryonic skin fibroblast (HESF) without (Fig. 1) or with prior colchicine (10 t~g/ml) treatment (Fig. 2), and an established human laryngeal carcinoma (HEp2) cell line (Fig. 3). Indirect immunofluorescence with fluorescein-conjugated class-specific sheep anti-human IgM (Wellcome Laboratories) as second antibody. Pre-treatment of the cultured monolayers with cytochalasin B (10 ~g/ml) which disorganizes the microffdaments [8] had no effect on the s t a g pattern. 204
Normals
+ EBV
- EBV
+ EBV
- EBV
6/11
0/11
5/8
0/8
Cells were exposed to a 1/10 dilution of the culture fluid from the B-958 marmoset cell line (half the dose required to transform 50% of cord blood lymphocytes in culture) for 60 min, washed in RPMI 1640 supplemented with 10% foetal calf serum and antibiotics, and then incubated in 2 ml vols. of the same medium. Results shown (number positive for antibody/ number tested) were obtained from such cultures. Six of the 11 rheumatoid cultures and 2 of the 8 normal cultures were T-lymphocyte-depleted. Four of the 6 positive rheumatoid and the 2 positive normal cell culture supernatants were from T-cell-depleted cultures. T-Depletion was achieved by SRBC rosetting in the conventional way. The T-cell concentration after separation was always below 10%.
Table 2 Comparative prevalence of anti-IF antibodies in culture supernatants of peripheral blood mononuclear cells from rheumatoid and normal subjects exposed to EBV, and in sera from the same subjects Number of samples
Rheumatoids
Total Normals
Total
Culture supernatants positive or negative for anti- IF antibody
Serapositive or negative for anti-IF antibody
4 2 3 2
+ + -
+
11
6*
7*
5
+
+
2
--
1
--
+
8
5*
1*
*No positive for anti-IF.
5. Discussion Thus, the polyclonal stimulating effect of EBV on 8 cells [7] in vitro may give rise to the production of anti-IF antibodies, similar to those occurring in vivo in IM and rheumatoid arthritis. If, as these results sug-
gest, autoreactive B-cells with acquired or inherent anti-IF specificity exist in both normal and rheumatoid subjects, the question arises whether the in vitro induction of anti-IF antibody is characteristic of stimulation by EBV, or whether anti-IF antibody can also be induced by other polyclonal B-cell activators in culture. We are now investigating this.
References [1] Lazaxides, E. (1980) Nature (London) 283,249-256. [2] Linder, E., Kurki, P. and Andersson, L. C. (1979) Clin. Immunol. Immunopath. 14, 41 i - 4 1 7 . [3] Milani, M. M., Ostmg, O. A., Badakere, S. S., Chandra, M., March, R. E. and Holborow, E. J. (1981) XVth Int. Congress Rheumatology, Abstract 0043. [4] Toh, B. H., Yildiz, A., Sotelo, J., Osung, O., Holborow, E. J., Kanakoudi, F. and Small, J. V. (1979) Clin. Exp. Immunol. 37, 76-82. [5] Osung, O., Chandra, M. and Holborow, E. J. (1982) Ann. Rheum. Dis. 41, 69-73. [6] Slaughter, L., Carson, D. A., Jensen, F. C., Holbrook, T. L. and Vaughan, J. H. (1978) J. Exp. Med. 148, 1429-34. [7] Nilsson, K. (1979) in: The Epstein-Barr Virus (Epstein, M. A., Achong, B. G., Eds.), p. 255, Springer-Verlag, New York. [8] Weber, K. and Osborn, M. (1979) in: Cell Motility: Molecules and Organization (l-Iatano, S., Ishikawa, H. and Sato, H., Eds.), pp. 483-490, University of Tokyo Press.
205
Elsevier Biomedical Press
IN VITRO INDUCTION OF ANTI-INTERMEDIATE FILAMENT ANTIBODY IN LYMPHOCYTE CULTURES BY E P S T E I N - B A R R V I R U S S. M. MORTAZAVI-MILANI*, H. E. STIERLE and E. J. HOLBOROW Bone and Joint Research Unit, The London Hospital Medical College, 2 5 - 2 9 Ash field Street, London E1 2AD, U.K.
(Received 4 August 1982) (Accepted 14 August 1982)
1. Summary Serum antibodies reactive with intermediate filaments of the cytoskeleton (anti-IF antibodies) are often present in infectious mononucleosis, some other viral diseases, and rheumatoid arthritis. The mechanism of their production is not known, but it is possible that the formation of this and other autoantibodies result from polyclonal activation of B-cells. Peripheral blood mononuclear cells from subjects with or without serum anti-IF antibody were therefore cultured in the presence or absence of Epstein-Barr virus (EBV). IgM anti-IF antibody was produced in both unfractionated and T-cell-depleted cultures, but not in the supernatants of the same cells cultured without added EBV.
2. Introduction Intermediate (7-11 nm) filaments are a widespread class of cytoskeletal structures in eukaryotic cells, distinct from both microf'llaments ( 5 - 6 nm) and microtubules ( 2 0 - 2 5 nm). Anti-IF antibodies are readily detected by indirect immunofluorescence, staining a fine network of filaments in the cytoplasm of cultured cells of different types [ 1]. They are present in infectious mononucleosis [2,3], some other acute viral infections [4], and in rheumatoid arthritis [5]. The mechanism of their production is unknown. We have separated blood mononuclear cells from * On sabbatical leave from the Department of Biology, University of Tehran, Tehxan, Iran.
the blood of subjects with or without anti-IF antibody, cultured them in the presence or absence of EBV and tested the supernatant culture fluids for anti-IF antibody.
3. Materials and methods Mononuclear cells separated from the blood of normal subjects and rheumatoid arthritis patients by gradient centrifugation on Lymphoprep (Nyegaard, Oslo) were divided into two lots for culture, one being exposed to EBV released into the fluid phase of transformed marmoset B95-8 cell cultures, the other being cultured without virus. T-Cell depletion was performed by removal of E-rosetting cells. Supernatants from transformed cell cultures were collected after 6 weeks, and tested after concentration (× 10-20). Supernatants of cultures without added EBV from the same subjects were collected at 4-day intervals for 14 days, pooled, concentrated as above and tested as controls. The culture supernatants were tested for the production of antibodies reactive with IF by indirect immunofluorescence on monolayers of cultured human foetal skin fibroblasts or HEp2 cells as substrates. Cells were seeded on to multispot slides after brief trypsinization of stock cultures and maintained in RPMI 1640 supplemented with 10% FCS and antibiotics until sub-confluency. After rinsing the slides in pre-warmed PBS (37°C) the cells were fixed in cold methanol (-20°C) for 10 min and thoroughly rinse d in PBS before sera or culture supernatants were applied. Positive culture fluids stained cytoplasmic arrays of filaments in both types of cell (Figs. 1 and 3) and in
0165-2478[82]0000-0000/$2.75 © 1982 Elsevier Biomedical Press
203
colchicine-treated fibroblasts perinuclear filamentous coils in typical intermediate filament pattern were seen (Fig. 2).
4. Results
Fig. 1.
Fig. 2.
Anti-IF antibody was present in supernatants from EBV-stimulated cultures of unfractionated lymphocytes (Table 1) from both rheumatoid patients and normals. Antibody was also present in T-depleted EBVstimulated lymphocyte culture supernatants. In two experiments anti-IF antibody was also found in the culture supernatant of umbilical cord blood lymphocytes exposed to EBV. Anti-IF antibody was in some cases found in EBV-stimulated cell cultures from subjects in whose sera anti-IF antibody was not detected (Table 2). Since it has been shown that in vitro EBV stimulation of rheumatoid or normal blood mononuclear cells induced IgM anti-IgG antibody (rheumatoid factor) [6], 4 culture fluids (2 rheumatoid and 2 normal) positive for anti-IF antibody were absorbed with heataggregated human IgG (63°C, 15 min, final concentration 10 mg/ml). This did not affect the positive staining for anti-IF antibody.
Table 1 Anti-IF antibody in culture supernatants Rheumatoids
Fig. 3. Figs. 1-3. Different staining patterns observed with culture fluids from EBV-stimulated cell cultures (×400). Human embryonic skin fibroblast (HESF) without (Fig. 1) or with prior colchicine (10 t~g/ml) treatment (Fig. 2), and an established human laryngeal carcinoma (HEp2) cell line (Fig. 3). Indirect immunofluorescence with fluorescein-conjugated class-specific sheep anti-human IgM (Wellcome Laboratories) as second antibody. Pre-treatment of the cultured monolayers with cytochalasin B (10 ~g/ml) which disorganizes the microffdaments [8] had no effect on the s t a g pattern. 204
Normals
+ EBV
- EBV
+ EBV
- EBV
6/11
0/11
5/8
0/8
Cells were exposed to a 1/10 dilution of the culture fluid from the B-958 marmoset cell line (half the dose required to transform 50% of cord blood lymphocytes in culture) for 60 min, washed in RPMI 1640 supplemented with 10% foetal calf serum and antibiotics, and then incubated in 2 ml vols. of the same medium. Results shown (number positive for antibody/ number tested) were obtained from such cultures. Six of the 11 rheumatoid cultures and 2 of the 8 normal cultures were T-lymphocyte-depleted. Four of the 6 positive rheumatoid and the 2 positive normal cell culture supernatants were from T-cell-depleted cultures. T-Depletion was achieved by SRBC rosetting in the conventional way. The T-cell concentration after separation was always below 10%.
Table 2 Comparative prevalence of anti-IF antibodies in culture supernatants of peripheral blood mononuclear cells from rheumatoid and normal subjects exposed to EBV, and in sera from the same subjects Number of samples
Rheumatoids
Total Normals
Total
Culture supernatants positive or negative for anti- IF antibody
Serapositive or negative for anti-IF antibody
4 2 3 2
+ + -
+
11
6*
7*
5
+
+
2
--
1
--
+
8
5*
1*
*No positive for anti-IF.
5. Discussion Thus, the polyclonal stimulating effect of EBV on 8 cells [7] in vitro may give rise to the production of anti-IF antibodies, similar to those occurring in vivo in IM and rheumatoid arthritis. If, as these results sug-
gest, autoreactive B-cells with acquired or inherent anti-IF specificity exist in both normal and rheumatoid subjects, the question arises whether the in vitro induction of anti-IF antibody is characteristic of stimulation by EBV, or whether anti-IF antibody can also be induced by other polyclonal B-cell activators in culture. We are now investigating this.
References [1] Lazaxides, E. (1980) Nature (London) 283,249-256. [2] Linder, E., Kurki, P. and Andersson, L. C. (1979) Clin. Immunol. Immunopath. 14, 41 i - 4 1 7 . [3] Milani, M. M., Ostmg, O. A., Badakere, S. S., Chandra, M., March, R. E. and Holborow, E. J. (1981) XVth Int. Congress Rheumatology, Abstract 0043. [4] Toh, B. H., Yildiz, A., Sotelo, J., Osung, O., Holborow, E. J., Kanakoudi, F. and Small, J. V. (1979) Clin. Exp. Immunol. 37, 76-82. [5] Osung, O., Chandra, M. and Holborow, E. J. (1982) Ann. Rheum. Dis. 41, 69-73. [6] Slaughter, L., Carson, D. A., Jensen, F. C., Holbrook, T. L. and Vaughan, J. H. (1978) J. Exp. Med. 148, 1429-34. [7] Nilsson, K. (1979) in: The Epstein-Barr Virus (Epstein, M. A., Achong, B. G., Eds.), p. 255, Springer-Verlag, New York. [8] Weber, K. and Osborn, M. (1979) in: Cell Motility: Molecules and Organization (l-Iatano, S., Ishikawa, H. and Sato, H., Eds.), pp. 483-490, University of Tokyo Press.
205