Immunology 1976 30 475
Lymphocyte nucleoli activation as a marker of autoimmune disorder development I.
OBSERVATIONS IN NZB MICE
LI DKA KORCAKOVA Research Centre of Organ Transplantation, Institute for Clinical and Experimental Medicine, Praha, Czechoslovakia J. ROVENSKY Research Institute of Rheumatology, Piesgiany, Czechoslovakia J. PEKA REK Institute of Sera and Vaccines, Praha, Czechoslovakia VERA HA9KOVA Research Centre of Organ Transplantation, Institute for Clinical and Experimental Medicine, Praha, Czechoslovakia
Received 29 September 1975; accepted for publication 7 October 1975
Summary. An increased number of lymphocytes with nucleoli synthesizing RNA was found in blood and lymph nodes of aged NZB mice who developed an autoimmune disease. This was not observed in young NZB mice and in 4ONX mice where no immune disorders were noted.
antigen, to which autoantibodies develop, was shown by the migration inhibition test in aged NZB mice (Pekairek, Svejcar, 2itnian, Rovensk' & Cebacauer, 1974). Delayed hypersensitivity may be manifested in vivo by lymphocyte nucleoli activation (Pekairek, Vepiekova and Doutlik, 1972; Korcakova, 1973; Nekvasil and Pekdrek, 1974) suggesting increased RNA synthesis (Busch and Smetana, 1970). Nucleolar activation in blood and lymph node lymphocytes in NZB mice before and after development of autoimmune disease is reported in this paper.
INTRODUCTION The New Zealand Black (NZB) mice spontaneously develop an auto-immune disease characterized by abnormalities in humoral and cellular immunity. In antibody formation, as compared with other strains of mice NZB mice exhibit hyper- as well as hyporesponsiveness, depending on the antigen used (Playfair, 1968; Baum, 1969; Morton and Siegel, 1969; Cerrotini, Lambert and Dixon, 1969; Talal, 1970; Blankwater, Levert and Hijmans, 1975). Cell-mediated immune functions, i.e. transplantation reactions, response to PHA and suppressor function are impaired in ageing NZB mice (Cantor, Asofsky and Talal, 1970; Talal, 1970; Rodey, Good and Yunis, 1971; Gerber, Hardin, Chused and Steinberg 1974). Despite this, delayed hypersensitivity to DNA
MATERIALS AND METHODS Animals NZB mice (Institute of Pharmacology, Slovak Academy of Sciences) and inbred 4ONX mice (Institute of Experimental Biology and Genetics, Czechoslovak Academy of Sciences), both 2-3 and 7-8 months old, were used. Correspondence: Dr J. Pekdrek, Department of Experimental Immunology, Institute of Sera and Vaccines, W. Pieck Street 108, Praha 10, Czechoslovakia.
475
Lidka Korc'akova et al.
476
Table 1. Numbers of white cells in the blood of ageing NZB mice and of 4ONX mice Group and mice 1 NZB 2 NZB 3 4ONX 4 4ONX
Age (months)/ no. of animals
Leucocytes (absolute no.)
Active lymphocytes
Lymphocytes (absolute no.)
Percentage
Absolute no. 395+ 129
2-3/15
5282+ 1507*
4205+ 1360
9 5+ 3-6
7-8/12
11,959+ 1560t
9939+ 1937t
20 9± 6-Ot
1790+ 417t
10,664
9758 (7654-12,672) 8741 (7482-10,964)
94 (7-14) 9.1 (6-14)
940 (571-1764) 814 (464-1260)
2-3/5
(8520-13,200)4 7-8/9
9293 (7960-12,320) * s.d.
t Significant difference from group 1 (P< 0 01).
I Minimal and maximal values are shown in parentheses. Haematological methods Blood was collected from the tail vein. The white blood picture was followed by usual haematological methods. Absolute numbers of lymphocytes in 1 mm3 were calculated from the percentage found in smears stained according to Pappenheim and from the total number of nucleated cells.
Lymph node cell isolation Cells from the right submandibular, axillary, inguinal and popliteal lymph nodes were released into drops of normal mouse serum and smears were prepared.
Nucleolar test Blood smears and smears of lymph node cells were stained with buffered toluidine blue, pH 5 (Smetana, Lejnar and Potmesil, 1969). One hundred lymphocytes were evaluated according to the morphology of their nucleoli. The cells with compact nucleoli and nucleoli with nucleolonemas, were evaluated as active (Busch and Smetana, 1970). Absolute numbers of active lymphocytes in 1 mm3 of blood were obtained from the values of the nucleolar test and from the number of all lymphocytes. Statistics The significance of the differences was determined by Student's t-test or by the Mann-Whitney U test.
Table 2. Percentage of active lymphocytes in lymph nodes in ageing NZB mice and in 4ONX mice
Group and mice 1 NZB 2 NZB 3
Lymph node
Age (months)/ no. of animals Submandibular
Axillary
Inguinal
Popliteal
2-3/15
8-0+2-5*
7-3+ 1-8
7 7+3 0
7-6+2-2
7-8/9
168+3-Ot
178+2-Ot
175+±3-t
94+2-9
2-3/5
4ONX 4 4ONX
7-8/9
11*6
8-4
9.4
70
(9-14)4
(5-11)
(7-14)
(5-8)
11-4
8-3
(7-14)
(7-10)
95 (7-11)
* s.d.
t Significant difference from group 1 (P< 0-01). t Minimal and maximal values are shown in parentheses.
8-7 (4-13)
Lymphocyte nucleoli activation. L RESULTS Counts of peripheral blood leucocytes and lymphocytes were significantly higher in NZB mice 7-8 months old when the auto-immune disorder had developed than in NZB mice aged 2-3 months. The number of active lymphocytes with nucleoli synthesizing RNA increased simultaneously (Table 1, groups 1 and 2). In older NZB mice, the percentage of active lymphocytes was also raised in the submandibular, axillary and inguinal lymph nodes, but not in the popliteal ones (Table 2, groups 1 and 2). No significant changes in numbers of white blood cells and no lymphocyte nucleoli activation were observed in 4ONX mice, which are not known to develop any auto-immune process during ageing.
DISCUSSION Increased numbers of lymphocytes with nucleoli synthesizing RNA were found in blood and in lymph nodes of NZB mice with developed autoimmune disease. The occurrence of active lymphocytes in all probability indicates the presence of cell-mediated immunity (Pekatrek, Vepfekova and Doutlik, 1972; Korcakova, 1973; Nekvasil and Pekafrek, 1974). The finding of these lymphocytes in aged NZB mice is in accord with the finding of delayed hypersensitivity to DNA antigen by means of the migration inhibition test (Peka6rek et al., 1974). Positive migration inhibition tests to DNA are found also in humans with systemic lupus erythematosus (Moulias, Goust, Reinert, Deville-Chabrolle Muller-Berat, Hereman and Godeau, 1972; Dormont, Sobel, Galanaud, Boelaert, Mery and Richet, 1972; Abe, Hara, Yamasaki and Homa, 1973; gvejcar et al., 1974). Such patients should be investigated for blood lymphocyte activation and whether the method used could serve as a practical clinical test. REFERENCES ABE T., HARA M., YAMASAKI K. & HOMA M. (1973) Cell
mediated immune response in systemic lupus erythematosus: in vitro cellular response to native DNA by macrophage inhibitory test. Arthr. Rheum. 16, 688. BAUM J. (1969) Increased 7S antibody response to sheep erythrocytes in the 2-month-old NZB mouse. Clin. exp. Immunol. 5, 251.
477
BLANKWATER M.-J., LEVERT L.A. & HIJMANS W. (1975) Age-related decline in the antibody response to E. coli lipopolysaccharide in New Zealand Black mice. Immunology, 28, 847. BUSCH H. & SMETANA K. (1970) The Nucleolus. Academic Press, New York. CEROTTINI J.C., LAMBERT P.H. & DIXON F.J. (1969) Comparison of the immune responsiveness of NZB and NZB x NZW Fl hybrid mice with that of other strains of mice. J. exp. Med. 130, 1093. CANTOR H., AsOFSKY R. & TALAL N. (1970) Synergy among lymphoid cells mediating the graft versus host response. I. Synergy in graft versus host reactions produced by cells from NZB/B1 mice. J. exp. Med. 131, 223. DORMONT J., SOBEL A., GALANAUD P., BOELAERT J., MERY
J.P. & RICHET G. (1972) Exploration de l'immunit6 cellulaire au cours du lupus 6rythemateux dissemin6. J. Urol. Nephrol. 78, 980. GERBER N.L., HARDIN J.A., CHUSED T.M. & STEINBERG A.D. (1974) Loss with age in NZB/W mice of thymic suppressor cells in the graft-vs-host reaction. J. Immunol. 113, 1618. KORCAKOvk L. (1973) Blood lymphocyte nuclei activation and skin allograft rejection in mice. Folia biol. (Praha), 19, 25. MORTON J.I. & SIEGEL B.V. (1969) Response of NZB mice to foreign antigen and development of autoimmune disease. J. reticuloendothel. Soc. 6, 78. MOULIAs R., GOUST J.M., REINERT P., DEVILLE-CHABROLLE
A., MULLER-BERAT C.N., HEREMAN C. & GODEAU P. (1972) Le test de migration en presence d'ADN des leucocytes au cours du lupus eryth6mateux diss6mine. Nouv. Presse med. 1, 1403. NEKVASIL M. & PEKAREK J. (1974) Activation of the peripheral lymphocytes in the course of the immune response to transplanted SV40-transformed tumour cells in hamsters. Immunology, 27, 159. PLAYFAIR J.H.L. (1968) Strain differences in the immune response of mice. I. The neonatal response to sheep red cells. Immunology, 15, 34. PEKAREK J., SVEJCAR J., ZITNAN D., ROVENSKY J. & CEBECAUER L. (1974) Spontaneous delayed hypersensitivity to DNA in NZB mice. Immunology, 27, 241. PEKAREK J., VEPkEKOVk A. & DOUTLfK S. (1972) Activation of peripheral lymphocytes in the course of experimental allergic encephalomyelitis in the guinea pig. Z. Immun.Forsch. 143, 139. RODEY G.E., GOOD R.A. & YUNIs E.J. (1971) Progressive loss in vitro of cellular immunity with ageing in strains of mice susceptible to autoimmune disease. Clin. exp. Immunol. 9, 305. SMETANA K., LEJNAR J. & POTMtAIL M. (1969) A further contribution to the demonstration of RNA and nucleoli in blood cells in smear preparations. Folia Haemat. 91, 4. SVEJCAR J., ROVENSKY J., PEKAREK J., ZITNIAN D. &
CEBECAUER L. (1974) Delayed hypersensitivity to DNA in systemic lupus erythematosus and related diseases. Z. Immun.-Forsch. 148, 244. TALAL N. (1970) Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthr. Rheum. 13, 887.
Lymphocyte nucleoli activation as a marker of autoimmune disorder development I.
OBSERVATIONS IN NZB MICE
LI DKA KORCAKOVA Research Centre of Organ Transplantation, Institute for Clinical and Experimental Medicine, Praha, Czechoslovakia J. ROVENSKY Research Institute of Rheumatology, Piesgiany, Czechoslovakia J. PEKA REK Institute of Sera and Vaccines, Praha, Czechoslovakia VERA HA9KOVA Research Centre of Organ Transplantation, Institute for Clinical and Experimental Medicine, Praha, Czechoslovakia
Received 29 September 1975; accepted for publication 7 October 1975
Summary. An increased number of lymphocytes with nucleoli synthesizing RNA was found in blood and lymph nodes of aged NZB mice who developed an autoimmune disease. This was not observed in young NZB mice and in 4ONX mice where no immune disorders were noted.
antigen, to which autoantibodies develop, was shown by the migration inhibition test in aged NZB mice (Pekairek, Svejcar, 2itnian, Rovensk' & Cebacauer, 1974). Delayed hypersensitivity may be manifested in vivo by lymphocyte nucleoli activation (Pekairek, Vepiekova and Doutlik, 1972; Korcakova, 1973; Nekvasil and Pekdrek, 1974) suggesting increased RNA synthesis (Busch and Smetana, 1970). Nucleolar activation in blood and lymph node lymphocytes in NZB mice before and after development of autoimmune disease is reported in this paper.
INTRODUCTION The New Zealand Black (NZB) mice spontaneously develop an auto-immune disease characterized by abnormalities in humoral and cellular immunity. In antibody formation, as compared with other strains of mice NZB mice exhibit hyper- as well as hyporesponsiveness, depending on the antigen used (Playfair, 1968; Baum, 1969; Morton and Siegel, 1969; Cerrotini, Lambert and Dixon, 1969; Talal, 1970; Blankwater, Levert and Hijmans, 1975). Cell-mediated immune functions, i.e. transplantation reactions, response to PHA and suppressor function are impaired in ageing NZB mice (Cantor, Asofsky and Talal, 1970; Talal, 1970; Rodey, Good and Yunis, 1971; Gerber, Hardin, Chused and Steinberg 1974). Despite this, delayed hypersensitivity to DNA
MATERIALS AND METHODS Animals NZB mice (Institute of Pharmacology, Slovak Academy of Sciences) and inbred 4ONX mice (Institute of Experimental Biology and Genetics, Czechoslovak Academy of Sciences), both 2-3 and 7-8 months old, were used. Correspondence: Dr J. Pekdrek, Department of Experimental Immunology, Institute of Sera and Vaccines, W. Pieck Street 108, Praha 10, Czechoslovakia.
475
Lidka Korc'akova et al.
476
Table 1. Numbers of white cells in the blood of ageing NZB mice and of 4ONX mice Group and mice 1 NZB 2 NZB 3 4ONX 4 4ONX
Age (months)/ no. of animals
Leucocytes (absolute no.)
Active lymphocytes
Lymphocytes (absolute no.)
Percentage
Absolute no. 395+ 129
2-3/15
5282+ 1507*
4205+ 1360
9 5+ 3-6
7-8/12
11,959+ 1560t
9939+ 1937t
20 9± 6-Ot
1790+ 417t
10,664
9758 (7654-12,672) 8741 (7482-10,964)
94 (7-14) 9.1 (6-14)
940 (571-1764) 814 (464-1260)
2-3/5
(8520-13,200)4 7-8/9
9293 (7960-12,320) * s.d.
t Significant difference from group 1 (P< 0 01).
I Minimal and maximal values are shown in parentheses. Haematological methods Blood was collected from the tail vein. The white blood picture was followed by usual haematological methods. Absolute numbers of lymphocytes in 1 mm3 were calculated from the percentage found in smears stained according to Pappenheim and from the total number of nucleated cells.
Lymph node cell isolation Cells from the right submandibular, axillary, inguinal and popliteal lymph nodes were released into drops of normal mouse serum and smears were prepared.
Nucleolar test Blood smears and smears of lymph node cells were stained with buffered toluidine blue, pH 5 (Smetana, Lejnar and Potmesil, 1969). One hundred lymphocytes were evaluated according to the morphology of their nucleoli. The cells with compact nucleoli and nucleoli with nucleolonemas, were evaluated as active (Busch and Smetana, 1970). Absolute numbers of active lymphocytes in 1 mm3 of blood were obtained from the values of the nucleolar test and from the number of all lymphocytes. Statistics The significance of the differences was determined by Student's t-test or by the Mann-Whitney U test.
Table 2. Percentage of active lymphocytes in lymph nodes in ageing NZB mice and in 4ONX mice
Group and mice 1 NZB 2 NZB 3
Lymph node
Age (months)/ no. of animals Submandibular
Axillary
Inguinal
Popliteal
2-3/15
8-0+2-5*
7-3+ 1-8
7 7+3 0
7-6+2-2
7-8/9
168+3-Ot
178+2-Ot
175+±3-t
94+2-9
2-3/5
4ONX 4 4ONX
7-8/9
11*6
8-4
9.4
70
(9-14)4
(5-11)
(7-14)
(5-8)
11-4
8-3
(7-14)
(7-10)
95 (7-11)
* s.d.
t Significant difference from group 1 (P< 0-01). t Minimal and maximal values are shown in parentheses.
8-7 (4-13)
Lymphocyte nucleoli activation. L RESULTS Counts of peripheral blood leucocytes and lymphocytes were significantly higher in NZB mice 7-8 months old when the auto-immune disorder had developed than in NZB mice aged 2-3 months. The number of active lymphocytes with nucleoli synthesizing RNA increased simultaneously (Table 1, groups 1 and 2). In older NZB mice, the percentage of active lymphocytes was also raised in the submandibular, axillary and inguinal lymph nodes, but not in the popliteal ones (Table 2, groups 1 and 2). No significant changes in numbers of white blood cells and no lymphocyte nucleoli activation were observed in 4ONX mice, which are not known to develop any auto-immune process during ageing.
DISCUSSION Increased numbers of lymphocytes with nucleoli synthesizing RNA were found in blood and in lymph nodes of NZB mice with developed autoimmune disease. The occurrence of active lymphocytes in all probability indicates the presence of cell-mediated immunity (Pekatrek, Vepfekova and Doutlik, 1972; Korcakova, 1973; Nekvasil and Pekafrek, 1974). The finding of these lymphocytes in aged NZB mice is in accord with the finding of delayed hypersensitivity to DNA antigen by means of the migration inhibition test (Peka6rek et al., 1974). Positive migration inhibition tests to DNA are found also in humans with systemic lupus erythematosus (Moulias, Goust, Reinert, Deville-Chabrolle Muller-Berat, Hereman and Godeau, 1972; Dormont, Sobel, Galanaud, Boelaert, Mery and Richet, 1972; Abe, Hara, Yamasaki and Homa, 1973; gvejcar et al., 1974). Such patients should be investigated for blood lymphocyte activation and whether the method used could serve as a practical clinical test. REFERENCES ABE T., HARA M., YAMASAKI K. & HOMA M. (1973) Cell
mediated immune response in systemic lupus erythematosus: in vitro cellular response to native DNA by macrophage inhibitory test. Arthr. Rheum. 16, 688. BAUM J. (1969) Increased 7S antibody response to sheep erythrocytes in the 2-month-old NZB mouse. Clin. exp. Immunol. 5, 251.
477
BLANKWATER M.-J., LEVERT L.A. & HIJMANS W. (1975) Age-related decline in the antibody response to E. coli lipopolysaccharide in New Zealand Black mice. Immunology, 28, 847. BUSCH H. & SMETANA K. (1970) The Nucleolus. Academic Press, New York. CEROTTINI J.C., LAMBERT P.H. & DIXON F.J. (1969) Comparison of the immune responsiveness of NZB and NZB x NZW Fl hybrid mice with that of other strains of mice. J. exp. Med. 130, 1093. CANTOR H., AsOFSKY R. & TALAL N. (1970) Synergy among lymphoid cells mediating the graft versus host response. I. Synergy in graft versus host reactions produced by cells from NZB/B1 mice. J. exp. Med. 131, 223. DORMONT J., SOBEL A., GALANAUD P., BOELAERT J., MERY
J.P. & RICHET G. (1972) Exploration de l'immunit6 cellulaire au cours du lupus 6rythemateux dissemin6. J. Urol. Nephrol. 78, 980. GERBER N.L., HARDIN J.A., CHUSED T.M. & STEINBERG A.D. (1974) Loss with age in NZB/W mice of thymic suppressor cells in the graft-vs-host reaction. J. Immunol. 113, 1618. KORCAKOvk L. (1973) Blood lymphocyte nuclei activation and skin allograft rejection in mice. Folia biol. (Praha), 19, 25. MORTON J.I. & SIEGEL B.V. (1969) Response of NZB mice to foreign antigen and development of autoimmune disease. J. reticuloendothel. Soc. 6, 78. MOULIAs R., GOUST J.M., REINERT P., DEVILLE-CHABROLLE
A., MULLER-BERAT C.N., HEREMAN C. & GODEAU P. (1972) Le test de migration en presence d'ADN des leucocytes au cours du lupus eryth6mateux diss6mine. Nouv. Presse med. 1, 1403. NEKVASIL M. & PEKAREK J. (1974) Activation of the peripheral lymphocytes in the course of the immune response to transplanted SV40-transformed tumour cells in hamsters. Immunology, 27, 159. PLAYFAIR J.H.L. (1968) Strain differences in the immune response of mice. I. The neonatal response to sheep red cells. Immunology, 15, 34. PEKAREK J., SVEJCAR J., ZITNAN D., ROVENSKY J. & CEBECAUER L. (1974) Spontaneous delayed hypersensitivity to DNA in NZB mice. Immunology, 27, 241. PEKAREK J., VEPkEKOVk A. & DOUTLfK S. (1972) Activation of peripheral lymphocytes in the course of experimental allergic encephalomyelitis in the guinea pig. Z. Immun.Forsch. 143, 139. RODEY G.E., GOOD R.A. & YUNIs E.J. (1971) Progressive loss in vitro of cellular immunity with ageing in strains of mice susceptible to autoimmune disease. Clin. exp. Immunol. 9, 305. SMETANA K., LEJNAR J. & POTMtAIL M. (1969) A further contribution to the demonstration of RNA and nucleoli in blood cells in smear preparations. Folia Haemat. 91, 4. SVEJCAR J., ROVENSKY J., PEKAREK J., ZITNIAN D. &
CEBECAUER L. (1974) Delayed hypersensitivity to DNA in systemic lupus erythematosus and related diseases. Z. Immun.-Forsch. 148, 244. TALAL N. (1970) Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthr. Rheum. 13, 887.
