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Immunoglobulin isotypes in plasma cells of normal and athymic mice
CELLULAR
IMMUNOLOGY
lmmunoglobulin
PAUL
Department
4,
343-351 (1979)
lsotypes in Plasma Cells of Normal and Athymic Mice1
WEISZ-CARRINGTON, A. FAYE SCHRATER, MICHAEL AND G. JEANETTE THORBECKE of Pathology,
NeM, York University New York, New Received
School of Medicine, York 10016
October
25,
550
E. LAMM,
First
Avenue,
1978
The distribution of plasma cells was determined in various lymphoid tissues and exocrine glands of athymic (nude) mice. Compared to values for normal mice, the total number of plasma cells in organs of athymic mice showed a variable decrease as follows: a%, small intestine; 29%, respiratory tree; 33%, spleen; 50%, lymph nodes; 75%, lactating mammary gland; 85%, Peyer’s patches; and 90%, parotid gland. Plasma cells containing IgG, or IgA showed the greatest decrease, whereas IgM-containing plasma cells were actually increased by 100% or more in most organs. In exocrine glands the absolute deficit of IgA-containing plasma cells was most marked in the parotid and lactating mammary gland, and least in the small intestine. All lymphoid tissues had a striking deficit in the absolute numbers of IgA as well as IgG, plasma cells. Total plasma cell numbers and their isotype distribution were similar for BALB/c +/+ (homozygous) and +/nu (heterozygous) mice.
INTRODUCTION As a result of the thymic dependence of B-cell maturation [reviewed in (l)], the athymic nude mouse has levels of serum immunoglobulins which differ markedly from those of normal mice of the same strain (2,3). Serum IgM levels of nude mice may be either normal or increased, whereas levels of IgG and IgA are usually decreased. The T-cell dependency of plasma cells synthesizing isotypes other than IgM is further suggested by studies of nude and normal mice treated with anti-p and anti-a antisera (4). These experiments showed that stable suppression of all immunoglobulin (Ig)2 classes with anti-p antisera, and selective suppression with anti-a and anti-y, antisera, were much easier to attain in nude than in normal mice. It has been observed by immunohistological methods that plasma cells making IgG and IgA are decreased in number in tissues of the nude mouse (5), whereas studies of surface Ig on circulating B cells show anormal distribution of the different isotypes (6,7). The presence in nude mice of normal lymphoid follicles which lack germinal centers (8,9) also suggests that virgin B cells are relatively normal but that their differentiation and/or proliferation is abnormal. Reconstitution of athymic 1 This investigation was supported by Grants AI-3076, CA-23885, CA-08627, AI-15071, and AI-05196 from the National Institutes of Health. ’ Abbreviations used: Ig, immunoglobulin; nu/nu, homozygous athymic; +/+, wild type; +/nu, heterozygous athymic; H&E, hematoxylin and eosin; TNP, 2,4,6-trinitrophenyl. 343 0008-8749/79/060343-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
344
WEISZ-CARRINGTON
ET AL.
mice with T cells results in development ofgerminal centers (.5,9, 10) and restores to normal levels the number of IgA plasma cells in the gut (5) and those antibody responses which are low in nude mice, i.e., primary responses to T-dependent antigens (11) and secondary responses to T-independent antigens (9). The apparent discrepancies with regard to quantitation of I3 cells with various surface Ig markers versus serum Ig isotypes in nude mice could be explained by an incomplete differentiation of B cells to Ig-secreting plasma cells for certain isotypes. Since a comprehensive study of the numbers of plasma cells in nude as compared to normal mice has not been reported, we have studied the distribution of plasma cells synthesizing the various Ig classes in lymphoid tissues of such mice. Since IgA is one of the deficient Ig isotypes in nude mice, we also examined exocrine gland and mucosal laminae propriae, sites where the majority of resident plasma cells in normal mice synthesize IgA. MATERIALS
AND METHODS
Mice. Six- to lo-week-old homozygous (nuinu) male and female mice on a BALB/c background were raised from heterozygous (+/nu) parents (fourth generation backcross) maintained in our animal facilities. Mice were housed in double air-flow chambers and supplied with clean food and bedding. To increase survival of nude neonates, mice were given 500 kg/ml Neo-Terramycin (Chas. Pfizer & Co., Inc., New York, N.Y.) in the drinking water from birth until 4 weeks of age. Thymic aplasia in nude mice was verified by histological analysis. Tissues. Mice were sacrificed by cervical dislocation. The organs were removed, embedded in Tissue-Tek II OCT compound (Lab Tek Products, Miles Laboratories, Inc., Naperville, Ill.), and then frozen at -30°C. Tissue sections (6-8 pm thick) were cut in a cryostat and fixed in acetone for 10 min. Immuno$horescence. Tissue sections were stained by indirect immunofluorescence (12). Rabbit antisera against murine IgM, IgGI, IgG,, IgGOb, and IgA (Litton Bionetics, Kensington, Md.) were used for the first incubation. After one IO-min rinse the sections were incubated with fluoresceinated goat anti-rabbit y-globulin (Behring Diagnostics, Somerville, N.J.). All antisera were checked for specificity as described previously (12). Sections from the organs taken from six nuinu, six +/+ , and four +/nu animals were examined with a Leitz Orthoplan microscope illuminated by a 200-W ultrahigh-pressure mercury lamp. Lactating mammary glands, 10 days postpartum, were included from three additional animals per group. Plasma cells in five random fields were counted with a 10x oil immersion objective and a 10x ocular. Only cells with definite cytoplasmic fluorescence were tallied. Hematoxylin and eosin (H&E)stained step sections of each organ were studied for general histology, and methyl green pyronin stains were also made for total plasma cell counts. RESULTS Normal
BALBIc
Mice (+I+)
Spleen and peripheral lymph nodes such as the axillary, lumbar, and popliteal, which do not drain secretory sites, contained more IgG- than IgM- or IgAproducing plasma cells (Table 1). The results for lumbar and popliteal nodes
PLASMA
CELL ISOTYPES
IN NORMAL
345
AND NUDE MICE
were analogous to those obtained for axillary nodes, and are, therefore, not included in Table 1. For each of the above organs, comparable numbers of cells synthesized IgG1, IgG*,, and IgG2,,. However, lymph nodes which drain mucosal or exocrine sites, such as the mesenteric node and the cervical node within the parotid gland, contained numerous IgA plasma cells. IgA plasma cells were also predominant in secretory sites such as the lactating mammary gland, parotid gland, and small intestinal and bronchial mucosae (Table 2). The isotype distribution of plasma cells in these sites agrees with previously reported data from studies of TABLE Isotype Distribution
Organ Spleen
of Plasma Cells in Various Lymphoid Tissues Determined by Indirect Immunofluorescence”
Isotype
BALBlc (+/+I
P Yl y2a y2b a
99k 445 43i392 99 2
CL Yl +a -db a
/* Yl y2a y2b *
Total Mesenteric node
Total
862 462 48k 362 735
125 19k 222 202 29k
132 6 2Ok 7 235 5 21k 4 292 12
172 102 12k 105 1295
F Yl +a r2b a
6 2 3 1 6
342 9 16i 8 19~ 6 15s 4 231 i- 70
5+ 2r 3+ 2+ 3t
3 6 5 3 6
(1Figures are average numbers of cells in five fields (10x) f SD. ’ Located within the parotid gland.
48% 11 12 0 4+ 2 4t 2 42 2 61
152 6 132 4 142 8 132 8 88k 10
32% 5 1-c 0 5i 1 4t 1 6% I 48
3 5 2 3 11
246
15
204 k 38 co.5 4+ 1 St 1 4-c 1
102
102 132 16-c 14-t 193 -c
5 0 0 0 I
Homozygote (nuinu)
217
143
315 k Yl y2a r2b a
5 8 9 7 6
289
178
Total Peyer’s patch
8 6 5 8 20
106
Total Cervical node”
Heterozygote (+/t-m)
324
Total Axillary node
I
7-c 2-c 2k 3+ 22 16
88 f 36 It 1 162 2 13t 4 21 t 15 139
2 0 0 0 1
co.5 It 0 It 0 co.5 co.5 2
346
WEISZ-CARRINGTON TABLE
ET AL.
2
Isotype Distribution of Plasma Cells Determined by Indirect Immunofluorescence Selected Tissues Containing Exocrine Epithelium” Organ Small intestine
Isotype I* Yl +a y2b a
Total Respiratory tree
CL Yl +a y2b a
CL Yl y2a y2b a
Total Lactating mammary gland
Total
Heterozygote (+/nu)
16 k 6 822 10 k 1 922 53 L 5 96
Total Parotid gland
BALBlc (+I+)
CL Yl +a y2b Ly
220 <0.5 ‘co.5 <0.5 22 k 7
17k 62 72 lo+ 45*
3 3 3 2 7
85 2k 1t lk co.5 l5k
in
Homozygote (nu/nu) 47 + 12 7+ 7+ 35 ?
29 0 4 3 15
97 0 0 0 5
6t 5 <0.5 co.5 <0.5 12 0
24
19
7
5kl 521 921 8kl 35 + 9
5+ 1 9k 1 9k 1 7k 1 42 2 12
5+ 1 -co.5 1k 0 <0.5 co.5
62
72
10 f 1 320 420 421 100 + 9 121
12? 3+ 4+ 3+ 842 106
6 1 1 0 0 1
27? co.5 I+ 1+ 2+
5 0 0 0
31
” Figures are average numbers of cells in five fields (10x) + SD.
humans and mice (12- 14). Homozygous (+/+) and heterozygous (+/nu) BALB/c mice had comparable numbers and isotype distribution of plasma cells. Homozygous nulnu Mice
The lymphoid organs showed decreased numbers of lymphocytes in the thymus-dependent areas, as is usual in nude mice, and a paucity of germinal centers (8, 9). Peyer’s patches were very poorly developed. The organ distribution of plasma cells in nude mice is shown in Tables 1 and 2 and Fig. 1. There was a marked deficit of IgG, and IgA plasma cells in most lymphoid organs. Although some lymphoid tissues and exocrine epithelia also had a deficit of IgG, plasma cells, they were present in near-normal numbers in the small intestine and mesenteric lymph node. There was an enormous deficit of IgA plasma cells in the lamina propria of exocrine epithelia as well, with the exception of the small intestine in which IgA plasma cells were present at almost normal levels (Table 2; Fig. 1). Cells containing IgG, were much less numerous than IgA cells in these
PLASMA
CELL ISOTYPES
IN NORMAL
AND NUDE MICE
347
Plasma Cell1sotype Ratlo 0,+I+ : “u/n”
lgG2a
IgGZb
FIG. 1. Ratios of plasma cells containing specific Ig isotypes in organs from normal (+/+) versus athymic (nu/nu) mice. Organs represented are spleen, axillary lymph node (AXLN), mesenteric lymph node (MLN), parotid gland (PAR. GL.), small intestine (SM. INT.), lung, and lactating mammary gland (MAM. GL.). Ratios were calculated from data in Tables 1 and 2.
locations in normal mice, and were essentially absent from mucosal linings in nude mice. In contrast, the values for IgM-containing plasma cells from nude mice were approximately two to four times those of normal mice in virtually all organs examined. Although these values compensated to some extent for the deficiency in IgG and IgA plasma cells, the total number of plasma cells remained below normal levels except in the small intestine (Fig. 2). Plasma cells also tended to be less mature morphologically in nude than in normal mice. Total
Plasma Cell Of +/+ : ““,“” 123456769
SPLEEN
m
AXLN
m
Ratio
FIG. 2. Ratios of total plasma cells in organs from normal (+/+) versus athymic (nu/nu) mice. Organs and ratios as in Fig. 1.
348
WEISZ-CARRINGTON
ET AL.
Of particular interest were the observations in three postparturient lo-day lactating nude mice. Relatively few IgA plasma cells were seen in the connective tissue surrounding the mammary gland acini in nu/nu compared to +/+ or +/nu lactating mice, which had 50 times as many such cells. The isotype of most of the plasma cells in the mammary glands of nude mice was IgM (Table 2). Occasional large IgM-containing cells were seen within the lumens of acini. In H&E-stained sections these cells were identified as plasma cells. These cells might be analogous to the IgA plasma cells which have been described in human milk (15). As could be predicted from the isotype distribution of plasma cells, the predominant intraepithelial Ig in the mammary glands of athymic mice was IgM, and not IgA as is normally seen (12). Other exocrine glands such as the parotid and bronchial glands showed similar results, with a predominantly IgM-secreting plasma cell population, and associated intraepithelial IgM. DISCUSSION The results presented here are in accord with and provide a cellular basis for the observations by other authors regarding levels of immunoglobulins in the serum of athymic mice. The cellular deficit observed for IgG, plasma cells was striking and agrees well with previous reports which showed that serum IgG, was the most deficient Ig class in sera of nude mice (5-8% of control values) (2,3). Correlations between the numbers of IgA plasma cells in different sites and levels of IgA in serum are more difficult to make because the relative contributions of IgA plasma cells in exocrine glands, mucous membranes, bone marrow, and peripheral lymphoid tissues to serum IgA have not been well defined. The total number of plasma cells found in the lamina propria of the small intestine was normal, possibly due to intense stimulation by gut-associated environmental antigens and mitogens, as well as to the impaired ability of IgM cells to differentiate into IgG and IgA plasma cells in the absence of T-cell help. The numbers of plasma cells containing IgG, agrees with the serum values reported for nude mice (2,3). The number of IgM plasma cells in nude mice is approximately 100% higher than in normal mice, a finding which is consistent with the normal to elevated serum IgM reported for nude mice (2, 3). In the absence of the thymus, the Ig isotype pattern as represented in plasma cells and in the serum shows marked changes. However, surface Ig of B cells in bone marrow (6) and thoracic duct (7) is reported to have a normal isotype distribution. These observations suggest that maturation of B cells to plasma cells rather than the switch from IgM to the synthesis of other Ig isotypes requires T-cell help. It has been suggested that maturation of virgin B cells in regard to surface Ig isotypes in fetal liver and neonatal bone marrow is thymus independent (6). There are several other aspects of the results which need to be considered. The first is the presence in nude mice of greater than normal numbers of IgM-synthesizing plasma cells which would seem to indicate that a majority of IgM-synthesizing cells need no T-cell help for maturation to plasma cells. A large proportion of these plasma cells must have been induced by environmental thymus-independent polyclonal B-cell activators, which are known to induce primarily IgM synthesis (16, 17). It is important to note that the original demonstration of the T-cell dependence of antibody production involved the primary response of mouse spleen cells to sheep erythrocytes (18, 19). Moreover,
PLASMA
CELL
ISOTYPES
IN NORMAL
AND
NUDE
MICE
349
T-dependent B-cell differentiation into secreting plasma cells is as dependent on T-cell help in the secondary 19s as in the secondary 7S antibody response (20). It is known, however, that T-independent antigens such as TNP-Ficoll can induce a sizeable IgM response with detectable IgGI, IgGz, and IgA components in nude mice (21). The cells responsible for the T-independent responses to polysaccharide antigens such as this might represent a separate subclass of B cells (22). It is possible that the plasma cells seen in nude mice represent an expansion of this subclass at the expense of “T-dependent” B cells and their progeny. The second aspect to be considered is the stringent thymus dependency for generation of IgG memory B cells, which has been studied in nude mice (9) and in thymectomized mice (23,24). Thus, while virgin B-cell generation may be relatively T-independent, memory B cell generation (9,25), germinal center formation (9, lo), as well as efficient IgG production (23-26) during the immune response are all thymus dependent. If the majority of surface Ig-bearing cells represent virgin B cells rather than memory B cells, similarity in this respect between B cells of nude and normal mice is to be expected (6). In contrast, plasma cells which synthesize those Ig classes represented in T-dependent responses would be relatively few in number. The present results suggest that IgG, and to a lesser extent IgA are foremost among the T-dependent isotypes. The other experimental mouse model in which serum IgM levels are much less reduced than serum IgG, or IgA is the gnotobiotic mouse where the deficiency is secondary to a lack of appropriate antigenic stimulation (27-29). The one major histological characteristic that germ-free animals share with nude mice is the absence of germinal center proliferation along the mucosal lining of the intestines (30,3 1). It is tempting to speculate that the absence of these T-dependent structures is somehow related to the profound deficiency in IgG,, IgGz, and IgA plasma cells in many locations. The conversion of IgM synthesis to IgG synthesis, which occurs at an accelerated rate during the immune response, may very well be linked to B-cell proliferation in these lymphoid structures. The lactating mammary gland in nude mice contained about one-quarter the total number of plasma cells seen in normal mice; of these, 87% contained IgM. Homing studies in normal mice showed that the precursors of IgA-synthesizing plasma cells in the lactating mammary gland are surface IgA-bearing cells which lack surface IgM (32). Whether surface IgA and/or some other cell surface component is required for B-cell localization in exocrine sites is not known. Further studies are needed to determine whether the IgM plasma cells observed in the lactating mammary glands of nude mice were derived from IgA-bearing cells which failed to complete their differentiation locally into IgA plasma cells, or whether cells can localize in the mammary gland by a “trapping” mechanism independent of IgA. The data presented here suggest that IgM should be the prevalent Ig class in the milk of nude mice. This is based on the observation that the majority of plasma cells in the mammary gland of athymic mice produce IgM. Furthermore, this isotype was the one most readily detected within glandular epithelial cells of lactating mammary glands rather than IgA which is normally found in these cells (12). This observation would be consistent with results from human studies wherein IgM often replaces IgA in secretions of IgA-deficient individuals (33). We have noticed that heterozygous (+/nu) mice suckled on homozygous (nu/nu) mothers do not thrive in a normal environment and in many cases succumb to
350
WEISZ-CARRINGTON
ET AL.
neonatal diarrhea. However, if these animals are nursed by a +/+ or +/nu mother, they remain healthy. This difference can be related to evidence that under normal conditions milk contains protective IgA antibodies which are directed toward antigens of gastrointestinal microorganisms. These antibodies are thought to be secreted by cells whose precursors originated in the gut-associated lymphoid tissue where they were sensitized prior to migration to the mammary gland (15,32,34,35). A deficiency of these cells in nude mice could account for a diminished transfer to the offspring, via the milk, of secretory IgA antibodies directed against intestinal pathogens. The characterization of plasma cells in the mammary glands of lactating nude and normal mice may offer a clue to the lineage of IgA plasma cells in the mouse. There is general agreement that these cells originate from precursors that formerly synthesized IgM [reviewed in (36)]. However, two schools of thought exist concerning the pathway of the switch (4, 37-39): (i) IgM-synthesizing precursor cells differentiate directly into IgA plasma cells, or (ii) IgM-synthesizing cells differentiate indirectly into IgA plasma cells via an IgG stage. Further experiments with this model, including the use of lactating nude mice, with and without thymic reconstitution, might provide pertinent information regarding the sequence of this switch. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Katz, D. H., and Benacerraf, B., Advan. Immunol. 15, 1, 1972. Luzzati, A. L., and Jacobson, E. B., Eur. J. Immunol. 2,473, 1972. Bankhurst, A. D., Lambert, P. H., and Miescher, P. A., Proc. Sot. Exp. Biol. Med. 148,501,197.5. Manning, D. D., and Jutila, J. W., Cell. Immunol. 14, 453, 1974. Guy-Grand, D., Griscelli, C., and Vassalli, P., J. Immunol. 115, 361, 1975. Abney, E. R., Cooper, M. D., Kearney, J. F., Lawton, A. R., and Parkhouse, R. M. E. ,J. Immunol. 120, 2041, 1978. Bankhurst, A. D., and Warner, N. L., Aust. .I. Exp. Biol. Med. Sci. 50, 661, 1972. De Sousa, M. A. B., Parrott, D. M. V., and Pantelouris, E. M., Clin. Exp. Immunol. 4,637, 1969. Jacobson, E. B., Caporale, L. H., and Thorbecke, G. J., Cell. Immunol. 13,416, 1974. De Sousa, M., and Pritchard, H., Immunology 26, 769, 1974. Kindred, B., Cell. Immunol. 17, 277, 1975. Weisz-Carrington, P., Roux, M. E., and Lamm, M. E., .I. Immunol. 119, 1306, 1977. Haaijman, J. J., Schuit, H. R. E., and Hijmans, W., Immunology 32, 427, 1977. Tourville, D. R., Adler, R. H., Bienenstock, J., and Tomasi, T. B., J. Exp. Med. 129,411, 1969. Goldblum, R. M., Ahlstedt, S., Carlsson, B., Hanson, L. A., Jodal, U., Lidin-Janson, G., and Sohl-Akerlund, A., Nature (London) 257, 797, 1975. Andersson, J., Sjoberg, O., and Moller, G., Eur. .I. Immunol. 2, 349, 1972. Greaves, M. F., and Janossy, G., Transplant. Rev. 14,76, 1973. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Exp. Biol. Med. 122, 1167, 1966. Miller, J. F. A. P., and Mitchell, G. F., J. Exp. Med. 128, 801, 1968. Romano, T. J., and G. J. Thorbecke, Cell. Immunol. 17, 240, 1975. Gershwin, M. E., Merchant, B., and Steinberg, A. D., Immunology 32, 327, 1977. Ahmed, A., Scher, I., Sharrow, S. O., Smith, A. H., Paul, W. E., Sachs, D. H., and Sell, K. W., J. Exp. Med. 145, 101, 1977. Davie, J. M., and Paul, W. E., J. Immunol. 113, 1438, 1974. Romano, T. J., and Thorbecke, G. J., J. Immunol. 115, 332, 1975. Braley-Mullen, H., J. Exp. Med. 147, 1824, 1978. Grumet, F. C., J. Exp. Med. 135, 110, 1972. Beneviste, J., Lespinats, G., and Salomon, J. C., Proc. Sot. Exp. Biol. Med. 130, 936, 1969. Beneviste, J., Lespinats, G., Adam, C., and Salomon, J. C., J. Immunol. 107, 1647, 1971.
PLASMA 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
CELL ISOTYPES
IN NORMAL
AND NUDE MICE
351
Stecher, V. J., and Thorbecke, G. J., Immunology 12, 475, 1967. Glimstedt, G., Acta Pathol. Microbial. Stand. Suppl. 30, 1936. Thorbecke, G. J., Ann. N. Y. Acad. Sci. 78, 237, 1959. Roux, M. E., McWilliams, M., Phillips-Quagliata, J. M., Weisz-Canington, P., and Lamm, M. E., .I. Exp. Med. 146, 1311, 1977. Brandtzaeg, P., Fjellanger, I., and Gjeruldsen, S. T., Science 160, 789, 1968. Montgomery, P. C., Rosner, B. R., and Cohn, J., Immunol. Commun. 3, 143, 1974. Cebra, J. J., Kamat, R., Gearhart, P., Robertson, S. M., and Tseng, J., Cold Spring Harbor Symp. Quant. Biol. 41, 201, 1977. Lamm, M. E., Advan. Immunol. 22, 223, 1976. Pierce, C. W., Solliday, S. M., and Asofsky, R., J. Exp. Med. 135, 698, 1972. Lawton, A. R., and Cooper, M. D., Contemp. Top. Immunobiol. 3, 193, 1974. Honjo, T., and Kataoka, T., Proc. Nat. Acad. Sci. USA 75, 2140, 1978.
IMMUNOLOGY
lmmunoglobulin
PAUL
Department
4,
343-351 (1979)
lsotypes in Plasma Cells of Normal and Athymic Mice1
WEISZ-CARRINGTON, A. FAYE SCHRATER, MICHAEL AND G. JEANETTE THORBECKE of Pathology,
NeM, York University New York, New Received
School of Medicine, York 10016
October
25,
550
E. LAMM,
First
Avenue,
1978
The distribution of plasma cells was determined in various lymphoid tissues and exocrine glands of athymic (nude) mice. Compared to values for normal mice, the total number of plasma cells in organs of athymic mice showed a variable decrease as follows: a%, small intestine; 29%, respiratory tree; 33%, spleen; 50%, lymph nodes; 75%, lactating mammary gland; 85%, Peyer’s patches; and 90%, parotid gland. Plasma cells containing IgG, or IgA showed the greatest decrease, whereas IgM-containing plasma cells were actually increased by 100% or more in most organs. In exocrine glands the absolute deficit of IgA-containing plasma cells was most marked in the parotid and lactating mammary gland, and least in the small intestine. All lymphoid tissues had a striking deficit in the absolute numbers of IgA as well as IgG, plasma cells. Total plasma cell numbers and their isotype distribution were similar for BALB/c +/+ (homozygous) and +/nu (heterozygous) mice.
INTRODUCTION As a result of the thymic dependence of B-cell maturation [reviewed in (l)], the athymic nude mouse has levels of serum immunoglobulins which differ markedly from those of normal mice of the same strain (2,3). Serum IgM levels of nude mice may be either normal or increased, whereas levels of IgG and IgA are usually decreased. The T-cell dependency of plasma cells synthesizing isotypes other than IgM is further suggested by studies of nude and normal mice treated with anti-p and anti-a antisera (4). These experiments showed that stable suppression of all immunoglobulin (Ig)2 classes with anti-p antisera, and selective suppression with anti-a and anti-y, antisera, were much easier to attain in nude than in normal mice. It has been observed by immunohistological methods that plasma cells making IgG and IgA are decreased in number in tissues of the nude mouse (5), whereas studies of surface Ig on circulating B cells show anormal distribution of the different isotypes (6,7). The presence in nude mice of normal lymphoid follicles which lack germinal centers (8,9) also suggests that virgin B cells are relatively normal but that their differentiation and/or proliferation is abnormal. Reconstitution of athymic 1 This investigation was supported by Grants AI-3076, CA-23885, CA-08627, AI-15071, and AI-05196 from the National Institutes of Health. ’ Abbreviations used: Ig, immunoglobulin; nu/nu, homozygous athymic; +/+, wild type; +/nu, heterozygous athymic; H&E, hematoxylin and eosin; TNP, 2,4,6-trinitrophenyl. 343 0008-8749/79/060343-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
344
WEISZ-CARRINGTON
ET AL.
mice with T cells results in development ofgerminal centers (.5,9, 10) and restores to normal levels the number of IgA plasma cells in the gut (5) and those antibody responses which are low in nude mice, i.e., primary responses to T-dependent antigens (11) and secondary responses to T-independent antigens (9). The apparent discrepancies with regard to quantitation of I3 cells with various surface Ig markers versus serum Ig isotypes in nude mice could be explained by an incomplete differentiation of B cells to Ig-secreting plasma cells for certain isotypes. Since a comprehensive study of the numbers of plasma cells in nude as compared to normal mice has not been reported, we have studied the distribution of plasma cells synthesizing the various Ig classes in lymphoid tissues of such mice. Since IgA is one of the deficient Ig isotypes in nude mice, we also examined exocrine gland and mucosal laminae propriae, sites where the majority of resident plasma cells in normal mice synthesize IgA. MATERIALS
AND METHODS
Mice. Six- to lo-week-old homozygous (nuinu) male and female mice on a BALB/c background were raised from heterozygous (+/nu) parents (fourth generation backcross) maintained in our animal facilities. Mice were housed in double air-flow chambers and supplied with clean food and bedding. To increase survival of nude neonates, mice were given 500 kg/ml Neo-Terramycin (Chas. Pfizer & Co., Inc., New York, N.Y.) in the drinking water from birth until 4 weeks of age. Thymic aplasia in nude mice was verified by histological analysis. Tissues. Mice were sacrificed by cervical dislocation. The organs were removed, embedded in Tissue-Tek II OCT compound (Lab Tek Products, Miles Laboratories, Inc., Naperville, Ill.), and then frozen at -30°C. Tissue sections (6-8 pm thick) were cut in a cryostat and fixed in acetone for 10 min. Immuno$horescence. Tissue sections were stained by indirect immunofluorescence (12). Rabbit antisera against murine IgM, IgGI, IgG,, IgGOb, and IgA (Litton Bionetics, Kensington, Md.) were used for the first incubation. After one IO-min rinse the sections were incubated with fluoresceinated goat anti-rabbit y-globulin (Behring Diagnostics, Somerville, N.J.). All antisera were checked for specificity as described previously (12). Sections from the organs taken from six nuinu, six +/+ , and four +/nu animals were examined with a Leitz Orthoplan microscope illuminated by a 200-W ultrahigh-pressure mercury lamp. Lactating mammary glands, 10 days postpartum, were included from three additional animals per group. Plasma cells in five random fields were counted with a 10x oil immersion objective and a 10x ocular. Only cells with definite cytoplasmic fluorescence were tallied. Hematoxylin and eosin (H&E)stained step sections of each organ were studied for general histology, and methyl green pyronin stains were also made for total plasma cell counts. RESULTS Normal
BALBIc
Mice (+I+)
Spleen and peripheral lymph nodes such as the axillary, lumbar, and popliteal, which do not drain secretory sites, contained more IgG- than IgM- or IgAproducing plasma cells (Table 1). The results for lumbar and popliteal nodes
PLASMA
CELL ISOTYPES
IN NORMAL
345
AND NUDE MICE
were analogous to those obtained for axillary nodes, and are, therefore, not included in Table 1. For each of the above organs, comparable numbers of cells synthesized IgG1, IgG*,, and IgG2,,. However, lymph nodes which drain mucosal or exocrine sites, such as the mesenteric node and the cervical node within the parotid gland, contained numerous IgA plasma cells. IgA plasma cells were also predominant in secretory sites such as the lactating mammary gland, parotid gland, and small intestinal and bronchial mucosae (Table 2). The isotype distribution of plasma cells in these sites agrees with previously reported data from studies of TABLE Isotype Distribution
Organ Spleen
of Plasma Cells in Various Lymphoid Tissues Determined by Indirect Immunofluorescence”
Isotype
BALBlc (+/+I
P Yl y2a y2b a
99k 445 43i392 99 2
CL Yl +a -db a
/* Yl y2a y2b *
Total Mesenteric node
Total
862 462 48k 362 735
125 19k 222 202 29k
132 6 2Ok 7 235 5 21k 4 292 12
172 102 12k 105 1295
F Yl +a r2b a
6 2 3 1 6
342 9 16i 8 19~ 6 15s 4 231 i- 70
5+ 2r 3+ 2+ 3t
3 6 5 3 6
(1Figures are average numbers of cells in five fields (10x) f SD. ’ Located within the parotid gland.
48% 11 12 0 4+ 2 4t 2 42 2 61
152 6 132 4 142 8 132 8 88k 10
32% 5 1-c 0 5i 1 4t 1 6% I 48
3 5 2 3 11
246
15
204 k 38 co.5 4+ 1 St 1 4-c 1
102
102 132 16-c 14-t 193 -c
5 0 0 0 I
Homozygote (nuinu)
217
143
315 k Yl y2a r2b a
5 8 9 7 6
289
178
Total Peyer’s patch
8 6 5 8 20
106
Total Cervical node”
Heterozygote (+/t-m)
324
Total Axillary node
I
7-c 2-c 2k 3+ 22 16
88 f 36 It 1 162 2 13t 4 21 t 15 139
2 0 0 0 1
co.5 It 0 It 0 co.5 co.5 2
346
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ET AL.
2
Isotype Distribution of Plasma Cells Determined by Indirect Immunofluorescence Selected Tissues Containing Exocrine Epithelium” Organ Small intestine
Isotype I* Yl +a y2b a
Total Respiratory tree
CL Yl +a y2b a
CL Yl y2a y2b a
Total Lactating mammary gland
Total
Heterozygote (+/nu)
16 k 6 822 10 k 1 922 53 L 5 96
Total Parotid gland
BALBlc (+I+)
CL Yl +a y2b Ly
220 <0.5 ‘co.5 <0.5 22 k 7
17k 62 72 lo+ 45*
3 3 3 2 7
85 2k 1t lk co.5 l5k
in
Homozygote (nu/nu) 47 + 12 7+ 7+ 35 ?
29 0 4 3 15
97 0 0 0 5
6t 5 <0.5 co.5 <0.5 12 0
24
19
7
5kl 521 921 8kl 35 + 9
5+ 1 9k 1 9k 1 7k 1 42 2 12
5+ 1 -co.5 1k 0 <0.5 co.5
62
72
10 f 1 320 420 421 100 + 9 121
12? 3+ 4+ 3+ 842 106
6 1 1 0 0 1
27? co.5 I+ 1+ 2+
5 0 0 0
31
” Figures are average numbers of cells in five fields (10x) + SD.
humans and mice (12- 14). Homozygous (+/+) and heterozygous (+/nu) BALB/c mice had comparable numbers and isotype distribution of plasma cells. Homozygous nulnu Mice
The lymphoid organs showed decreased numbers of lymphocytes in the thymus-dependent areas, as is usual in nude mice, and a paucity of germinal centers (8, 9). Peyer’s patches were very poorly developed. The organ distribution of plasma cells in nude mice is shown in Tables 1 and 2 and Fig. 1. There was a marked deficit of IgG, and IgA plasma cells in most lymphoid organs. Although some lymphoid tissues and exocrine epithelia also had a deficit of IgG, plasma cells, they were present in near-normal numbers in the small intestine and mesenteric lymph node. There was an enormous deficit of IgA plasma cells in the lamina propria of exocrine epithelia as well, with the exception of the small intestine in which IgA plasma cells were present at almost normal levels (Table 2; Fig. 1). Cells containing IgG, were much less numerous than IgA cells in these
PLASMA
CELL ISOTYPES
IN NORMAL
AND NUDE MICE
347
Plasma Cell1sotype Ratlo 0,+I+ : “u/n”
lgG2a
IgGZb
FIG. 1. Ratios of plasma cells containing specific Ig isotypes in organs from normal (+/+) versus athymic (nu/nu) mice. Organs represented are spleen, axillary lymph node (AXLN), mesenteric lymph node (MLN), parotid gland (PAR. GL.), small intestine (SM. INT.), lung, and lactating mammary gland (MAM. GL.). Ratios were calculated from data in Tables 1 and 2.
locations in normal mice, and were essentially absent from mucosal linings in nude mice. In contrast, the values for IgM-containing plasma cells from nude mice were approximately two to four times those of normal mice in virtually all organs examined. Although these values compensated to some extent for the deficiency in IgG and IgA plasma cells, the total number of plasma cells remained below normal levels except in the small intestine (Fig. 2). Plasma cells also tended to be less mature morphologically in nude than in normal mice. Total
Plasma Cell Of +/+ : ““,“” 123456769
SPLEEN
m
AXLN
m
Ratio
FIG. 2. Ratios of total plasma cells in organs from normal (+/+) versus athymic (nu/nu) mice. Organs and ratios as in Fig. 1.
348
WEISZ-CARRINGTON
ET AL.
Of particular interest were the observations in three postparturient lo-day lactating nude mice. Relatively few IgA plasma cells were seen in the connective tissue surrounding the mammary gland acini in nu/nu compared to +/+ or +/nu lactating mice, which had 50 times as many such cells. The isotype of most of the plasma cells in the mammary glands of nude mice was IgM (Table 2). Occasional large IgM-containing cells were seen within the lumens of acini. In H&E-stained sections these cells were identified as plasma cells. These cells might be analogous to the IgA plasma cells which have been described in human milk (15). As could be predicted from the isotype distribution of plasma cells, the predominant intraepithelial Ig in the mammary glands of athymic mice was IgM, and not IgA as is normally seen (12). Other exocrine glands such as the parotid and bronchial glands showed similar results, with a predominantly IgM-secreting plasma cell population, and associated intraepithelial IgM. DISCUSSION The results presented here are in accord with and provide a cellular basis for the observations by other authors regarding levels of immunoglobulins in the serum of athymic mice. The cellular deficit observed for IgG, plasma cells was striking and agrees well with previous reports which showed that serum IgG, was the most deficient Ig class in sera of nude mice (5-8% of control values) (2,3). Correlations between the numbers of IgA plasma cells in different sites and levels of IgA in serum are more difficult to make because the relative contributions of IgA plasma cells in exocrine glands, mucous membranes, bone marrow, and peripheral lymphoid tissues to serum IgA have not been well defined. The total number of plasma cells found in the lamina propria of the small intestine was normal, possibly due to intense stimulation by gut-associated environmental antigens and mitogens, as well as to the impaired ability of IgM cells to differentiate into IgG and IgA plasma cells in the absence of T-cell help. The numbers of plasma cells containing IgG, agrees with the serum values reported for nude mice (2,3). The number of IgM plasma cells in nude mice is approximately 100% higher than in normal mice, a finding which is consistent with the normal to elevated serum IgM reported for nude mice (2, 3). In the absence of the thymus, the Ig isotype pattern as represented in plasma cells and in the serum shows marked changes. However, surface Ig of B cells in bone marrow (6) and thoracic duct (7) is reported to have a normal isotype distribution. These observations suggest that maturation of B cells to plasma cells rather than the switch from IgM to the synthesis of other Ig isotypes requires T-cell help. It has been suggested that maturation of virgin B cells in regard to surface Ig isotypes in fetal liver and neonatal bone marrow is thymus independent (6). There are several other aspects of the results which need to be considered. The first is the presence in nude mice of greater than normal numbers of IgM-synthesizing plasma cells which would seem to indicate that a majority of IgM-synthesizing cells need no T-cell help for maturation to plasma cells. A large proportion of these plasma cells must have been induced by environmental thymus-independent polyclonal B-cell activators, which are known to induce primarily IgM synthesis (16, 17). It is important to note that the original demonstration of the T-cell dependence of antibody production involved the primary response of mouse spleen cells to sheep erythrocytes (18, 19). Moreover,
PLASMA
CELL
ISOTYPES
IN NORMAL
AND
NUDE
MICE
349
T-dependent B-cell differentiation into secreting plasma cells is as dependent on T-cell help in the secondary 19s as in the secondary 7S antibody response (20). It is known, however, that T-independent antigens such as TNP-Ficoll can induce a sizeable IgM response with detectable IgGI, IgGz, and IgA components in nude mice (21). The cells responsible for the T-independent responses to polysaccharide antigens such as this might represent a separate subclass of B cells (22). It is possible that the plasma cells seen in nude mice represent an expansion of this subclass at the expense of “T-dependent” B cells and their progeny. The second aspect to be considered is the stringent thymus dependency for generation of IgG memory B cells, which has been studied in nude mice (9) and in thymectomized mice (23,24). Thus, while virgin B-cell generation may be relatively T-independent, memory B cell generation (9,25), germinal center formation (9, lo), as well as efficient IgG production (23-26) during the immune response are all thymus dependent. If the majority of surface Ig-bearing cells represent virgin B cells rather than memory B cells, similarity in this respect between B cells of nude and normal mice is to be expected (6). In contrast, plasma cells which synthesize those Ig classes represented in T-dependent responses would be relatively few in number. The present results suggest that IgG, and to a lesser extent IgA are foremost among the T-dependent isotypes. The other experimental mouse model in which serum IgM levels are much less reduced than serum IgG, or IgA is the gnotobiotic mouse where the deficiency is secondary to a lack of appropriate antigenic stimulation (27-29). The one major histological characteristic that germ-free animals share with nude mice is the absence of germinal center proliferation along the mucosal lining of the intestines (30,3 1). It is tempting to speculate that the absence of these T-dependent structures is somehow related to the profound deficiency in IgG,, IgGz, and IgA plasma cells in many locations. The conversion of IgM synthesis to IgG synthesis, which occurs at an accelerated rate during the immune response, may very well be linked to B-cell proliferation in these lymphoid structures. The lactating mammary gland in nude mice contained about one-quarter the total number of plasma cells seen in normal mice; of these, 87% contained IgM. Homing studies in normal mice showed that the precursors of IgA-synthesizing plasma cells in the lactating mammary gland are surface IgA-bearing cells which lack surface IgM (32). Whether surface IgA and/or some other cell surface component is required for B-cell localization in exocrine sites is not known. Further studies are needed to determine whether the IgM plasma cells observed in the lactating mammary glands of nude mice were derived from IgA-bearing cells which failed to complete their differentiation locally into IgA plasma cells, or whether cells can localize in the mammary gland by a “trapping” mechanism independent of IgA. The data presented here suggest that IgM should be the prevalent Ig class in the milk of nude mice. This is based on the observation that the majority of plasma cells in the mammary gland of athymic mice produce IgM. Furthermore, this isotype was the one most readily detected within glandular epithelial cells of lactating mammary glands rather than IgA which is normally found in these cells (12). This observation would be consistent with results from human studies wherein IgM often replaces IgA in secretions of IgA-deficient individuals (33). We have noticed that heterozygous (+/nu) mice suckled on homozygous (nu/nu) mothers do not thrive in a normal environment and in many cases succumb to
350
WEISZ-CARRINGTON
ET AL.
neonatal diarrhea. However, if these animals are nursed by a +/+ or +/nu mother, they remain healthy. This difference can be related to evidence that under normal conditions milk contains protective IgA antibodies which are directed toward antigens of gastrointestinal microorganisms. These antibodies are thought to be secreted by cells whose precursors originated in the gut-associated lymphoid tissue where they were sensitized prior to migration to the mammary gland (15,32,34,35). A deficiency of these cells in nude mice could account for a diminished transfer to the offspring, via the milk, of secretory IgA antibodies directed against intestinal pathogens. The characterization of plasma cells in the mammary glands of lactating nude and normal mice may offer a clue to the lineage of IgA plasma cells in the mouse. There is general agreement that these cells originate from precursors that formerly synthesized IgM [reviewed in (36)]. However, two schools of thought exist concerning the pathway of the switch (4, 37-39): (i) IgM-synthesizing precursor cells differentiate directly into IgA plasma cells, or (ii) IgM-synthesizing cells differentiate indirectly into IgA plasma cells via an IgG stage. Further experiments with this model, including the use of lactating nude mice, with and without thymic reconstitution, might provide pertinent information regarding the sequence of this switch. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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351
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