Virchows Arch. B Cell Path. 18,331--335 (1975) 9 by Springer-Verlag 1975
Leucocyte Specific Antinuclear Antibodies Preparation and Fluorescence Study I. A. Lampert, and D. J. Evans Department of ttistopathology, t~oyal Postgraduate Medical School, Hammersmith Hospital, London C. J. Chesterton Department of Biochemistry, King's College, London l~cceived March 3, 1975 Summary. Tissue and species specific anti-nuclear sera detectable by immunofluorescence were produced by immunising rabbits with D.N.A.-non-histone protein extract from human leucocytes. This is a potentially valuable method for the morphological study of differentiation. Key words: Nucleoproteins - - Leucocytes - - Anti-nuclear factor - - Immunofluorescence.
Introduction I t is well known that the genetic information in cells resides in the D.N.A., and that in any one species it is identical in all diploid cells. I t therefore follows that some other component in the cell nucleus must be responsible for controlling the differentiation and function of the cell. Although Bustin and Stollar (1972) have observed cell and species specificity in the F 1 Histone nucleoprotein fraction, most investigators have observed a striking similarity between the histories present in different cells and tissues (Baserga and Stein, 1971). The non-histone nuclear proteins have been found by many workers, using standard analytical techniques, to have cell and species specificity (Baserga and Stein, 1971), although it has been suggested that some of the differences are more quantitative than qualitative (Elgin st al., 1974). Several groups have employed immunological techniques and have shown cell and species specificity for the non-histone nuclear proteins (Chytil and Spelsberg, 1971 ; Zardi, Lin and Baserga, 1973 ; Wakabayashi, Wang and Hnilica, 1974). Their technique has involved raising anti-sera and testing specificity by a complement fixation test. A criticism of this method is that the antigens could be contaminated by cytoplasmic components (Elgin et al., 1974). In this study we have raised antibodies to human polymorph and lymphocyte non-histone nuclear proteins. Using an immunofluorescence technique we have demonstrated that the human polymorph and the human lymphocyte each has a specific intranuclear antigen.
332
I.A. Lampert et al. Materials and Methods
Antigen Leucocytes were obtained in large quantity via a cell separator unit from a patient suffering from pro-lymphocytic leukaemia. The differential cell count was--pro- and mature lymphocytes 94.4%, neutrophils 3.9%, eosinophils 0.8 %, monocytes 0.6%, basophils 0.1%, blasts 0.2%. No attempt was made to separate the individual cell lines. Nuclei were isolated following 0.5% Nonidet Pd0 (Shell Chemicals) treatment. Cytoplasmic contamination was checked with phase contrast microscopy. A D.N.A. non-histone nucleoprotein complex was isolated by the method of Chytil and Spelsberg (1971).
Immunisation Schedule Weekly subcutaneous injections of nucleoprotein equivalent to 1 mg of D.N.A. were given in Freund's complete adjuvant to groups of rabbits for six weeks. Sera were obtained two weeks following the last injection. Rabbits injected with Freund's adjuvant alone were used as control.
Antibody Testing Indirect immunofluorescencc was carried out using the following re-agents: Undiluted anti-sera and anti-sera diluted 1:10 in Phosphate Buffered Saline pH 7.0 (PBS) and sheep anti-rabbit serum conjugated with fluorescein isothiocyanate (Wellcome). Substrates: Human blood smears (unfixed and fixed in cold ethanol) from the patient and four normal subjects. Rat peripheral blood and marrow Cryostat sections of the following tissues: Human: Kidney, skin, decidua, trophoblasts, thyroid, myometrium, endometrium, ccto- and endocervix, pituitary, parathyroid and pancreas (the latter containing an adenoearcinoma). Rat: Liver, kidney, spleen, thymus, small intestine. Prior to sectioning all tissues were snap frozen in Arcton 12 (I.C.I.) pre-cooled in liquid Nitrogen. 5 ~tm sections were cut in a cryostat microtome and mounted on glass slides. After staining all slides were examined in a Leitz Ortholux UV microscope using a Hb0200 mercury vapour lamp, fluorescein interference filter, and a BG38 red suppressor, incident illumination and a K510 barrier filter.
Antibody Absorption Polymorphs from a normal human donor were separated from whole blood by centrifugation through a Ficoll-Hypaque gradient [12 parts 9% Ficoll (Pharmacia) to 5 parts 34 % sodium diatrizoate]. Chromatin was obtained from nuclei isolated by Nonidet treatment and suspended in 1/100 Saline Sodium Citrate (SSC). Anti-sera were diluted in 1:8 in 1/100 SSC containing 33 ~zg D.N.A. in whole chromatin and were mixed overnight at 4~ As control, anti-sera were diluted 1:8 in 1/100 SSC and mixed overnight at 4~ Chromatin was removed by centrifugation. Following the absorption, sera were tested by indirect immunofluorescence on smears of patient's and normal blood.
Results The results of the immunofluorescence are illustrated in Table 1. As will be seen there are antibodies to the nuclei of h u m a n polymorphs, monocytes a n d l y m p h o c y t e s o n l y - - t h i s includes both the n o r m a l a n d the p a t i e n t ' s l y m p h o cytes. I n h u m a n tissues s t a i n i n g was restricted to the nuclei of polymorphs a n d l y m p h o c y t e s in the blood vessels a n d in the i n f l a m m a t o r y e x u d a t c a b o u t the pancreatic carcinoma. There was no nuclear s t a i n i n g in a n y of the rat tissues studied.
Leucocyte Specific Antinuclear Antibodies
333
Table 1. Immunofluorescence staining of nuclei with undiluted sera and sera diluted 1:10 in PBS Patients blood smear
Polymorphs and lymphocytes (mature and immature)
Normal blood smears
Polymorphs, lymphocytes and monocytes
Human kidney sections pancreas
No tissue staining Staining of polymorphs in inflammatory cxudate only
skin decidua trophoblast eeto- and endocervix endometrium myometrium pituitary thyroid, parathyroid
No staining No staining No staining No staining No staining No staining No staining No staining
AlL rat tissues negative viz. peripheral blood, marrow, liver, kidney, spleen and thymus, small intestine.
In serial dilutions of the best anti-serum, polymorph nucleofluorescence was noted up to a dilution of 1/160, and lymphocyte nucleofluorescence up to a dilution of 1/40. Lymphocyte nuclei showed a finely granular fluorescent staining accentuated on or near the nuclear membrane (Fig. la). The appearance of the polymorph nuclei differed in the unfixed and ethanol-fixed smears. In the former the nuclear staining was arranged in coarse clumps; this clumping was less evident following ethanol fixation (compare Fig. 1 b and c).
Absorption Following absorption with polymorph whole chromatin, polymorph nuclear staining was weak, whereas lymphocyte staining was un-altered. Dilution of the serum in 1/100 SSC produced staining which was similar to that found with serum in an equal dilution of PBS. Discussion
The immunising antigen was derived from a mixed population of leucocytes and anti-nuclear factors were produced to these cell Lines only. The absorption of the polymorph staining antibody with polymorph chromatin indicates that the serum contains a mixture of antibodies of distinct specificity, i.e. polymorph anti-nuclear factor and lymphocytic anti-nuclear factor. The complement fixation test used by previous investigators presents various difficulties; polynucleotides may bind complement fraction Clq; anti complementary activity is often a problem (Chytil and Spelsberg, 1971 ; Zardi, Lin and Baserga, 1973 ; Wakabayashi, Wang and Hnilica, 1974) ; relatively large quantities
334
I . A . L~mpert et al.
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Leucocyte Specific Antinuelear Antibodies
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of antigen must be purified for use in the test and most important of all, localisation of antigen within the cell is uncertain. Immunofluorescence presents none of the above difficulties. Since the method of antigen preparation used here effectively removes histones (Chytil and Spelsberg, 1971), the specificity observed must reside in the nonhistone nueleoprotein components. The non-histone protein fraction is believed to include the regulators of gene function and it is tempting to speculate t h a t the sera are directed against regulator proteins. The nature of the staining is of some interest. The antigen is seen throughout the nucleus in the form of clumps which in polymorphs are large and irregular (this clumping is obscured if fixation is used). Studies by Wakabayashi, Wang and Hniliea (1974) suggest that this antigen contains relatively few different proteins. The widespread distribution of the antigen(s) suggest that they are unlikely to be regulating a single gene. I t could mean that we are dealing with a regulator with widespread activity which determines the differentiation of the cell, or alternatively, with a nuclear structural component, unique to a given cell, but having no regulatory activity. This study confirms the presence, within the non-histone nucleoproteins, of cell and species specific antigens and shows that it is possible to raise specific anti-sera to these antigens. These anti-sera can be used in standard immunofluorescence tests and can identify cells by their nuclear staining. This provides a simple and objective method of assessing differentiation, which is of potential value not only to the cell biologist but also to the haematologist and oncologist. We should like to thank the members of the Anaemia Unit at the Royal Postgraduate Medical School who made the blood ceils available to us, and to Miss Susan van Noorden for tissue sections.
References Baserga, R., Stein, G. : Nuclear acidic proteins and cell proliferation. Fed. Proc. 30, 1752-1759 (1971) Bustin, M., Stoller, R. D.: Immunochemical specificity in lysine rich histone subfractions. J. biol. Chem. 247, 5716-5721 (1972) Chytil, F., Spelsberg, T. C. : Tissue differences in antigenic properties of non-histone protein D.N.A. complexes. Nature (Lond.) New Biol. 233, 215-218 (1971) Elgin, S. C. R., Boyd, J. B., Hood, L. E., Wray, W., Wu, F. C.: A prologue to the study of non-histone chromosomal proteins. Cold Spring Harbor Symposium on Quantitative Biology, Vol. XXXVIII. Chromosome Structure and Function, p. 821-833, Cold Spring Harbor Laboratory (1974) Wakabayashi, K., Wang, S., Hnilica, L. S.: Immuno-specificity of non-histone proteins in chromatin. Biochemistry (Wash.) 13, i027-1032 (1974) Zardi, L., Lin, J. C., Baserga, g.: Immunospeeificity to non-histone chromosomal proteins of antichromatin antibodies. Nature (Lond.) New Biol. 245, 211-213 (1973) Dr. D. J. Evans Department of Histopathology Royal Postgraduate Medical School Hammersmith Hospital Du Cane Road London W. 12, England
Leucocyte Specific Antinuclear Antibodies Preparation and Fluorescence Study I. A. Lampert, and D. J. Evans Department of ttistopathology, t~oyal Postgraduate Medical School, Hammersmith Hospital, London C. J. Chesterton Department of Biochemistry, King's College, London l~cceived March 3, 1975 Summary. Tissue and species specific anti-nuclear sera detectable by immunofluorescence were produced by immunising rabbits with D.N.A.-non-histone protein extract from human leucocytes. This is a potentially valuable method for the morphological study of differentiation. Key words: Nucleoproteins - - Leucocytes - - Anti-nuclear factor - - Immunofluorescence.
Introduction I t is well known that the genetic information in cells resides in the D.N.A., and that in any one species it is identical in all diploid cells. I t therefore follows that some other component in the cell nucleus must be responsible for controlling the differentiation and function of the cell. Although Bustin and Stollar (1972) have observed cell and species specificity in the F 1 Histone nucleoprotein fraction, most investigators have observed a striking similarity between the histories present in different cells and tissues (Baserga and Stein, 1971). The non-histone nuclear proteins have been found by many workers, using standard analytical techniques, to have cell and species specificity (Baserga and Stein, 1971), although it has been suggested that some of the differences are more quantitative than qualitative (Elgin st al., 1974). Several groups have employed immunological techniques and have shown cell and species specificity for the non-histone nuclear proteins (Chytil and Spelsberg, 1971 ; Zardi, Lin and Baserga, 1973 ; Wakabayashi, Wang and Hnilica, 1974). Their technique has involved raising anti-sera and testing specificity by a complement fixation test. A criticism of this method is that the antigens could be contaminated by cytoplasmic components (Elgin et al., 1974). In this study we have raised antibodies to human polymorph and lymphocyte non-histone nuclear proteins. Using an immunofluorescence technique we have demonstrated that the human polymorph and the human lymphocyte each has a specific intranuclear antigen.
332
I.A. Lampert et al. Materials and Methods
Antigen Leucocytes were obtained in large quantity via a cell separator unit from a patient suffering from pro-lymphocytic leukaemia. The differential cell count was--pro- and mature lymphocytes 94.4%, neutrophils 3.9%, eosinophils 0.8 %, monocytes 0.6%, basophils 0.1%, blasts 0.2%. No attempt was made to separate the individual cell lines. Nuclei were isolated following 0.5% Nonidet Pd0 (Shell Chemicals) treatment. Cytoplasmic contamination was checked with phase contrast microscopy. A D.N.A. non-histone nucleoprotein complex was isolated by the method of Chytil and Spelsberg (1971).
Immunisation Schedule Weekly subcutaneous injections of nucleoprotein equivalent to 1 mg of D.N.A. were given in Freund's complete adjuvant to groups of rabbits for six weeks. Sera were obtained two weeks following the last injection. Rabbits injected with Freund's adjuvant alone were used as control.
Antibody Testing Indirect immunofluorescencc was carried out using the following re-agents: Undiluted anti-sera and anti-sera diluted 1:10 in Phosphate Buffered Saline pH 7.0 (PBS) and sheep anti-rabbit serum conjugated with fluorescein isothiocyanate (Wellcome). Substrates: Human blood smears (unfixed and fixed in cold ethanol) from the patient and four normal subjects. Rat peripheral blood and marrow Cryostat sections of the following tissues: Human: Kidney, skin, decidua, trophoblasts, thyroid, myometrium, endometrium, ccto- and endocervix, pituitary, parathyroid and pancreas (the latter containing an adenoearcinoma). Rat: Liver, kidney, spleen, thymus, small intestine. Prior to sectioning all tissues were snap frozen in Arcton 12 (I.C.I.) pre-cooled in liquid Nitrogen. 5 ~tm sections were cut in a cryostat microtome and mounted on glass slides. After staining all slides were examined in a Leitz Ortholux UV microscope using a Hb0200 mercury vapour lamp, fluorescein interference filter, and a BG38 red suppressor, incident illumination and a K510 barrier filter.
Antibody Absorption Polymorphs from a normal human donor were separated from whole blood by centrifugation through a Ficoll-Hypaque gradient [12 parts 9% Ficoll (Pharmacia) to 5 parts 34 % sodium diatrizoate]. Chromatin was obtained from nuclei isolated by Nonidet treatment and suspended in 1/100 Saline Sodium Citrate (SSC). Anti-sera were diluted in 1:8 in 1/100 SSC containing 33 ~zg D.N.A. in whole chromatin and were mixed overnight at 4~ As control, anti-sera were diluted 1:8 in 1/100 SSC and mixed overnight at 4~ Chromatin was removed by centrifugation. Following the absorption, sera were tested by indirect immunofluorescence on smears of patient's and normal blood.
Results The results of the immunofluorescence are illustrated in Table 1. As will be seen there are antibodies to the nuclei of h u m a n polymorphs, monocytes a n d l y m p h o c y t e s o n l y - - t h i s includes both the n o r m a l a n d the p a t i e n t ' s l y m p h o cytes. I n h u m a n tissues s t a i n i n g was restricted to the nuclei of polymorphs a n d l y m p h o c y t e s in the blood vessels a n d in the i n f l a m m a t o r y e x u d a t c a b o u t the pancreatic carcinoma. There was no nuclear s t a i n i n g in a n y of the rat tissues studied.
Leucocyte Specific Antinuclear Antibodies
333
Table 1. Immunofluorescence staining of nuclei with undiluted sera and sera diluted 1:10 in PBS Patients blood smear
Polymorphs and lymphocytes (mature and immature)
Normal blood smears
Polymorphs, lymphocytes and monocytes
Human kidney sections pancreas
No tissue staining Staining of polymorphs in inflammatory cxudate only
skin decidua trophoblast eeto- and endocervix endometrium myometrium pituitary thyroid, parathyroid
No staining No staining No staining No staining No staining No staining No staining No staining
AlL rat tissues negative viz. peripheral blood, marrow, liver, kidney, spleen and thymus, small intestine.
In serial dilutions of the best anti-serum, polymorph nucleofluorescence was noted up to a dilution of 1/160, and lymphocyte nucleofluorescence up to a dilution of 1/40. Lymphocyte nuclei showed a finely granular fluorescent staining accentuated on or near the nuclear membrane (Fig. la). The appearance of the polymorph nuclei differed in the unfixed and ethanol-fixed smears. In the former the nuclear staining was arranged in coarse clumps; this clumping was less evident following ethanol fixation (compare Fig. 1 b and c).
Absorption Following absorption with polymorph whole chromatin, polymorph nuclear staining was weak, whereas lymphocyte staining was un-altered. Dilution of the serum in 1/100 SSC produced staining which was similar to that found with serum in an equal dilution of PBS. Discussion
The immunising antigen was derived from a mixed population of leucocytes and anti-nuclear factors were produced to these cell Lines only. The absorption of the polymorph staining antibody with polymorph chromatin indicates that the serum contains a mixture of antibodies of distinct specificity, i.e. polymorph anti-nuclear factor and lymphocytic anti-nuclear factor. The complement fixation test used by previous investigators presents various difficulties; polynucleotides may bind complement fraction Clq; anti complementary activity is often a problem (Chytil and Spelsberg, 1971 ; Zardi, Lin and Baserga, 1973 ; Wakabayashi, Wang and Hnilica, 1974) ; relatively large quantities
334
I . A . L~mpert et al.
~2
~• @
.
0 @ 0 "8 ~
o
~,~ e
o
@
o 9
.~
~
Leucocyte Specific Antinuelear Antibodies
335
of antigen must be purified for use in the test and most important of all, localisation of antigen within the cell is uncertain. Immunofluorescence presents none of the above difficulties. Since the method of antigen preparation used here effectively removes histones (Chytil and Spelsberg, 1971), the specificity observed must reside in the nonhistone nueleoprotein components. The non-histone protein fraction is believed to include the regulators of gene function and it is tempting to speculate t h a t the sera are directed against regulator proteins. The nature of the staining is of some interest. The antigen is seen throughout the nucleus in the form of clumps which in polymorphs are large and irregular (this clumping is obscured if fixation is used). Studies by Wakabayashi, Wang and Hniliea (1974) suggest that this antigen contains relatively few different proteins. The widespread distribution of the antigen(s) suggest that they are unlikely to be regulating a single gene. I t could mean that we are dealing with a regulator with widespread activity which determines the differentiation of the cell, or alternatively, with a nuclear structural component, unique to a given cell, but having no regulatory activity. This study confirms the presence, within the non-histone nucleoproteins, of cell and species specific antigens and shows that it is possible to raise specific anti-sera to these antigens. These anti-sera can be used in standard immunofluorescence tests and can identify cells by their nuclear staining. This provides a simple and objective method of assessing differentiation, which is of potential value not only to the cell biologist but also to the haematologist and oncologist. We should like to thank the members of the Anaemia Unit at the Royal Postgraduate Medical School who made the blood ceils available to us, and to Miss Susan van Noorden for tissue sections.
References Baserga, R., Stein, G. : Nuclear acidic proteins and cell proliferation. Fed. Proc. 30, 1752-1759 (1971) Bustin, M., Stoller, R. D.: Immunochemical specificity in lysine rich histone subfractions. J. biol. Chem. 247, 5716-5721 (1972) Chytil, F., Spelsberg, T. C. : Tissue differences in antigenic properties of non-histone protein D.N.A. complexes. Nature (Lond.) New Biol. 233, 215-218 (1971) Elgin, S. C. R., Boyd, J. B., Hood, L. E., Wray, W., Wu, F. C.: A prologue to the study of non-histone chromosomal proteins. Cold Spring Harbor Symposium on Quantitative Biology, Vol. XXXVIII. Chromosome Structure and Function, p. 821-833, Cold Spring Harbor Laboratory (1974) Wakabayashi, K., Wang, S., Hnilica, L. S.: Immuno-specificity of non-histone proteins in chromatin. Biochemistry (Wash.) 13, i027-1032 (1974) Zardi, L., Lin, J. C., Baserga, g.: Immunospeeificity to non-histone chromosomal proteins of antichromatin antibodies. Nature (Lond.) New Biol. 245, 211-213 (1973) Dr. D. J. Evans Department of Histopathology Royal Postgraduate Medical School Hammersmith Hospital Du Cane Road London W. 12, England
