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Comparison of the sensitivity of the protein A plaque assay and the immunofluorescence assay for detection of immunoglobulin producing cells
Journal of Immunological Methods, 56 (1983) 117-124 Elsevier Biomedical Press
117
Comparison of the Sensitivity of the Protein A Plaque Assay and the Immunofluorescence Assay for Detection of Immunoglobulin Producing Cells A. V a n O u d e n a r e n *, J.J. H a a i j m a n a n d R. B e n n e r Department of Cell Biology and Genetics, Erasmus University, Rotterdam. and Institute for Experimental Gerontology, Organization for Health Research TNO, Rijswijk, The Netherlands (Received 3 March 1982. accepted I July 1982)
This paper reports a comparative study on the sensitivity of the protein A plaque assay and the cytoplasmic immunofluorescence assay for detection of immunoglobulin (lg) producing cells in cell suspensions of routine lymphoid organs. The protein A plaque assay was found to detect as many or several times more lg producing cells than the immunofluorescence assay, depending on the age and antigenic load of the mice, and upon the lg class and organ studied. Key words: cytoplasmic immunofluorescence - - mouse lg-secreting cells - - protein A plaque assay
Introduction In vivo immunoglobulin (Ig) production is usually estimated by quantitating one of the following: (a) the serum Ig concentrations; (b) the numbers of cytoplasmic Ig-positive cells in the various lymphoid organs by immunofluorescence; and (c) the numbers of Ig-secreting cells in the various lymphoid organs by applying appropriate plaque assays. Each of these methods has its advantages and limitations. Serum Ig level determinations have the advantage of revealing the overall activity of the humoral immune system, that is, the contribution to all lymphoid organs and tissues taken together. Such determinations may be done reliably and relatively easily in Mancini immunodiffusion plates (Mancini et al., 1965), by rocket electrophoresis (Laurell, 1972), or by enzyme- and radioimmunoassay, provided the antisera and the standard preparations are properly defined and of good quality. A disadvantage of serum Ig level measurements, however, is that a large part of the synthesized Ig is secreted (Orlans et al., 1975; Osebold et al., 1975; Tomasi, 1976). The proportion of secreted Ig is variable for the different Ig (sub)classes. Also the * Correspondence to: A. Van Oudenaren, Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
118
different pool size and half-life of the various lg (sub)classes (Spiegelberg, 1974) hampers the straightforward use of this parameter as a clue to Ig production. Direct quantitation of the cells producing circulating and secretory Ig overcomes these problems. Morphologically, such cells are characterized by an increased amount of basophylic cytoplasm. Their morphology can range from lymphoblast to plasma cell. Many of these cells may be easily enumerated by means of immunofluorescence with heterologous antibodies specific for the Ig produced (Hijmans et al., 1969). Cells producing and secreting Ig may be assayed functionally in the reverse plaque assay (Molinaro and Dray, 1974), and in the protein A plaque assay (Gronowicz et al., 1976). Both assays make use of target sheep erythrocytes (SRBC) to which heterologous anti-Ig antibodies are coupled. In the present paper we compare the sensitivity of immunofluorescence assay and protein A plaque assay in detecting Ig producing cells from various lymphoid organs of mice not intentionally immunized. This was done for young as well as old mice, and for specific pathogen-free as well as germ-free mice. The data show that the protein A plaque assay detects more Ig producing cells than the immunofluorescence assay. The ratio of the values obtained with both assays was found to depend upon the age and antigenic load of the mice, and upon the Ig class and organ studied.
Materials and Methods
Mice
Female specific pathogen-free BALB/c mice, 8- and 100-week-old, and female germ-free B A L B / c mice, 100-week-old, were purchased from the Radiobiological Institute TNO, Rijswijk, The Netherlands. All mice were free of lymphoreticular malignancies. Cells
The preparation of cell suspensions from lymphoid organs has been described in detail elsewhere (Benner et al., 198 l c). The cell suspensions were prepared, washed and resuspended in balanced salt solution and, for use in the immunofluorescence assay, appropriately diluted in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin (BSA; Poviet, Amsterdam). The final pH of the PBS-BSA solution was adjusted to 7.8. Protein A plaque assay
IgM-, IgG- and IgA-secreting cells were assayed by the hemolytic plaque assay as described by Gronowicz et al. (1976), with some modifications. Staphylococcus aureus protein A (Pharmacia, Uppsala) was coupled to SRBC with CrC13 • 6H20 as described by Van Oudenaren et al. (1981). The protein A-coated SRBC were used within 3 days of preparation. The protein A plaque assay was performed in BSS in Cunningham chambers as described by Lefkovits and Cosenza (1979). Each chamber contained 100 ~tl of appropriately diluted cell suspension mixed with 20 #1 of an
119 optimal amount of diluted guinea pig complement, purified over a Sepharose-protein A column (Pharmacia) as described by Van Oudenaren et al. (1981), 15 pl of an optimal amount of diluted specific rabbit anti-mouse IgM, IgG or IgA antiserum and 25/~1 of a 10% suspension of protein A-coated SRBC. The rabbit antisera used were purified over a Sepharose-protein A column as described by Goding (1978), in order to isolate the protein A-binding fraction of Ig. The chambers were incubated at 37°C for 5 h. The Ig secreting plaque-forming cells (PFC) were counted under a dissecting microscope by dark field illumination.
Visualization of cytoplasmic Ig-containing cells Cytocentrifuge preparations of a known number of cells were prepared according to Vossen et al. (1976). The technique for the visualization of cytoplasmic Ig-containing cells (C-Ig cells) has been described in detail by Hijmans et al. (1969). Slides were fixed for 15 min at - 2 0 ° C in acid ethanol (5 parts of acetic acid to 95 parts of ethanol) and washed for 30 rain in PBS. Incubations with unlabeled and fluorescent antisera were for 30 min in humidified chamber. Excess conjugate was removed with one wash of PBS. Preparations were mounted in buffered glycerol (1 part of PBS to 9 parts of glycerol; Merck-Schuchard, Mtinchen) and sealed with paraffin. A fluorescein-conjugated goat antiserum directed against mouse Ig (GAMIg/FITC, lot no. 2-873; Nordic Immunological Laboratories, Tilburg) was used to determine the total number of C-Ig cells per slide. Cytoplasmic-Ig positive cells were easily distinguished from pre-B cells, the latter having only a small rim of cytoplasmic Ig, and showing faint staining in immunofluorescence (Raft et al., 1976). From the number of C-Ig positive cells per slide, the number of cells spun down per slide and the cell yield per organ, we calculated the total number of C-Ig cells per organ. The Ig class distribution of the C-Ig cells was determined according to Hijmans et al. (1969) by means of combinations of rhodamine and fluorescein labeled antisera specific for the heavy chains of IgM, IgG and IgA.
Antisera All antisera used were of rabbit origin, except the FITC-conjugated goat antiserum used to determine the total number of C-Ig cells per slide. The rhodamine and fluorescein labeled class specific antisera were prepared, purified, and generously supplied by Dr. J. R~idl and Miss P. Van den Berg, from the Institute for Experimental Gerontology, TNO. The antisera fulfilled the specificity criteria described elsewhere (Bloemmen et al., 1976). The class-specific antisera used in the protein A plaque assay were purchased from Litton-Bionetics, Kensington, MD. Whether or not both sets of antisera recognized the same cells was investigated by testing both antisera specific for a certain Ig class on the same cytocentrifuge preparation, the one directly labeled and the other indirectly labeled with a different fluorochrome. These comparative studies revealed at least 95% overlap for the anti-IgM, anti-IgG and anti-IgA rabbit antisera.
120
Calculation of the number of positive cells in the bone marrow Bone marrow PFC and C-Ig cells were determined in the femoral marrow. Following Benner et al. (1981c) we adopted a conversion factor of 7.9 to calculate the number of positive cells in the total bone marrow from the number found in 2 femurs.
Results and Discussion
Ig producing cells occur in all lymphoid organs, independently of whether or not the individuals have been intentionally immunized (Van Furth et al., 1966; Benner et ai., 1981b). In mice, the actual number of Ig-producing cells per organ differs between the different lymphoid organs (Table I), and is dependent upon antigenic load and age (Table II). In young adult mice most Ig producing cells occur in spleen, lymph nodes and bone marrow. Peyer's patches and thymus contain only small numbers of Ig producing cells (Benner et al., 1981b). The total number of Ig producing cells per mouse increases with increasing age. This is predominantly due to the fact that the number of Ig producing cells in the bone marrow increases markedly with age (Haaijman et al., 1979; Benner et al., 1981a). Enumerations of the lg producing cells by immunofluorescence and by the protein A plaque assay do not always give similar frequencies. The protein A plaque assay often gives a higher number than immunofluorescence. In the individual mice studied, the ratio of results by the 2 assays ranged from 0.8 to about 9, and was dependent on the lymphoid organ and Ig class studied (Table I). The discrepancy cannot be due to the quality of the antisera used, since comparative studies of both sets of antisera revealed an overlap of at least 95% in the immunofluorescence assay. In the spleen of 8-week-old B A L B / c mice the protein A plaque assay and the immunofluorescence assay gave equal numbers of IgM and IgG producing cells. However, the protein A plaque assay gave almost 3 times as many lgA producing cells as the immunofluorescence assay. For the other lymphoid organs this ratio was usually higher than 1.0, especially for IgA, but in some instances also for IgM and lgG (Table I). The relative sensitivity of the protein A plaque assay and the immunofluorescence assay was also determined on bone marrow cells from mice of different ages and under different antigenic loads (i.e., germ-free and specific pathogen-free mice). The most striking observation was that in the marrow of 100-week-old B A L B / c mice the protein A plaque assay detected 8.6 times as many IgA producing cells as the immunofluorescence assay, and that relatively high ratios were found for both IgG and IgA producing cells in the marrow of 100-week-old germ free mice (Table II). It is remarkable that the discrepancy between the 2 assays in old mice is greater for lgG and IgA than for IgM. This correlates with the degree to which the production of these Ig classes is dependent on T cell help (Bankhurst et al., 1975; Van Muiswinkel and Van Soest, 1975), and the age-related dysfunction of the T cell system (Makinodan et al., 1977; Krogsrud and Perkins, 1977). It suggests that the
Fiuoresc.
Plaques
Fluoresc.
Plaques
Fluoresc.
7.9 (6.0-11) 8.5 (5.9-12)
14 (13-16) 6.3 (5.2-7.6)
(44-48) 20 (17-24)
113 b (100-127) I 17 (98-140)
IgM
0.9
2.2
}
}
i.0 ¢
2.5
/
'[
/
1.6
1.2
(45-59) 15 (10-23)
54 (47-62) 35 (30-4 0
3.5
1.5
} 52 }
(98-137) 73 (57-92)
164 (136-198) i 41 ( 121- 170)
IgG
II (8.3-14) 2.8 (1.8-4.2)
22 (17-29) 3.1 (2.4-4.1)
55 (52-58) 21 (18-24) 2.6
} 3.9
/ 7.1 J
/
}
71 (64-78) 26 (18-38)
90 (79-102) 44 (39-51)
221 (196-250) 114 (101-129)
354 (314-399) 287 (248-331)
2.7
77 (66-89) 29 (25-33)
}
Total
lgA
• lg producing cells were simultaneously assayed by immunofluorescence and the protein A plaque assay. b Figures represent the geometric mean + I S.E.M. ( x 10-3) of the number of positive cells per whole organ (n = 6). ¢ Ratio of the number of lg producing cells found in the protein A plaque assay to number found by immunofluorescence.
Peyer's patches
Mesenteric lymph nodes
Bone marrow
Plaques
Spleen
Fluoresc.
Assay •
Organ
2.7
2.1
1.9
1.2
INCIDENCE OF BACKGROUND IgM, lgG AND lgA PRODUCING CELLS ( × 10- 3) IN THE SPLEEN. BONE MARROW, MESENTER1C LYMPH NODES AND PEYER'S PATCHES OF 8-WEEK-OLD SPECIFIC PATHOGEN-FREE (SPF) BALB/c MICE AS DETERMINED BY THE PROTEIN A PLAQUE ASSAY AND IMMUNOFLUORESCENCE
TABLE I
8
100
100
BALB/c
BALB/c
BALB/c
GF
SPF
SPF
Status a
Fluoresc.
Plaues
Fluoresc.
(961- I 650) 1 561 ( 1 240-1 967)
(1004-1682) 1 223 ( 1 016-1 473)
FI uoresc.
Paques
50c (44-58) 20 (17-24)
IgM
Plaques
Assay b
/
1
}
0.8
1.1
2.5
1S92
(1 220-2078) 475 (332-680)
2320
(1916-2809) 1342 ( 1 188-1 517)
116 (98-137) 73 (57-92)
lgG
/
1
}
3.4
1.7
1.6
49
(348-504) 69 (60 81 )
(1426-3074) 245 ( 188-318)
55 (52-58) 21 ( I 8- 24)
IgA
/
/
}
6.1
8.6
2.6
3270
(2478-4315) 2 105 ( I 658-2672)
573
(5219-6254) 2810 (2566-3075)
221 (196 250) I 14 ( 101 - 129)
Total
SPF, specific pathogen-free; GF, germ-free. b Ig producing cells in the bone marrow of mice were simultaneously assayed by immunofluorescence and the protein A plaque assay. " Figures represent the geometric mean -}- I S.E.M. ( x 10-3) of the number of positive cells per whole bone marrow (n = 6). d Ratio between the number of Ig producing cells found in the protein A plaque assay, and by immunofluore.~ence.
Age (weeks)
Mice
1.6
2.0
1.9
INCIDENCE OF B A C K G R O U N D IgM, IgG AND IgA P R O D U C I N G CELLS ( × 1 0 3) IN THE BONE MARROW OF BALB/c MICE AS DETERMINED BY THE PROTEIN A PLAQUE ASSAY A N D I M M U N O F L U O R E S C E N C E
TABLE II
123
extent of the discrepancy between the numbers of Ig producing cells given by the 2 assays is related to the degree of requirement for and/or the dysfunction of the T cell system, a view that is supported by comparable results of experiments on athymic nude mice (Benner et al., 1982). The underlying cause of the discrepancy between the numbers of Ig producing cells revealed by the 2 assays is probably that in immunofluorescence only plasmablasts and plasma cells are indentifiable with certainty. However, earlier stages of the plasmacytic series are also able to produce and secrete Ig (Melchers and Andersson, 1974), and are thus able to produce plaques. Although these findings suggest that the protein A plaque assay is more reliable for measuring activity of the humoral immune system than the immunofluorescence assay, another factor should also be taken into account. This is that the amount of Ig released per Ig producing cell increases during maturation (Melchers and Andersson, 1974). Thus, immature Ig producing cells release less Ig than more mature cells like plasma cells and thus contribute less to total Ig synthesis. Ideally, the numbers of Ig producing cells of the various lymphoid organs should correlate with the Ig concentrations in the serum. In man, comparative studies have shown such positive correlations for the various Ig classes and subclasses (Hijmans, 1975; Turesson, 1975), when Ig producing cells were enumerated by immunofluorescence assay. In mice, such studies do not reveal a simple linear correlation (J.J. Haaijman, unpublished). Studies in mice with the protein A plaque assay instead of the immunofluorescence assay have not as yet been performed.
Acknowledgements We thank Dr. J. R~dl and Ms. P. Van den Berg for the fluorescent conjugates used in this study, and Ms. Cary Meijerink for typing the manuscript.
References Bankhurst, A.D., P.H. Lambert and P.A. Miescher, 1975, Proc. Soc. Exp. Biol. Med. 148, 501. Benner, R., W. Hijmans and J.J. Haaijman, 1981a, Clin. Exp. lmmunol. 46, 1. Benner, R., A.-M. Rijnbeck, R.R. Bcrnab6, C. Martincz-Aionso and A. Coutinho, 1981b, Immunobiology 158, 225. Bcnner, R., A. Van Oudenaren and G. Koch, 1981c, in: Immunological Methods, Vol. 2, cds. 1. Lefkovits and B. Pernis (Academic Press, New York) p. 247. Benner, R., A. Van Oudenaren, M. Bj6rklund, F. lvars and D. Holmbcrg, 1982, Immunology Today 3, 243. Bloemmen, F.J., J. R~dl, J.J. Haaijman, P. Van den Berg, H.R.E. Schuit and W. l-lijmans, 1976, J. Immunol. Methods I0, 337. Goding, J., 1978, J. Immunol. Methods 20, 241. Gronowicz, E., A. Coutinho and F. Melchers, 1976, Eur. J. Immunol. 6, 588. Haaijman, J.J.,J. Slingerland-Teunissen,R. Benner and A. Van Oudenaren, 1979, Immunology 36, 271. Hijmans, W., 1975, in: The Immunological Basis of Connective Tissue Disorders, ed. L.G. Silvestri (North-Holland, Amsterdam) p. 203.
124 Hijmans, W., H.R.E. Schuit and F. Klein. 1969, Clin. Exp. immunol. 4, 457. Krogsrud, R.L. and E.H. Perkins, 1977, J. Immunol. 118, 1607. Laurell, C.B., 1972, Scan& J. Clin. Lab. Invest. 29, Suppl. 124, 21. Lefkovits. I. and H. Cosenza. 1979, in: Immunological Methods, Vol. 1, eds. 1. Lefkovits and B. Pernis (Academic Press, New York) p. 277. Makinodan, T., R.A. Good and M.M.B. Kay, 1977, in: Comprehensive Immunology, Vol. I, eds. "I. Makinodan and E. Yunis (Plenum Medical Book Co., New York) p. 9. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistr3' 2, 235. Melchers, F. and J. Andersson. 1974, Eur. J. Immunol. 4, 181. Molinaro, G.A. and S. Dray, 1974, Nature (London)248, 515. Orlans, E.. J. Peppard, J. Reynolds and J. Hall, 1975, J. Exp. Med. 147, 588. Osebold, J.W., N.I. Medin and Y.C. Zee, 1975, Infect. lmmun. 12. 1141. Raff, M.C., M. Megson, J.J.T. Owen and M.D. Cooper, 1976, Nature (London) 259, 224. Spiegelberg. H.L.. 1974, Adv. Immunol. 19, 259. Tomasi, T.B., 1976, in: The Immune System of Secretions, eds. A.G. Osier and L. Weiss (Prentice Hall. E n g l e w ~ d Cliffs, N J) p. 57. Turesson, I., 1976, Acta Med. Scand. 199, 293. Van Furth, R., H.R.E. Schuit and W. Hijmans, 1966, Immunology II, 19. Van Muiswinkel, W.B. and P.L. Van Soest, 1975, Immunology 28, 287. Van Oudenaren, A., H. Hooijkaas and R. Benner. 1981, J. lmmunol. Methods 43, 219. Vosmn, J.M.J.J., R.E. Langlois van der Berg, H.R.E. Schuit and W. Hijmans, 1976, J. Immunol. Methods 13, 71.
117
Comparison of the Sensitivity of the Protein A Plaque Assay and the Immunofluorescence Assay for Detection of Immunoglobulin Producing Cells A. V a n O u d e n a r e n *, J.J. H a a i j m a n a n d R. B e n n e r Department of Cell Biology and Genetics, Erasmus University, Rotterdam. and Institute for Experimental Gerontology, Organization for Health Research TNO, Rijswijk, The Netherlands (Received 3 March 1982. accepted I July 1982)
This paper reports a comparative study on the sensitivity of the protein A plaque assay and the cytoplasmic immunofluorescence assay for detection of immunoglobulin (lg) producing cells in cell suspensions of routine lymphoid organs. The protein A plaque assay was found to detect as many or several times more lg producing cells than the immunofluorescence assay, depending on the age and antigenic load of the mice, and upon the lg class and organ studied. Key words: cytoplasmic immunofluorescence - - mouse lg-secreting cells - - protein A plaque assay
Introduction In vivo immunoglobulin (Ig) production is usually estimated by quantitating one of the following: (a) the serum Ig concentrations; (b) the numbers of cytoplasmic Ig-positive cells in the various lymphoid organs by immunofluorescence; and (c) the numbers of Ig-secreting cells in the various lymphoid organs by applying appropriate plaque assays. Each of these methods has its advantages and limitations. Serum Ig level determinations have the advantage of revealing the overall activity of the humoral immune system, that is, the contribution to all lymphoid organs and tissues taken together. Such determinations may be done reliably and relatively easily in Mancini immunodiffusion plates (Mancini et al., 1965), by rocket electrophoresis (Laurell, 1972), or by enzyme- and radioimmunoassay, provided the antisera and the standard preparations are properly defined and of good quality. A disadvantage of serum Ig level measurements, however, is that a large part of the synthesized Ig is secreted (Orlans et al., 1975; Osebold et al., 1975; Tomasi, 1976). The proportion of secreted Ig is variable for the different Ig (sub)classes. Also the * Correspondence to: A. Van Oudenaren, Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
118
different pool size and half-life of the various lg (sub)classes (Spiegelberg, 1974) hampers the straightforward use of this parameter as a clue to Ig production. Direct quantitation of the cells producing circulating and secretory Ig overcomes these problems. Morphologically, such cells are characterized by an increased amount of basophylic cytoplasm. Their morphology can range from lymphoblast to plasma cell. Many of these cells may be easily enumerated by means of immunofluorescence with heterologous antibodies specific for the Ig produced (Hijmans et al., 1969). Cells producing and secreting Ig may be assayed functionally in the reverse plaque assay (Molinaro and Dray, 1974), and in the protein A plaque assay (Gronowicz et al., 1976). Both assays make use of target sheep erythrocytes (SRBC) to which heterologous anti-Ig antibodies are coupled. In the present paper we compare the sensitivity of immunofluorescence assay and protein A plaque assay in detecting Ig producing cells from various lymphoid organs of mice not intentionally immunized. This was done for young as well as old mice, and for specific pathogen-free as well as germ-free mice. The data show that the protein A plaque assay detects more Ig producing cells than the immunofluorescence assay. The ratio of the values obtained with both assays was found to depend upon the age and antigenic load of the mice, and upon the Ig class and organ studied.
Materials and Methods
Mice
Female specific pathogen-free BALB/c mice, 8- and 100-week-old, and female germ-free B A L B / c mice, 100-week-old, were purchased from the Radiobiological Institute TNO, Rijswijk, The Netherlands. All mice were free of lymphoreticular malignancies. Cells
The preparation of cell suspensions from lymphoid organs has been described in detail elsewhere (Benner et al., 198 l c). The cell suspensions were prepared, washed and resuspended in balanced salt solution and, for use in the immunofluorescence assay, appropriately diluted in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin (BSA; Poviet, Amsterdam). The final pH of the PBS-BSA solution was adjusted to 7.8. Protein A plaque assay
IgM-, IgG- and IgA-secreting cells were assayed by the hemolytic plaque assay as described by Gronowicz et al. (1976), with some modifications. Staphylococcus aureus protein A (Pharmacia, Uppsala) was coupled to SRBC with CrC13 • 6H20 as described by Van Oudenaren et al. (1981). The protein A-coated SRBC were used within 3 days of preparation. The protein A plaque assay was performed in BSS in Cunningham chambers as described by Lefkovits and Cosenza (1979). Each chamber contained 100 ~tl of appropriately diluted cell suspension mixed with 20 #1 of an
119 optimal amount of diluted guinea pig complement, purified over a Sepharose-protein A column (Pharmacia) as described by Van Oudenaren et al. (1981), 15 pl of an optimal amount of diluted specific rabbit anti-mouse IgM, IgG or IgA antiserum and 25/~1 of a 10% suspension of protein A-coated SRBC. The rabbit antisera used were purified over a Sepharose-protein A column as described by Goding (1978), in order to isolate the protein A-binding fraction of Ig. The chambers were incubated at 37°C for 5 h. The Ig secreting plaque-forming cells (PFC) were counted under a dissecting microscope by dark field illumination.
Visualization of cytoplasmic Ig-containing cells Cytocentrifuge preparations of a known number of cells were prepared according to Vossen et al. (1976). The technique for the visualization of cytoplasmic Ig-containing cells (C-Ig cells) has been described in detail by Hijmans et al. (1969). Slides were fixed for 15 min at - 2 0 ° C in acid ethanol (5 parts of acetic acid to 95 parts of ethanol) and washed for 30 rain in PBS. Incubations with unlabeled and fluorescent antisera were for 30 min in humidified chamber. Excess conjugate was removed with one wash of PBS. Preparations were mounted in buffered glycerol (1 part of PBS to 9 parts of glycerol; Merck-Schuchard, Mtinchen) and sealed with paraffin. A fluorescein-conjugated goat antiserum directed against mouse Ig (GAMIg/FITC, lot no. 2-873; Nordic Immunological Laboratories, Tilburg) was used to determine the total number of C-Ig cells per slide. Cytoplasmic-Ig positive cells were easily distinguished from pre-B cells, the latter having only a small rim of cytoplasmic Ig, and showing faint staining in immunofluorescence (Raft et al., 1976). From the number of C-Ig positive cells per slide, the number of cells spun down per slide and the cell yield per organ, we calculated the total number of C-Ig cells per organ. The Ig class distribution of the C-Ig cells was determined according to Hijmans et al. (1969) by means of combinations of rhodamine and fluorescein labeled antisera specific for the heavy chains of IgM, IgG and IgA.
Antisera All antisera used were of rabbit origin, except the FITC-conjugated goat antiserum used to determine the total number of C-Ig cells per slide. The rhodamine and fluorescein labeled class specific antisera were prepared, purified, and generously supplied by Dr. J. R~idl and Miss P. Van den Berg, from the Institute for Experimental Gerontology, TNO. The antisera fulfilled the specificity criteria described elsewhere (Bloemmen et al., 1976). The class-specific antisera used in the protein A plaque assay were purchased from Litton-Bionetics, Kensington, MD. Whether or not both sets of antisera recognized the same cells was investigated by testing both antisera specific for a certain Ig class on the same cytocentrifuge preparation, the one directly labeled and the other indirectly labeled with a different fluorochrome. These comparative studies revealed at least 95% overlap for the anti-IgM, anti-IgG and anti-IgA rabbit antisera.
120
Calculation of the number of positive cells in the bone marrow Bone marrow PFC and C-Ig cells were determined in the femoral marrow. Following Benner et al. (1981c) we adopted a conversion factor of 7.9 to calculate the number of positive cells in the total bone marrow from the number found in 2 femurs.
Results and Discussion
Ig producing cells occur in all lymphoid organs, independently of whether or not the individuals have been intentionally immunized (Van Furth et al., 1966; Benner et ai., 1981b). In mice, the actual number of Ig-producing cells per organ differs between the different lymphoid organs (Table I), and is dependent upon antigenic load and age (Table II). In young adult mice most Ig producing cells occur in spleen, lymph nodes and bone marrow. Peyer's patches and thymus contain only small numbers of Ig producing cells (Benner et al., 1981b). The total number of Ig producing cells per mouse increases with increasing age. This is predominantly due to the fact that the number of Ig producing cells in the bone marrow increases markedly with age (Haaijman et al., 1979; Benner et al., 1981a). Enumerations of the lg producing cells by immunofluorescence and by the protein A plaque assay do not always give similar frequencies. The protein A plaque assay often gives a higher number than immunofluorescence. In the individual mice studied, the ratio of results by the 2 assays ranged from 0.8 to about 9, and was dependent on the lymphoid organ and Ig class studied (Table I). The discrepancy cannot be due to the quality of the antisera used, since comparative studies of both sets of antisera revealed an overlap of at least 95% in the immunofluorescence assay. In the spleen of 8-week-old B A L B / c mice the protein A plaque assay and the immunofluorescence assay gave equal numbers of IgM and IgG producing cells. However, the protein A plaque assay gave almost 3 times as many lgA producing cells as the immunofluorescence assay. For the other lymphoid organs this ratio was usually higher than 1.0, especially for IgA, but in some instances also for IgM and lgG (Table I). The relative sensitivity of the protein A plaque assay and the immunofluorescence assay was also determined on bone marrow cells from mice of different ages and under different antigenic loads (i.e., germ-free and specific pathogen-free mice). The most striking observation was that in the marrow of 100-week-old B A L B / c mice the protein A plaque assay detected 8.6 times as many IgA producing cells as the immunofluorescence assay, and that relatively high ratios were found for both IgG and IgA producing cells in the marrow of 100-week-old germ free mice (Table II). It is remarkable that the discrepancy between the 2 assays in old mice is greater for lgG and IgA than for IgM. This correlates with the degree to which the production of these Ig classes is dependent on T cell help (Bankhurst et al., 1975; Van Muiswinkel and Van Soest, 1975), and the age-related dysfunction of the T cell system (Makinodan et al., 1977; Krogsrud and Perkins, 1977). It suggests that the
Fiuoresc.
Plaques
Fluoresc.
Plaques
Fluoresc.
7.9 (6.0-11) 8.5 (5.9-12)
14 (13-16) 6.3 (5.2-7.6)
(44-48) 20 (17-24)
113 b (100-127) I 17 (98-140)
IgM
0.9
2.2
}
}
i.0 ¢
2.5
/
'[
/
1.6
1.2
(45-59) 15 (10-23)
54 (47-62) 35 (30-4 0
3.5
1.5
} 52 }
(98-137) 73 (57-92)
164 (136-198) i 41 ( 121- 170)
IgG
II (8.3-14) 2.8 (1.8-4.2)
22 (17-29) 3.1 (2.4-4.1)
55 (52-58) 21 (18-24) 2.6
} 3.9
/ 7.1 J
/
}
71 (64-78) 26 (18-38)
90 (79-102) 44 (39-51)
221 (196-250) 114 (101-129)
354 (314-399) 287 (248-331)
2.7
77 (66-89) 29 (25-33)
}
Total
lgA
• lg producing cells were simultaneously assayed by immunofluorescence and the protein A plaque assay. b Figures represent the geometric mean + I S.E.M. ( x 10-3) of the number of positive cells per whole organ (n = 6). ¢ Ratio of the number of lg producing cells found in the protein A plaque assay to number found by immunofluorescence.
Peyer's patches
Mesenteric lymph nodes
Bone marrow
Plaques
Spleen
Fluoresc.
Assay •
Organ
2.7
2.1
1.9
1.2
INCIDENCE OF BACKGROUND IgM, lgG AND lgA PRODUCING CELLS ( × 10- 3) IN THE SPLEEN. BONE MARROW, MESENTER1C LYMPH NODES AND PEYER'S PATCHES OF 8-WEEK-OLD SPECIFIC PATHOGEN-FREE (SPF) BALB/c MICE AS DETERMINED BY THE PROTEIN A PLAQUE ASSAY AND IMMUNOFLUORESCENCE
TABLE I
8
100
100
BALB/c
BALB/c
BALB/c
GF
SPF
SPF
Status a
Fluoresc.
Plaues
Fluoresc.
(961- I 650) 1 561 ( 1 240-1 967)
(1004-1682) 1 223 ( 1 016-1 473)
FI uoresc.
Paques
50c (44-58) 20 (17-24)
IgM
Plaques
Assay b
/
1
}
0.8
1.1
2.5
1S92
(1 220-2078) 475 (332-680)
2320
(1916-2809) 1342 ( 1 188-1 517)
116 (98-137) 73 (57-92)
lgG
/
1
}
3.4
1.7
1.6
49
(348-504) 69 (60 81 )
(1426-3074) 245 ( 188-318)
55 (52-58) 21 ( I 8- 24)
IgA
/
/
}
6.1
8.6
2.6
3270
(2478-4315) 2 105 ( I 658-2672)
573
(5219-6254) 2810 (2566-3075)
221 (196 250) I 14 ( 101 - 129)
Total
SPF, specific pathogen-free; GF, germ-free. b Ig producing cells in the bone marrow of mice were simultaneously assayed by immunofluorescence and the protein A plaque assay. " Figures represent the geometric mean -}- I S.E.M. ( x 10-3) of the number of positive cells per whole bone marrow (n = 6). d Ratio between the number of Ig producing cells found in the protein A plaque assay, and by immunofluore.~ence.
Age (weeks)
Mice
1.6
2.0
1.9
INCIDENCE OF B A C K G R O U N D IgM, IgG AND IgA P R O D U C I N G CELLS ( × 1 0 3) IN THE BONE MARROW OF BALB/c MICE AS DETERMINED BY THE PROTEIN A PLAQUE ASSAY A N D I M M U N O F L U O R E S C E N C E
TABLE II
123
extent of the discrepancy between the numbers of Ig producing cells given by the 2 assays is related to the degree of requirement for and/or the dysfunction of the T cell system, a view that is supported by comparable results of experiments on athymic nude mice (Benner et al., 1982). The underlying cause of the discrepancy between the numbers of Ig producing cells revealed by the 2 assays is probably that in immunofluorescence only plasmablasts and plasma cells are indentifiable with certainty. However, earlier stages of the plasmacytic series are also able to produce and secrete Ig (Melchers and Andersson, 1974), and are thus able to produce plaques. Although these findings suggest that the protein A plaque assay is more reliable for measuring activity of the humoral immune system than the immunofluorescence assay, another factor should also be taken into account. This is that the amount of Ig released per Ig producing cell increases during maturation (Melchers and Andersson, 1974). Thus, immature Ig producing cells release less Ig than more mature cells like plasma cells and thus contribute less to total Ig synthesis. Ideally, the numbers of Ig producing cells of the various lymphoid organs should correlate with the Ig concentrations in the serum. In man, comparative studies have shown such positive correlations for the various Ig classes and subclasses (Hijmans, 1975; Turesson, 1975), when Ig producing cells were enumerated by immunofluorescence assay. In mice, such studies do not reveal a simple linear correlation (J.J. Haaijman, unpublished). Studies in mice with the protein A plaque assay instead of the immunofluorescence assay have not as yet been performed.
Acknowledgements We thank Dr. J. R~dl and Ms. P. Van den Berg for the fluorescent conjugates used in this study, and Ms. Cary Meijerink for typing the manuscript.
References Bankhurst, A.D., P.H. Lambert and P.A. Miescher, 1975, Proc. Soc. Exp. Biol. Med. 148, 501. Benner, R., W. Hijmans and J.J. Haaijman, 1981a, Clin. Exp. lmmunol. 46, 1. Benner, R., A.-M. Rijnbeck, R.R. Bcrnab6, C. Martincz-Aionso and A. Coutinho, 1981b, Immunobiology 158, 225. Bcnner, R., A. Van Oudenaren and G. Koch, 1981c, in: Immunological Methods, Vol. 2, cds. 1. Lefkovits and B. Pernis (Academic Press, New York) p. 247. Benner, R., A. Van Oudenaren, M. Bj6rklund, F. lvars and D. Holmbcrg, 1982, Immunology Today 3, 243. Bloemmen, F.J., J. R~dl, J.J. Haaijman, P. Van den Berg, H.R.E. Schuit and W. l-lijmans, 1976, J. Immunol. Methods I0, 337. Goding, J., 1978, J. Immunol. Methods 20, 241. Gronowicz, E., A. Coutinho and F. Melchers, 1976, Eur. J. Immunol. 6, 588. Haaijman, J.J.,J. Slingerland-Teunissen,R. Benner and A. Van Oudenaren, 1979, Immunology 36, 271. Hijmans, W., 1975, in: The Immunological Basis of Connective Tissue Disorders, ed. L.G. Silvestri (North-Holland, Amsterdam) p. 203.
124 Hijmans, W., H.R.E. Schuit and F. Klein. 1969, Clin. Exp. immunol. 4, 457. Krogsrud, R.L. and E.H. Perkins, 1977, J. Immunol. 118, 1607. Laurell, C.B., 1972, Scan& J. Clin. Lab. Invest. 29, Suppl. 124, 21. Lefkovits. I. and H. Cosenza. 1979, in: Immunological Methods, Vol. 1, eds. 1. Lefkovits and B. Pernis (Academic Press, New York) p. 277. Makinodan, T., R.A. Good and M.M.B. Kay, 1977, in: Comprehensive Immunology, Vol. I, eds. "I. Makinodan and E. Yunis (Plenum Medical Book Co., New York) p. 9. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistr3' 2, 235. Melchers, F. and J. Andersson. 1974, Eur. J. Immunol. 4, 181. Molinaro, G.A. and S. Dray, 1974, Nature (London)248, 515. Orlans, E.. J. Peppard, J. Reynolds and J. Hall, 1975, J. Exp. Med. 147, 588. Osebold, J.W., N.I. Medin and Y.C. Zee, 1975, Infect. lmmun. 12. 1141. Raff, M.C., M. Megson, J.J.T. Owen and M.D. Cooper, 1976, Nature (London) 259, 224. Spiegelberg. H.L.. 1974, Adv. Immunol. 19, 259. Tomasi, T.B., 1976, in: The Immune System of Secretions, eds. A.G. Osier and L. Weiss (Prentice Hall. E n g l e w ~ d Cliffs, N J) p. 57. Turesson, I., 1976, Acta Med. Scand. 199, 293. Van Furth, R., H.R.E. Schuit and W. Hijmans, 1966, Immunology II, 19. Van Muiswinkel, W.B. and P.L. Van Soest, 1975, Immunology 28, 287. Van Oudenaren, A., H. Hooijkaas and R. Benner. 1981, J. lmmunol. Methods 43, 219. Vosmn, J.M.J.J., R.E. Langlois van der Berg, H.R.E. Schuit and W. Hijmans, 1976, J. Immunol. Methods 13, 71.