Immunology 1976 31 921
Development of immunoglobulin and antibody-synthesizing cells after immunization with different doses of antigen J. C. ANTOINE, CHRISTINE PETIT & S. AVRAMEAS Unite'd'Immunocytochimie, De'partement de Biologie Moliculaire, Institut Pasteur, Paris, France
Received 15 April 1976; acceptedfor publication 13 May 1976
Summary. The kinetics of development of antibodysynthesizing cells and of cells synthesizing immunoglobulins without detectable antibody function were studied in rats immunized with different doses (0-1, 1, 10, 100 mg) of horse radish peroxidase, bovine serum albumin, human serum albumin, hen ovalbumin, or human IgG, which had been deaggregated or heat-aggregated. Each antigen was injected once or twice as a solution in saline. Antibody and immunoglobulin-producing cells were detected in draining lymph nodes by immunohistochemical staining. In the primary response a few antibody-synthesizing cells were found whatever the dose injected. No increase or some increase was found with the amount of antigen injected, according to the protein used, but with all doses of antigen injected, the population of cells remained small, except with human IgG where a relatively high number of positive cells was detected even after injection of 1 mg of antigen. In the secondary response a few antibody-forming cells were also detected with the lower doses of antigen, but this population increased after boosting with 100 mg of antigen. With human IgG a greater number of positive cells was induced with all the doses tested. A correlation between the number of cells synthesizing immunoglobulins without antibody funcCorrespondence: Dr J. C. Antoine, Unit6 d'Immunocytochimie, D6partement de Biologie Molculaire, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
921
tion and the amount of antigen injected was observed in the primary and secondary responses. The relative size of these two populations varied with the stage of immunity of the animals. In the primary response, the population ofcellssynthesizingimmunoglobulins without antibody function was larger than the population of antibody-forming cells. The same was true in the secondary response, but if after a booster injection the level of antibody-synthesizing cells exceeded that reached in the primary response, the increase of cells synthesizing Ig without antibody function was smaller than the increase in antibodyforming cells. In general the more immunogenic an antigen was, the smaller was the ratio between antibody-forming cells and cells producing immunoglobulin without antibody function.
INTRODUCTION In a previous paper we have shown that after primary immunization with peroxidase emulsified in Freund's complete or incomplete adjuvant the first immunoglobulin-synthesizing cells which appeared in the draining lymph nodes did not contain molecules with detectable antibody function. This population of cells was progressively replaced by antibody-forming cells. In the secondary response, antibody-producing cells were predominant, and they were not preceded by cells synthesizing immunoglobulins without antibody function (Antoine & Avrameas, 1976).
J. C. Antoine, Christine Petit & S. Avrameas
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In the present paper, in order to examine whether the appearance of cells synthesizing immunoglobulins without antibody function was associated with the particular antigen used (peroxidase) or with the presence of Freund's adjuvant, we immunized rats with various antigens dissolved in saline. Furthermore, to explore the influence of the amount of antigen injected on the relative proportion of these two populations of cells, various doses were used for each antigen. In these studies, we also tried to test if immunoglobulins without detectable antibody function were very low affinity antibodies. We found that after inoculation, and independently of the antigen employed, immunoglobulin-synthesizing cells always appeared. These cells appeared alone or with antibody-synthesizing cells, and their number increased with the amount of antigen injected. Thus it appears that the development of antibody-forming cells and of cells containing immunoglobulin without antibody function is a general characteristic of the immune response.
MATERIALS AND METHODS Animals Five-month-old OFA rats of both sexes, bred in specific pathogen-free conditions were used. Antigens
Peroxidase (PO) RZ = 3 was purchased from Boerhinger, Mannheim, Germany. Crystallized human serum albumin (HSA) was obtained from Miles Laboratories, Slough, Bucks. Crystallized bovine serum albumin (BSA) was obtained from Sigma Chemical Company, St Louis, Missouri. Five times crystallized hen ovalbumin (OVA) was a gift from Dr P. A. Cazenave (Institut Pasteur, Paris, France). Human IgG were prepared from Cohn's fraction II (Miles Laboratories, Slough, England) which had been further purified by chromatography on DEAE ion-exchange cellulose (grade DE 52, Whatman, Maidstone, Kent) following the procedure described by Fahey & Terry (1973). In some experiments Human IgG was prepared from human serum by salt fractionation, chromatography on DEAE-cellulose and molecular filtration through Sephadex G-200 (Pharmacia, Uppsala, Sweden). Tolerogenic (deaggregated) and immunogenic (heat-aggregated) physical forms of human IgG were prepared following the methods described by Chiller, Habicht & Weigle (1971).
All these above antigens were tested by immunoelectrophoresis with antisera obtained in rabbits hyperimmunized with these antigens and used subsequently to inoculate the rats. Antisera against PO, HSA, OVA and IgG revealed only a single precipitation line. Antisera against BSA revealed a strong precipitation line and a second very faint line. Immunizations
Immunization was carried out by injecting s.c. in both hind footpads 0-1, 1, 10, or 100 mg of antigen dissolved in 0-4 ml of saline made with pyrogen-free water. Ten to 15 days after the primary injection the animals received by the same route the same dose of antigen. Animals were killed 7 days and 10-13 days after the first injection and 5-7 days after the secondary injection. The tertiary response to peroxidase was also studied in animals receiving 0-1 or 10 mg of antigen in saline 38 days after the primary injection and 23 days after the secondary injection. Animals were killed either after the primary injection or 5 or 14 days after the second inoculation, or 5 days after the third injection. In each experiment control rats were injected once, twice, or thrice with 0 4 ml of saline. Preparation of cells At least two rats immunized with the different antigens were studied on each day and for each dose examined. Animals were killed by ether. The popliteal lymph nodes were removed, weighed and dissociated in cold Hanks's medium (Institut Pasteur, Paris, France). Cells were centrifuged at 200 g for 7 min, resuspended in 1 ml of Hanks's medium, counted and cytocentrifuged as described previously (Antoine & Avrameas, 1976). Preparation of antisera and ofpurified antibodies
Rabbit and sheep anti-rat immunoglobulin and rabbit anti-hen ovalbumin antisera were prepared as described earlier (Gonatas, Antoine, Stieber & Avrameas, 1972). Purified rabbit and sheep antibodies against rat Fab or IgG and rabbit antibodies against ovalbumin were prepared following procedures already described (Antoine & Avrameas, 1973, 1974). Fab fragments from sheep anti-rat immunoglobulin antibodies were prepared by papain digestion (Porter, 1959) according to a method
Ig- and Ab-synthesizing cell development described earlier (Antoine, Avrameas, Gonatas, Stieber & Gonatas, 1974).
Labelling of antigens and of antibodies Antigens and purified antibodies were labelled with peroxidase by a two-step procedure (Avrameas & Ternynck, 1971). Labelled proteins were filtered on a 95 x 26 cm Sephadex G-200 column equilibrated with 0-1 M Tris, HCl buffer, pH 8, 1 M NaCl to eliminate free proteins and free peroxidase. Detection of immunoglobulin- and antibody-synthesizing cells After cytocentrifugation, cells were left overnight, then fixed for 15 min in 0-2 M cacodylate buffer, pH 7 4, containing 4 per cent paraformaldehyde, washed three times in PBS and incubated with the different conjugates. To detect cells synthesizing antibodies against peroxidase, slides were incubated for 1 h with a 100 ug/ml solution of PO in PBS. Cells synthesizing antibodies against BSA, HSA and human IgG were detected by incubating slides for 2 h with a 250 pg/ml solution of the corresponding antigen labelled with PO and diluted in normal rat serum. To detect cells synthesizing antibodies against OVA, slides were incubated for 1 h with a 100 ,ug/ml solution of OVA in normal rat serum, washed and then incubated for 2 h with a 250 ,ug/ml solution of peroxidase-labelled rabbit anti-OVA antibodies in normal rat serum. To detect cells synthesizing immunoglobulins, the preparations were incubated for 2 h with a 250 ,ug/ml solution in PBS of POlabelled anti-rat immunoglobulin or anti-rat Fab antibodies or with a 125 pg/ml solution of sheep Fab fragments of anti-rat immunoglobulin antibodies conjugated with PO. Staining was done according to the method of Graham & Karnovsky (1966). After staining the total number of nucleated cells and of positive cells was calculated. Results were expressed as the percentage of the positive cells, or as the total number of positive cells per lymph node (Antoine & Avrameas, 1976).
RESULTS For each antigen we give the results obtained 7 days after a primary injection and 5 days after a booster injection (Tables 1 and 2). We also studied the response 10-15 days after a primary injection. However, at this time, whatever the doses employed,
923
there was a very low, often undetectable number of antibody and Ig-producing cells. It appears therefore that protein antigens injected in a soluble form in saline induced a very sharp primary immune response, and we do not report here results 10-15 days after primary inoculation. In the following, we call 'immunoglobulin-synthesizing cells' the cells producing immunoglobulin without detectable antibody function.
Peroxidase After one injection of various doses of PO, the weight and the number of cells of the lymph nodes increased with the amount of antigen injected. A very small amount of antibody-producing cells was detected after one injection of 0-1, 1, or 10 mg of PO. After two or three injections larger numbers of antibody-producing cells were found in animals injected with 0-1 mg of antigen. A progressive increase of immunoglobulin-producing cells was noted with increasing amounts of antigen injected. 5 days after secondary injection of 1 mg or 10 mg of antigen, the number of positive cells reached the level attained 7 days after the first injection. An increase in this population of cells was noted after two or three injections of a dose of 0 1 mg (Fig. 1). Bovine serum albumin Only the primary immune response was studied with this antigen. An increase in the weight of the lymph nodes was found after injection of the antigen but not as a function of the dose of antigen injected; a very slight increase in the cellular content of the nodes was found. No increase or a very slight increase as a function of the dose of antigen was noted in the amount of antibody-producing cells. The number of Ig-synthesizing cells increased with the amount of antigen inoculated (Fig. 2). Human serum albumin During the primary immune response no increase of the lymph node weight was observed whatever quantity of antigen was injected. The same was true for the cellular content of the nodes. In the secondary response an increase of the lymph node weight and cellular content was observed only with the 100 mg dose. A very low number of antibody-producing cells was found 7 days after the first injection of 1,
J. C. Antoine, Christine Petit & S. Avrameas
924
Table 1. Antibody and immunoglobulin-synthesizing cells in rat lymph nodes draining footpads injected with antigens 7 days previously Ig Ag Igc (7)
Cells/lymph Antigen
node
Ab (per cent)
Ig (per cent)
nb Ab
nb Ig
(1)
(2)
(3)
(4)
(5)
(6)
21 x 106 6-75x l0 33x 106 4-35x 106
0 0 0-018 0-013
0-1 0-3 0-5 1-5
0 0 1-2x 102 5-6x 102
4x l10 1-75x 106
0 0
0-1 0-4
0 0
4x 102 6 6x 103
17
Bovine serum albumin (expt 1) (mg) C 0-9+0-3x106 1-6+0-7x 106 0-1 1 1-2+0-3x 106 14+0 6x 106 10
0 0-03+0 0-02+ 0-02 0-04+ 0-02
0-12+ 0-02 0-04+ 0-06 0-35+0-13 1-05+0-07
0 45+2-1x 102 1-5+2-1x 102 50+ 1-4x 102
1l+0.lx 103 2-9+4-1 x102 3-8+0-3x 103 1-4+0-5x 104
0-2+ 0-3 3-5+0-3 12-7+ 4-6
Bovine serum albumin (expt 2) (mg) 2-7+0-5x l10 C 8x 104 0.1 1-35x 106 1 55x 105 10 1-9+ l x 106 100
0 0 0 0-007 0-03+ 0-01
0-06+ 0-04 0-27 0-16 0-37
0 0 0 38-5 4-9+0 4x 102
1-6+ 1-2x 102 2-2x 102 2 2x 103 2x 103 2-4+2-4x 104
1-4 13-7 12-5 151-7+ 147-4
0 0
0-05+ 0-02 0-02+0 0-05+ 0-01 0-28 0-17+ 0-06
0 0
4-0+0-3x 102 2-5+0-9x 102 8-9+8-6x 102 6-4x 103 2-9+ 1-8x 103
0-6+ 0-2 2-2_2-1 16 7-2+ 4-6
1-5+0-9x 102 6-2+0-3x 10 64+5x 10 6+5x 102 3-7+0-7x 103
0-4+ 000-4+ 0 4 3-8+ 3-1 24+4
Peroxidase (expt 1) (mg) C 0-1 1 10
Peroxidase (expt 2) (mg) C 1
Human serum albumin (mg) C 8-0+2-5x l0 1-3+0-5x 106 0-1 1-5+ 1-3x106 1 2-2x 106 10 1-6+0-4x106 100
Ovalbumin (mg) C 0-1 1 10 100
Deaggregated human IgG (mg) C 0-1 1 10
1-4+ 1-6x 102 1-35x 102 2-7+ 1-8x 102
0-04+ 001
0
3l1+0-4x l10
0 0 0
0.0- 00
0
6-7+0-7x iO0 17+0 4x 106
0-002+ 0 003 0-017+ 0-016
0-23+ 0-04
3 6+0-9x l0 4-7+2-4x l0
7+2x l10
0
13+Ox 106
0-035+ 0-021 0-055+ 0-063 0-15+ 007
2-4+0-7x 106 8+8x l10
Aggregated human IgG (expt 1) (mg) C 001 1 8
0-007+0-004 0-006 0-02+ 0-02
1-15+0-6
1-8+0-5x 105 8+6x 105 2 5+0 5x 106 5 3+0 8x 106
0 006+ 004 0 06+ 0°00 0-4+ 03
0-02± 0-01 0-08+ 0-06
009+ 0°00 0°14+0-08 0-11+0-13 0-4+ 0 3
0 06+0-06 02+0-2
0-09+0-06 0-10+0-07
2-3x 2x 1-65x 6 5x
0 13+ 19 2-5+2-5x 102
0 4-5+2-7x 102 1.5+ l-9X 103
1-6+ l-9x
103
0 3-8+0-Ox 102 1-6+ 0 5 x 103 2-3+2x 104
103 103 104 104
0-9 7-1 28
6-7+ 1-8x 102 1-8+ 1-1 x 103
30+_3-9x 103 2-1+1-1x 103
9-6+7-5x 10 1-3+0-6x 103 2-2+ 1-9x 103
6-6+4-8x
103
2-7+ 1-7 4-5+ 5-9 3-1+ 1-7
13-4+ 6-1 23-1+20-3 68-5+ 50-2
Ig- and Ab-synthesizing cell development
925
Table 1. (cont.)
Antigen
(1) Aggregated human IgG (expt 2) (mg) C 0-1 1 10 22 39 Aggregated human lgG (expt 3) (mg) C 25 Aggregated human lgG (expt 4) (mg) C 50
Cells/lymph node
Ab (per cent)
Ig (per cent)
nb Ab
nb Ig
Ig Ag Igc
(2)
(3)
(4)
(5)
(6)
(7)
0
0-07
0-16+0-04 0 07+000 0-28+0-21 0-15+ 0-10 07 0-45
0 75+4 22+3Ox 103 1 1+ 16x 102 7-1 x 103 3 3x 103
2-2+ 11 x 103 17+0 4x 103 34+25x 103 3 4+2 9x 103 2 7x 104 2x 104
0-7+0-2 1-6+ 11 1-6+ 1-2 12-3 9
8 6x 106
0 0-05
04 0 16
0 4-3x 103
5-6x 103 18x 104
2-5
49x 105 1 8x 107
0 0-24
0-24 0-66
0 4 3x 104
12x 103 1l2x 105
14+08x 106 2 2+0 3x 106
12+0Ox 106 2 3+0 Ox 106 3-9x 106 4-4x 106
14x106
0-003+0-000 0-19+0-25 0-005+0-007 0-18
100
Seven days after the primary injection, animals immunized with different doses of PO, BSA, HSA, OVA, deaggregated or heat-aggregated human IgG were killed. Antibody and immunoglobulin-synthesizing cells were detected by immunoenzymological methods in the draining lymph nodes. Column 1: antigen doses injected. C: control rats which received 0 4 ml of saline. Column 2: total number of cells per lymph node. Column 3: percentage of antibody-synthesizing cells. Column 4: percentage of cells producing immunoglobulin without antibody function. Column 5: number of antibody-synthesizing cells per lymph node. Column 6: number of cells producing Ig without antibody function per lymph node. Column 7: ratio between the total number of cells producing immunoglobulin without antibody function found in animals immunized with various doses of antigen and the total number of immunoglobulinsynthesizing cells found in lymph nodes of animals inoculated with saline alone.
10 or 100 mg of HSA. In the secondary response no or only a few antibody-positive cells were detected except at the 100 mg dose, where a much greater number were found. The number of immunoglobulinproducing cells progressively increased with the quantity of antigen injected both for the primary and the secondary response. With 01, 1 and 10 mg of antigen where approximately the same number of antibody-producing cells was present after one or two stimuli, no increase of the Ig-synthesizing cells was also noted. With the 100 mg dose the two populations of cells increased after the booster injection. The ratio between the number of antibodycontaining cells 5 days after a second injection and 7 days after primary inoculation was 430, while the same ratio for the immunoglobulin-synthesizing cells was 38. Ovalbumin
There was no significant variation in the weight of
the nodes with the amount of antigen injected. In the primary response the total number of cells per lymph node was approximately the same whatever the amount of antigen injected, except in the animals stimulated with 100 mg of ovalbumin, where cells were five times more numerous than in the control nodes. In the secondary response an increase of the lymph node cells correlated with the quantity of antigen the animals received. At day 7 antibodysynthesizing cells were detected only with 10 mg and 100 mg of ovalbumin. The number of immunoglobulin-producing cells was the same as or lower in rats receiving 0-1 or 1 mg than in control rats, and higher than rats receiving 10 and 100 mg. In animals stimulated with 10 mg, the ratio between numbers of antibody-producing cells detected at day 5 after a boosting and those present at day 7 after one injection was 1-6. The same ratio calculated for the 100 mg dose was 32. For the immunoglobulin-synthesizing cells the ratios were respectively 2 2 and 15.
926
J. C. Antoine, Christine Petit & S. Avrameas
Table 2. Antibody and immunoglobulin-synthesizing cells in rat lymph nodes 5 days after secondary injection of antigens
Cells/lymph nb Ig
IgAg lgc
(4)
nb Ab (5)
(6)
(7)
0 0-03 0-008 0
0-07 0-17 0-44 1-6
0 1-3x 103 2-2x 102 0
1-4x 103 71x 103 12x 104 6 9x 104
5-1 8-6 46
0 0-003+0 004 0 0-005+0-007 0-65+0-07
0-10+0 00 0-06+0-02 0-09+0-02 0-21+0 06 0-7+0-6
0 33+47 0 75+10 8 6+7-6x 104
1 7x 103 6 5+09x 102 1 0+0 3x 103 3-3+0 8x 103
0 0 0-006+0-001 0-001+0-002 0-165+0-005
0-08+0-01 0-05+0-00 0-04+0-00 0-07+0-03
2 8+1 1x102 5-4+ 1-6x 102 2-4+0-Ox 02
1-1+±00
0 0 37+3 21+ 29 7-9+1 7x 103
1O+0-5x 103 5-3+1-0x 104
1-9+0-6 0-9+0-0 3-6+ 1-8 186+36
0 1-1+0 3 3-1+0-7 5-9+2-1
0-15+0-07 0-9+0-2 0-9+0-6 3-4+1-5
0 3-5+2 7x 104 1-7+ 1-2x O 7-7+4-7x l0
1-3+0-7x 103 2 8+2-Ox 104 5 2+5 3x 104 4-2+0-3x 104
21-7+15-0 40-4+40-8 327+27
0 1-5+ 15 04+0 1 0 9+0-4
0-2+0-0 0-6+0-2 1 7+0-0 1-8+0-8
0
27+24x 104 1-7+1 4x 104 1l2+0-2x i05
1-2+0 3x103 1 1+0-6x104 8 3+8-1x 104 25+0 4x i05
9-3+5 1 69-2+67-1 206-7+33-0
Antigen
node
Ab (per cent)
Ig (per cent)
(1)
(2)
(3)
Peroxidase (expt 1) (mg) C 0.1 1 10 Human serum albumin
2x106 4-2x 106 2-7x 106 4 3x106
(mg) 11+06x 106 106 106
C 0 1 I 10 100
1 0+0 2x 1.I-+01 x 1-6+0-1 x 1 2+ lOx
Ovalbumin (mg) C 0-1 1 10 100
Deaggregated human IgG (mg) C 0-1 1 10
Aggregated human IgG (expt 2) (mg) C 0-1 1 10
106 106
3-5+0-5x105 1-0+0-2x 106 6-4+0-7x l0 1-3+00x 106 4-8+0 9x106
1-1+ l-Ox 2-9+1-5x 5-3+2-8x 1-3+0-4x
106 106 106 10
4 8+0 1xIO 1-9+0 2x 106 5 0+4 8x 106 1l5+0 5x l0
ll-+1-4x 10
0 3+0-0 0-6+0-2 1-9+0-5 68+82
Ten to 15 days after the primary injection animals received various doses of antigen as indicated in column 1, and lymph nodes were taken 5 days later. The same dose was inoculated in primary and secondary immunization except in the animals which had received 0-1 mg of aggregated IgG in the first inoculation and I mg in the second inoculation. For the explanations of columns 2-6: see legend to Table 1.
Deaggregated human IgG No correlations were found between the size and the total number of cells of the lymph nodes and the amount of
IgG inoculated during the primary
In secondary response both size and total numbers of lymph node cells increased with the
response.
quantity of antigen. After one injection of 01, 1 or 10 mg of IgG a small number of antibodyproducing cells appeared with all the three doses. The same was true for the immunoglobulin-synthesizing cells. After a booster injection there was a large increase in the size of the two populations of
cells at all doses tested. In both populations, the increase was a function of the amount of antigen injected. The ratio between the number of positive cells found at 5 days after boosting and 7 days after primary injection was calculated for the two populations. For the antibody population these ratios were 77, 113, and 480 for 01 mg, 1 mg and 10 mg respectively. The ratios for the Ig population were respectively 24, 23 and 300.
Heat-aggregated human IgG Both weight of lymph nodes and total number of
Ig- and Ab-synthesizing cell development
927
primary and secondary response and the ratios obtained for the antibody population were 360, 7-7 and 1140 for the 0 1, 1 and 10 mg doses respectively. The same ratios calculated for the immunoglobulin population were 36 and 113 for the 1 and 10 mg doses respectively. E
-
21 7
1301 42 Days
after antigen
Figure 1. Primary, secondary and tertiary immune responses of rats stimulated with peroxidase given as a solution in saline in the hind footpads (expt 1). On each day one rat was killed for each dose studied. Arrows indicate the time when the two booster injections were given. Animals which were used in secondary and tertiary responses received the same dose as in the first injection. Kinetics of development of antibody-producing cells in rats stimulated with: 0-1 mg (A); 1 mg (a); and 10 mg (0) of peroxidase. Kinetics of development of cells synthesizing Ig without antibody function in rats injected with: 0-1 mg (A); 1 mg (E); and 10 mg (0) of peroxidase. (v) Immunoglobulin-synthesizing cells in control rats injected once, twice or three times with saline alone.
cells rose progressively with the dose of antigen injected in the first and second inoculations. In the first experiment the number ofantibody and immunoglobulin-synthesizing cells present per lymph node was a function of the quantity of antigen injected. In the second experiment antibody-synthesizing cells were detected with all the doses tested, but high numbers were obtained only with the higher doses (22 and 39 mg). Approximately the same number of immunoglobulin-synthesizing cells was obtained in the control rats and in animals injected with 0-1, 1 and 10 mg. The higher doses of antigen injected led to the appearance of cells synthesizing Ig without detectable antibody activity. These two populations of cells were also very numerous with the doses 25 and 50 mg (expts 3 and 4, Table 1). In experiment 2, a secondary immune response was studied. A high number of both types of cells was revealed, depending on the quantity of antigen used to boost. The size of the two populations of cells was calculated in the
DISCUSSION The purpose of this paper was to study the kinetics of development of two populations of immunocytes: one synthesizing antibodies, the other synthesizing immunoglobulin without detectable antibody function after injection of different doses of various kinds of protein antigen without adjuvant, as a solution in saline. With all the antigens there was correlation between the size of the population of immunoglobulin-synthesizing cells and the amount of antigen injected. It seems therefore that this is a general characteristic of the immune response, but the threshold where these cells began to appear varied from an antigen to another. The population of antibody-synthesizing cells appeared with the same dose of some antigens as that needed to produce immunoglobulin-synthesizing cells, but low doses of others produced antibody-synthesizing cells without immunoglobulin-synthesizing cells. In the secondary response the threshold of antigen needed to give antibody and immunoglobulin-synthesizing cells was the same as or lower than that in primary response. In the primary response, the size of the population of antibody-forming cells did not vary or only varied slightly with the amount of antigen injected. In all cases the size of this population remained very small, except with human IgG where a greater number of positive cells was detected after injection of small doses. In the secondary response, at lower doses there was no increase in the population of antibody-forming cells, except with aggregated or deaggregated human IgG, where an increase of this population was noted after injection of a small dose of antigen. From comparisons of the sizes of the two populations the following observations can be made: in the primary response, the population of immunoglobulin-synthesizing cells is larger than the population of antibody-forming cells, except with IgG where the two populations are not very different in size. In the secondary response, at low doses, immunoglobulin-synthesizing cells are more numerous than antibody-forming cells, while at high
928
J. C. Antoine, Christine Petit & S. Avrameas
(a )
( b)
1-5
I. C-
SZ
(7
xL
aL)
"I
E, 0
a)
a)
cr CL.
la_
0
0-
-0
0-5 V
z
C
0.1
---f
10
BSA (mg)
Figure 2. Primary immune responses of rats stimulated with bovine serum albumin given as a solution in saline in the hind footpads (expt 1). Each point represents the arithmetic means of the percentage of positive cells (a) or of the number of positive cells per lymph node (b) detected separately in two rats 7 days after injection. Standard errors are shown by vertical bars. Percentage (a) or total number (b) of antibody-synthesizing cells as a function of the dose injected (0). Percentage (a) or total number (b) of cells containing immunoglobulin without antibody function as a function of the dose injected (0). C = control rats injected once with saline alone.
doses this difference diminishes. With human IgG as antigen the population of immunoglobulinsynthesizing cells is only slightly higher or even smaller than the population of antibody-producing cells. In secondary responses, when the antibodyproducing cells increase in number, immunoglobulinsynthesizing cells also increase, but less than the former. In cases where the population of antibodysynthesizing cells reached the same size in both primary and secondary responses, the number of immunoglobulin-containing cells also did not vary. These observations indicate that there is a close linkage between these two populations. It seems that the immunoglobulin-synthesizing cells can appear and rapidly disappear two or three successive times without the concomitant appearance of a high number of antibody-synthesizing cells. When this occurred, the level reached by both populations of cells was practically identical after either one or two injections, indicating that probably no memory cells were induced during this
time. With high doses of antigen, proliferation of both populations was noted, indicating that in this case, memory cells were induced after the first injection of antigen. It appears also that the more immunogenic an antigen (human IgG or high doses of the other antigens) the smaller was the ratio between cells synthesizing immunoglobulin without antibody function and antibody-forming cells. We were intrigued to find no great differences between the two physical forms of human IgG, one known to easily induce tolerance and the other to be a potent immunogen inducing a high immune response when injected without adjuvant. Are immunoglobulins without detectable antibody function very low affinity antibodies? From the results reported here, it is difficult to definitely eliminate this hypothesis. It was observed by Werblin et al. (Werblin, Kim, Quagliata & Siskind, 1973) and by Kim & Siskind (1974) that after immunization of rabbits and mice with DNP-bovine gammaglobulin
AIg- and Ab-synthesizing cell development emulsified in Freund's complete adjuvant, the amount of low affinity antibodies was relatively constant whatever the quantity of antigen injected and whatever the time after immunization. Furthermore, by using DNP-haemocyanin in the absence of adjuvants, Mond, Kim & Siskind (1974) found that low dose of antigen induced higher affinity antibody than a larger dose. Despite the fact that with low doses of antigen we have sometimes detected antibody-forming cells without detectable immunoglobulin-synthesizing cells, their number was always very small. On the other hand we have sometimes observed cells producing only immunoglobulin without antibody function in high amount. Furthermore, when we increased the quantity of antigen injected, both populations rose and the ratio between immunoglobulin-producing cells and antibody-synthesizing cells did not increase and sometimes even decreased, indicating either that no population is preferentially selected in the range of doses tested, or that antibody-forming cells proliferate more than immunoglobulin-producing cells when the dose of antigen is increased. If immunoglobulin without antibody function were low affinity antibody, it would be expected that cells synthesizing these molecules would preferentially proliferate when the dose of antigen injected is increased. This did not occur, and indeed sometimes the contrary result was obtained. Are our present results compatible with our previously formulated hypothesis that all or a part of the population of cells synthesizing Ig without antibody function are precursors of the antibodyforming cells (Miller, Ternynck & Avrameas, 1974, 1975; Antoine & Avrameas, 1976)? In the experiments reported here we found that the number of the immunoglobulin-synthesizing cells was strictly dependent on the amount of antigen injected, and in general, that these cells were the first to appear in substantial numbers at low doses of antigen. Also, from the results obtained with the secondary response it seems that immunoglobulin-synthesizing cells may differentiate from memory cells, unlike what happens in animals stimulated a second time with antigen in adjuvant (Antoine & Avrameas, 1976). Finally the fact that after a booster injection antibody-synthesizing cells increased more than cells producing immunoglobulin without antibody function may be explained as development of the former population by a recruitment from the latter.
929
ACKNOWLEDGMENTS We should like to thank Dr P. A. Cazenave for his generous gift of hen ovalbumin and Mr Andre Chedeville for his excellent technical assistance. This work was supported by research grant A.T.P. CNRS n' 2111.
REFERENCES ANTOINE J.C. & AVRAMEAS S. (1973) Etude de la formation des anticorps anti-tyrosinase au cours de la reponse immunitaire chez le Lapin. I. ttude cellulaire. Ann. Immunol. (Inst. Pasteur), 124C, 121. ANTOINE J.C. & AVRAMEAS S. (1974) Surface immunoglobulins of rat immunocytes: quantitation and fate of cell-bound peroxidase-labelled antibody and Fab fragment. Europ. J. Immunol. 4, 468. ANTOINE J.C. & AVRAMEAS S. (1976) Correlations between immunoglobulin and antibody-synthesizing cells during primary and secondary immune responses of rats immunized with peroxidase. Immunology, 30, 537. ANTOINE J.C., AVRAMEAS S., GONATAs N.K., STIEBER A. & GONATAS J.O. (1974) Plasma membrane and internalized immunoglobulins of lymph node cells studied with conjugates of antibody or its Fab fragment with horseradish peroxidase. J. Cell Biol. 63, 12. AVRAMEAS S. & TERNYNCK T. (1971) Peroxidase-labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry, 9, 1175. CHILLER J.M., HABICHT G.S. & WEIGLE W.O. (1971) Kinetic differences in unresponsiveness of thymus and bone marrow cells. Science, 171, 813. FAHEY J.L. & TERRY E.W. (1973) Ion exchange chromatography and gel filtration. Handbook of Experimental Immunology, 2nd edn (ed. by D. M. Weir), p. 7.1. Blackwell Scientific Publications, Oxford. GONATAs N.K., ANTOINE J.C., STIEBER A. & AVRAMEAS S. (1972) Surface immunoglobulins of thymus and lymph node cells demonstrated by the peroxidase coupling technique. Lab. Invest. 26, 253. GRAHAM R. & KARNOVSKY M.S. (1966) The early stages of adsorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291. KIM Y.T. & SISKIND G.W. (1974) Studies on the control of antibody synthesis. VI. Effect of antigen dose and time after immunization on antibody affinity and heterogeneity in the mouse. Clin. exp. Immunol. 17, 329. MILLER H.R.P., TERNYCK T. & AVRAMEAS S. (1974) A comparison of specific antibody and immunoglobulinsynthesizing immunocytes in peroxidase stimulated lymph nodes. Ann. Immunol. (Inst. Pasteur), 125C, 231. MILLER H.R.P., TERNYNCK T. & AVRAMEAS S. (1975) Synthesis of antibody and immunoglobulins without detectable antibody function in cells responding to horse radish peroxidase. J. Immunol. 114, 626. MOND J., KiM Y.T. & SISKIND G.W. (1974) Studies on the
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control of antibody synthesis. V. Effect of nonspecific modification of the magnitude of the immune response on the affinity of the antibody synthesized. J. Immunol. 112, 1255. PORTER R.R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem. J. 73, 119.
SISKIND G.W. & BENACERRAF B. (1969) Cell selection by antigen in the immune response. Advanc. Immunol. 10, 1. WERBLIN T.P., KIM Y.T., QUAGLIATA F. & SISKIND G.W. (1973) Studies on the control of antibody synthesis. HII. Changes in heterogeneity of antibody affinity during the course of the immune response. Immunology, 24, 477.
Development of immunoglobulin and antibody-synthesizing cells after immunization with different doses of antigen J. C. ANTOINE, CHRISTINE PETIT & S. AVRAMEAS Unite'd'Immunocytochimie, De'partement de Biologie Moliculaire, Institut Pasteur, Paris, France
Received 15 April 1976; acceptedfor publication 13 May 1976
Summary. The kinetics of development of antibodysynthesizing cells and of cells synthesizing immunoglobulins without detectable antibody function were studied in rats immunized with different doses (0-1, 1, 10, 100 mg) of horse radish peroxidase, bovine serum albumin, human serum albumin, hen ovalbumin, or human IgG, which had been deaggregated or heat-aggregated. Each antigen was injected once or twice as a solution in saline. Antibody and immunoglobulin-producing cells were detected in draining lymph nodes by immunohistochemical staining. In the primary response a few antibody-synthesizing cells were found whatever the dose injected. No increase or some increase was found with the amount of antigen injected, according to the protein used, but with all doses of antigen injected, the population of cells remained small, except with human IgG where a relatively high number of positive cells was detected even after injection of 1 mg of antigen. In the secondary response a few antibody-forming cells were also detected with the lower doses of antigen, but this population increased after boosting with 100 mg of antigen. With human IgG a greater number of positive cells was induced with all the doses tested. A correlation between the number of cells synthesizing immunoglobulins without antibody funcCorrespondence: Dr J. C. Antoine, Unit6 d'Immunocytochimie, D6partement de Biologie Molculaire, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
921
tion and the amount of antigen injected was observed in the primary and secondary responses. The relative size of these two populations varied with the stage of immunity of the animals. In the primary response, the population ofcellssynthesizingimmunoglobulins without antibody function was larger than the population of antibody-forming cells. The same was true in the secondary response, but if after a booster injection the level of antibody-synthesizing cells exceeded that reached in the primary response, the increase of cells synthesizing Ig without antibody function was smaller than the increase in antibodyforming cells. In general the more immunogenic an antigen was, the smaller was the ratio between antibody-forming cells and cells producing immunoglobulin without antibody function.
INTRODUCTION In a previous paper we have shown that after primary immunization with peroxidase emulsified in Freund's complete or incomplete adjuvant the first immunoglobulin-synthesizing cells which appeared in the draining lymph nodes did not contain molecules with detectable antibody function. This population of cells was progressively replaced by antibody-forming cells. In the secondary response, antibody-producing cells were predominant, and they were not preceded by cells synthesizing immunoglobulins without antibody function (Antoine & Avrameas, 1976).
J. C. Antoine, Christine Petit & S. Avrameas
922
In the present paper, in order to examine whether the appearance of cells synthesizing immunoglobulins without antibody function was associated with the particular antigen used (peroxidase) or with the presence of Freund's adjuvant, we immunized rats with various antigens dissolved in saline. Furthermore, to explore the influence of the amount of antigen injected on the relative proportion of these two populations of cells, various doses were used for each antigen. In these studies, we also tried to test if immunoglobulins without detectable antibody function were very low affinity antibodies. We found that after inoculation, and independently of the antigen employed, immunoglobulin-synthesizing cells always appeared. These cells appeared alone or with antibody-synthesizing cells, and their number increased with the amount of antigen injected. Thus it appears that the development of antibody-forming cells and of cells containing immunoglobulin without antibody function is a general characteristic of the immune response.
MATERIALS AND METHODS Animals Five-month-old OFA rats of both sexes, bred in specific pathogen-free conditions were used. Antigens
Peroxidase (PO) RZ = 3 was purchased from Boerhinger, Mannheim, Germany. Crystallized human serum albumin (HSA) was obtained from Miles Laboratories, Slough, Bucks. Crystallized bovine serum albumin (BSA) was obtained from Sigma Chemical Company, St Louis, Missouri. Five times crystallized hen ovalbumin (OVA) was a gift from Dr P. A. Cazenave (Institut Pasteur, Paris, France). Human IgG were prepared from Cohn's fraction II (Miles Laboratories, Slough, England) which had been further purified by chromatography on DEAE ion-exchange cellulose (grade DE 52, Whatman, Maidstone, Kent) following the procedure described by Fahey & Terry (1973). In some experiments Human IgG was prepared from human serum by salt fractionation, chromatography on DEAE-cellulose and molecular filtration through Sephadex G-200 (Pharmacia, Uppsala, Sweden). Tolerogenic (deaggregated) and immunogenic (heat-aggregated) physical forms of human IgG were prepared following the methods described by Chiller, Habicht & Weigle (1971).
All these above antigens were tested by immunoelectrophoresis with antisera obtained in rabbits hyperimmunized with these antigens and used subsequently to inoculate the rats. Antisera against PO, HSA, OVA and IgG revealed only a single precipitation line. Antisera against BSA revealed a strong precipitation line and a second very faint line. Immunizations
Immunization was carried out by injecting s.c. in both hind footpads 0-1, 1, 10, or 100 mg of antigen dissolved in 0-4 ml of saline made with pyrogen-free water. Ten to 15 days after the primary injection the animals received by the same route the same dose of antigen. Animals were killed 7 days and 10-13 days after the first injection and 5-7 days after the secondary injection. The tertiary response to peroxidase was also studied in animals receiving 0-1 or 10 mg of antigen in saline 38 days after the primary injection and 23 days after the secondary injection. Animals were killed either after the primary injection or 5 or 14 days after the second inoculation, or 5 days after the third injection. In each experiment control rats were injected once, twice, or thrice with 0 4 ml of saline. Preparation of cells At least two rats immunized with the different antigens were studied on each day and for each dose examined. Animals were killed by ether. The popliteal lymph nodes were removed, weighed and dissociated in cold Hanks's medium (Institut Pasteur, Paris, France). Cells were centrifuged at 200 g for 7 min, resuspended in 1 ml of Hanks's medium, counted and cytocentrifuged as described previously (Antoine & Avrameas, 1976). Preparation of antisera and ofpurified antibodies
Rabbit and sheep anti-rat immunoglobulin and rabbit anti-hen ovalbumin antisera were prepared as described earlier (Gonatas, Antoine, Stieber & Avrameas, 1972). Purified rabbit and sheep antibodies against rat Fab or IgG and rabbit antibodies against ovalbumin were prepared following procedures already described (Antoine & Avrameas, 1973, 1974). Fab fragments from sheep anti-rat immunoglobulin antibodies were prepared by papain digestion (Porter, 1959) according to a method
Ig- and Ab-synthesizing cell development described earlier (Antoine, Avrameas, Gonatas, Stieber & Gonatas, 1974).
Labelling of antigens and of antibodies Antigens and purified antibodies were labelled with peroxidase by a two-step procedure (Avrameas & Ternynck, 1971). Labelled proteins were filtered on a 95 x 26 cm Sephadex G-200 column equilibrated with 0-1 M Tris, HCl buffer, pH 8, 1 M NaCl to eliminate free proteins and free peroxidase. Detection of immunoglobulin- and antibody-synthesizing cells After cytocentrifugation, cells were left overnight, then fixed for 15 min in 0-2 M cacodylate buffer, pH 7 4, containing 4 per cent paraformaldehyde, washed three times in PBS and incubated with the different conjugates. To detect cells synthesizing antibodies against peroxidase, slides were incubated for 1 h with a 100 ug/ml solution of PO in PBS. Cells synthesizing antibodies against BSA, HSA and human IgG were detected by incubating slides for 2 h with a 250 pg/ml solution of the corresponding antigen labelled with PO and diluted in normal rat serum. To detect cells synthesizing antibodies against OVA, slides were incubated for 1 h with a 100 ,ug/ml solution of OVA in normal rat serum, washed and then incubated for 2 h with a 250 ,ug/ml solution of peroxidase-labelled rabbit anti-OVA antibodies in normal rat serum. To detect cells synthesizing immunoglobulins, the preparations were incubated for 2 h with a 250 ,ug/ml solution in PBS of POlabelled anti-rat immunoglobulin or anti-rat Fab antibodies or with a 125 pg/ml solution of sheep Fab fragments of anti-rat immunoglobulin antibodies conjugated with PO. Staining was done according to the method of Graham & Karnovsky (1966). After staining the total number of nucleated cells and of positive cells was calculated. Results were expressed as the percentage of the positive cells, or as the total number of positive cells per lymph node (Antoine & Avrameas, 1976).
RESULTS For each antigen we give the results obtained 7 days after a primary injection and 5 days after a booster injection (Tables 1 and 2). We also studied the response 10-15 days after a primary injection. However, at this time, whatever the doses employed,
923
there was a very low, often undetectable number of antibody and Ig-producing cells. It appears therefore that protein antigens injected in a soluble form in saline induced a very sharp primary immune response, and we do not report here results 10-15 days after primary inoculation. In the following, we call 'immunoglobulin-synthesizing cells' the cells producing immunoglobulin without detectable antibody function.
Peroxidase After one injection of various doses of PO, the weight and the number of cells of the lymph nodes increased with the amount of antigen injected. A very small amount of antibody-producing cells was detected after one injection of 0-1, 1, or 10 mg of PO. After two or three injections larger numbers of antibody-producing cells were found in animals injected with 0-1 mg of antigen. A progressive increase of immunoglobulin-producing cells was noted with increasing amounts of antigen injected. 5 days after secondary injection of 1 mg or 10 mg of antigen, the number of positive cells reached the level attained 7 days after the first injection. An increase in this population of cells was noted after two or three injections of a dose of 0 1 mg (Fig. 1). Bovine serum albumin Only the primary immune response was studied with this antigen. An increase in the weight of the lymph nodes was found after injection of the antigen but not as a function of the dose of antigen injected; a very slight increase in the cellular content of the nodes was found. No increase or a very slight increase as a function of the dose of antigen was noted in the amount of antibody-producing cells. The number of Ig-synthesizing cells increased with the amount of antigen inoculated (Fig. 2). Human serum albumin During the primary immune response no increase of the lymph node weight was observed whatever quantity of antigen was injected. The same was true for the cellular content of the nodes. In the secondary response an increase of the lymph node weight and cellular content was observed only with the 100 mg dose. A very low number of antibody-producing cells was found 7 days after the first injection of 1,
J. C. Antoine, Christine Petit & S. Avrameas
924
Table 1. Antibody and immunoglobulin-synthesizing cells in rat lymph nodes draining footpads injected with antigens 7 days previously Ig Ag Igc (7)
Cells/lymph Antigen
node
Ab (per cent)
Ig (per cent)
nb Ab
nb Ig
(1)
(2)
(3)
(4)
(5)
(6)
21 x 106 6-75x l0 33x 106 4-35x 106
0 0 0-018 0-013
0-1 0-3 0-5 1-5
0 0 1-2x 102 5-6x 102
4x l10 1-75x 106
0 0
0-1 0-4
0 0
4x 102 6 6x 103
17
Bovine serum albumin (expt 1) (mg) C 0-9+0-3x106 1-6+0-7x 106 0-1 1 1-2+0-3x 106 14+0 6x 106 10
0 0-03+0 0-02+ 0-02 0-04+ 0-02
0-12+ 0-02 0-04+ 0-06 0-35+0-13 1-05+0-07
0 45+2-1x 102 1-5+2-1x 102 50+ 1-4x 102
1l+0.lx 103 2-9+4-1 x102 3-8+0-3x 103 1-4+0-5x 104
0-2+ 0-3 3-5+0-3 12-7+ 4-6
Bovine serum albumin (expt 2) (mg) 2-7+0-5x l10 C 8x 104 0.1 1-35x 106 1 55x 105 10 1-9+ l x 106 100
0 0 0 0-007 0-03+ 0-01
0-06+ 0-04 0-27 0-16 0-37
0 0 0 38-5 4-9+0 4x 102
1-6+ 1-2x 102 2-2x 102 2 2x 103 2x 103 2-4+2-4x 104
1-4 13-7 12-5 151-7+ 147-4
0 0
0-05+ 0-02 0-02+0 0-05+ 0-01 0-28 0-17+ 0-06
0 0
4-0+0-3x 102 2-5+0-9x 102 8-9+8-6x 102 6-4x 103 2-9+ 1-8x 103
0-6+ 0-2 2-2_2-1 16 7-2+ 4-6
1-5+0-9x 102 6-2+0-3x 10 64+5x 10 6+5x 102 3-7+0-7x 103
0-4+ 000-4+ 0 4 3-8+ 3-1 24+4
Peroxidase (expt 1) (mg) C 0-1 1 10
Peroxidase (expt 2) (mg) C 1
Human serum albumin (mg) C 8-0+2-5x l0 1-3+0-5x 106 0-1 1-5+ 1-3x106 1 2-2x 106 10 1-6+0-4x106 100
Ovalbumin (mg) C 0-1 1 10 100
Deaggregated human IgG (mg) C 0-1 1 10
1-4+ 1-6x 102 1-35x 102 2-7+ 1-8x 102
0-04+ 001
0
3l1+0-4x l10
0 0 0
0.0- 00
0
6-7+0-7x iO0 17+0 4x 106
0-002+ 0 003 0-017+ 0-016
0-23+ 0-04
3 6+0-9x l0 4-7+2-4x l0
7+2x l10
0
13+Ox 106
0-035+ 0-021 0-055+ 0-063 0-15+ 007
2-4+0-7x 106 8+8x l10
Aggregated human IgG (expt 1) (mg) C 001 1 8
0-007+0-004 0-006 0-02+ 0-02
1-15+0-6
1-8+0-5x 105 8+6x 105 2 5+0 5x 106 5 3+0 8x 106
0 006+ 004 0 06+ 0°00 0-4+ 03
0-02± 0-01 0-08+ 0-06
009+ 0°00 0°14+0-08 0-11+0-13 0-4+ 0 3
0 06+0-06 02+0-2
0-09+0-06 0-10+0-07
2-3x 2x 1-65x 6 5x
0 13+ 19 2-5+2-5x 102
0 4-5+2-7x 102 1.5+ l-9X 103
1-6+ l-9x
103
0 3-8+0-Ox 102 1-6+ 0 5 x 103 2-3+2x 104
103 103 104 104
0-9 7-1 28
6-7+ 1-8x 102 1-8+ 1-1 x 103
30+_3-9x 103 2-1+1-1x 103
9-6+7-5x 10 1-3+0-6x 103 2-2+ 1-9x 103
6-6+4-8x
103
2-7+ 1-7 4-5+ 5-9 3-1+ 1-7
13-4+ 6-1 23-1+20-3 68-5+ 50-2
Ig- and Ab-synthesizing cell development
925
Table 1. (cont.)
Antigen
(1) Aggregated human IgG (expt 2) (mg) C 0-1 1 10 22 39 Aggregated human lgG (expt 3) (mg) C 25 Aggregated human lgG (expt 4) (mg) C 50
Cells/lymph node
Ab (per cent)
Ig (per cent)
nb Ab
nb Ig
Ig Ag Igc
(2)
(3)
(4)
(5)
(6)
(7)
0
0-07
0-16+0-04 0 07+000 0-28+0-21 0-15+ 0-10 07 0-45
0 75+4 22+3Ox 103 1 1+ 16x 102 7-1 x 103 3 3x 103
2-2+ 11 x 103 17+0 4x 103 34+25x 103 3 4+2 9x 103 2 7x 104 2x 104
0-7+0-2 1-6+ 11 1-6+ 1-2 12-3 9
8 6x 106
0 0-05
04 0 16
0 4-3x 103
5-6x 103 18x 104
2-5
49x 105 1 8x 107
0 0-24
0-24 0-66
0 4 3x 104
12x 103 1l2x 105
14+08x 106 2 2+0 3x 106
12+0Ox 106 2 3+0 Ox 106 3-9x 106 4-4x 106
14x106
0-003+0-000 0-19+0-25 0-005+0-007 0-18
100
Seven days after the primary injection, animals immunized with different doses of PO, BSA, HSA, OVA, deaggregated or heat-aggregated human IgG were killed. Antibody and immunoglobulin-synthesizing cells were detected by immunoenzymological methods in the draining lymph nodes. Column 1: antigen doses injected. C: control rats which received 0 4 ml of saline. Column 2: total number of cells per lymph node. Column 3: percentage of antibody-synthesizing cells. Column 4: percentage of cells producing immunoglobulin without antibody function. Column 5: number of antibody-synthesizing cells per lymph node. Column 6: number of cells producing Ig without antibody function per lymph node. Column 7: ratio between the total number of cells producing immunoglobulin without antibody function found in animals immunized with various doses of antigen and the total number of immunoglobulinsynthesizing cells found in lymph nodes of animals inoculated with saline alone.
10 or 100 mg of HSA. In the secondary response no or only a few antibody-positive cells were detected except at the 100 mg dose, where a much greater number were found. The number of immunoglobulinproducing cells progressively increased with the quantity of antigen injected both for the primary and the secondary response. With 01, 1 and 10 mg of antigen where approximately the same number of antibody-producing cells was present after one or two stimuli, no increase of the Ig-synthesizing cells was also noted. With the 100 mg dose the two populations of cells increased after the booster injection. The ratio between the number of antibodycontaining cells 5 days after a second injection and 7 days after primary inoculation was 430, while the same ratio for the immunoglobulin-synthesizing cells was 38. Ovalbumin
There was no significant variation in the weight of
the nodes with the amount of antigen injected. In the primary response the total number of cells per lymph node was approximately the same whatever the amount of antigen injected, except in the animals stimulated with 100 mg of ovalbumin, where cells were five times more numerous than in the control nodes. In the secondary response an increase of the lymph node cells correlated with the quantity of antigen the animals received. At day 7 antibodysynthesizing cells were detected only with 10 mg and 100 mg of ovalbumin. The number of immunoglobulin-producing cells was the same as or lower in rats receiving 0-1 or 1 mg than in control rats, and higher than rats receiving 10 and 100 mg. In animals stimulated with 10 mg, the ratio between numbers of antibody-producing cells detected at day 5 after a boosting and those present at day 7 after one injection was 1-6. The same ratio calculated for the 100 mg dose was 32. For the immunoglobulin-synthesizing cells the ratios were respectively 2 2 and 15.
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J. C. Antoine, Christine Petit & S. Avrameas
Table 2. Antibody and immunoglobulin-synthesizing cells in rat lymph nodes 5 days after secondary injection of antigens
Cells/lymph nb Ig
IgAg lgc
(4)
nb Ab (5)
(6)
(7)
0 0-03 0-008 0
0-07 0-17 0-44 1-6
0 1-3x 103 2-2x 102 0
1-4x 103 71x 103 12x 104 6 9x 104
5-1 8-6 46
0 0-003+0 004 0 0-005+0-007 0-65+0-07
0-10+0 00 0-06+0-02 0-09+0-02 0-21+0 06 0-7+0-6
0 33+47 0 75+10 8 6+7-6x 104
1 7x 103 6 5+09x 102 1 0+0 3x 103 3-3+0 8x 103
0 0 0-006+0-001 0-001+0-002 0-165+0-005
0-08+0-01 0-05+0-00 0-04+0-00 0-07+0-03
2 8+1 1x102 5-4+ 1-6x 102 2-4+0-Ox 02
1-1+±00
0 0 37+3 21+ 29 7-9+1 7x 103
1O+0-5x 103 5-3+1-0x 104
1-9+0-6 0-9+0-0 3-6+ 1-8 186+36
0 1-1+0 3 3-1+0-7 5-9+2-1
0-15+0-07 0-9+0-2 0-9+0-6 3-4+1-5
0 3-5+2 7x 104 1-7+ 1-2x O 7-7+4-7x l0
1-3+0-7x 103 2 8+2-Ox 104 5 2+5 3x 104 4-2+0-3x 104
21-7+15-0 40-4+40-8 327+27
0 1-5+ 15 04+0 1 0 9+0-4
0-2+0-0 0-6+0-2 1 7+0-0 1-8+0-8
0
27+24x 104 1-7+1 4x 104 1l2+0-2x i05
1-2+0 3x103 1 1+0-6x104 8 3+8-1x 104 25+0 4x i05
9-3+5 1 69-2+67-1 206-7+33-0
Antigen
node
Ab (per cent)
Ig (per cent)
(1)
(2)
(3)
Peroxidase (expt 1) (mg) C 0.1 1 10 Human serum albumin
2x106 4-2x 106 2-7x 106 4 3x106
(mg) 11+06x 106 106 106
C 0 1 I 10 100
1 0+0 2x 1.I-+01 x 1-6+0-1 x 1 2+ lOx
Ovalbumin (mg) C 0-1 1 10 100
Deaggregated human IgG (mg) C 0-1 1 10
Aggregated human IgG (expt 2) (mg) C 0-1 1 10
106 106
3-5+0-5x105 1-0+0-2x 106 6-4+0-7x l0 1-3+00x 106 4-8+0 9x106
1-1+ l-Ox 2-9+1-5x 5-3+2-8x 1-3+0-4x
106 106 106 10
4 8+0 1xIO 1-9+0 2x 106 5 0+4 8x 106 1l5+0 5x l0
ll-+1-4x 10
0 3+0-0 0-6+0-2 1-9+0-5 68+82
Ten to 15 days after the primary injection animals received various doses of antigen as indicated in column 1, and lymph nodes were taken 5 days later. The same dose was inoculated in primary and secondary immunization except in the animals which had received 0-1 mg of aggregated IgG in the first inoculation and I mg in the second inoculation. For the explanations of columns 2-6: see legend to Table 1.
Deaggregated human IgG No correlations were found between the size and the total number of cells of the lymph nodes and the amount of
IgG inoculated during the primary
In secondary response both size and total numbers of lymph node cells increased with the
response.
quantity of antigen. After one injection of 01, 1 or 10 mg of IgG a small number of antibodyproducing cells appeared with all the three doses. The same was true for the immunoglobulin-synthesizing cells. After a booster injection there was a large increase in the size of the two populations of
cells at all doses tested. In both populations, the increase was a function of the amount of antigen injected. The ratio between the number of positive cells found at 5 days after boosting and 7 days after primary injection was calculated for the two populations. For the antibody population these ratios were 77, 113, and 480 for 01 mg, 1 mg and 10 mg respectively. The ratios for the Ig population were respectively 24, 23 and 300.
Heat-aggregated human IgG Both weight of lymph nodes and total number of
Ig- and Ab-synthesizing cell development
927
primary and secondary response and the ratios obtained for the antibody population were 360, 7-7 and 1140 for the 0 1, 1 and 10 mg doses respectively. The same ratios calculated for the immunoglobulin population were 36 and 113 for the 1 and 10 mg doses respectively. E
-
21 7
1301 42 Days
after antigen
Figure 1. Primary, secondary and tertiary immune responses of rats stimulated with peroxidase given as a solution in saline in the hind footpads (expt 1). On each day one rat was killed for each dose studied. Arrows indicate the time when the two booster injections were given. Animals which were used in secondary and tertiary responses received the same dose as in the first injection. Kinetics of development of antibody-producing cells in rats stimulated with: 0-1 mg (A); 1 mg (a); and 10 mg (0) of peroxidase. Kinetics of development of cells synthesizing Ig without antibody function in rats injected with: 0-1 mg (A); 1 mg (E); and 10 mg (0) of peroxidase. (v) Immunoglobulin-synthesizing cells in control rats injected once, twice or three times with saline alone.
cells rose progressively with the dose of antigen injected in the first and second inoculations. In the first experiment the number ofantibody and immunoglobulin-synthesizing cells present per lymph node was a function of the quantity of antigen injected. In the second experiment antibody-synthesizing cells were detected with all the doses tested, but high numbers were obtained only with the higher doses (22 and 39 mg). Approximately the same number of immunoglobulin-synthesizing cells was obtained in the control rats and in animals injected with 0-1, 1 and 10 mg. The higher doses of antigen injected led to the appearance of cells synthesizing Ig without detectable antibody activity. These two populations of cells were also very numerous with the doses 25 and 50 mg (expts 3 and 4, Table 1). In experiment 2, a secondary immune response was studied. A high number of both types of cells was revealed, depending on the quantity of antigen used to boost. The size of the two populations of cells was calculated in the
DISCUSSION The purpose of this paper was to study the kinetics of development of two populations of immunocytes: one synthesizing antibodies, the other synthesizing immunoglobulin without detectable antibody function after injection of different doses of various kinds of protein antigen without adjuvant, as a solution in saline. With all the antigens there was correlation between the size of the population of immunoglobulin-synthesizing cells and the amount of antigen injected. It seems therefore that this is a general characteristic of the immune response, but the threshold where these cells began to appear varied from an antigen to another. The population of antibody-synthesizing cells appeared with the same dose of some antigens as that needed to produce immunoglobulin-synthesizing cells, but low doses of others produced antibody-synthesizing cells without immunoglobulin-synthesizing cells. In the secondary response the threshold of antigen needed to give antibody and immunoglobulin-synthesizing cells was the same as or lower than that in primary response. In the primary response, the size of the population of antibody-forming cells did not vary or only varied slightly with the amount of antigen injected. In all cases the size of this population remained very small, except with human IgG where a greater number of positive cells was detected after injection of small doses. In the secondary response, at lower doses there was no increase in the population of antibody-forming cells, except with aggregated or deaggregated human IgG, where an increase of this population was noted after injection of a small dose of antigen. From comparisons of the sizes of the two populations the following observations can be made: in the primary response, the population of immunoglobulin-synthesizing cells is larger than the population of antibody-forming cells, except with IgG where the two populations are not very different in size. In the secondary response, at low doses, immunoglobulin-synthesizing cells are more numerous than antibody-forming cells, while at high
928
J. C. Antoine, Christine Petit & S. Avrameas
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Figure 2. Primary immune responses of rats stimulated with bovine serum albumin given as a solution in saline in the hind footpads (expt 1). Each point represents the arithmetic means of the percentage of positive cells (a) or of the number of positive cells per lymph node (b) detected separately in two rats 7 days after injection. Standard errors are shown by vertical bars. Percentage (a) or total number (b) of antibody-synthesizing cells as a function of the dose injected (0). Percentage (a) or total number (b) of cells containing immunoglobulin without antibody function as a function of the dose injected (0). C = control rats injected once with saline alone.
doses this difference diminishes. With human IgG as antigen the population of immunoglobulinsynthesizing cells is only slightly higher or even smaller than the population of antibody-producing cells. In secondary responses, when the antibodyproducing cells increase in number, immunoglobulinsynthesizing cells also increase, but less than the former. In cases where the population of antibodysynthesizing cells reached the same size in both primary and secondary responses, the number of immunoglobulin-containing cells also did not vary. These observations indicate that there is a close linkage between these two populations. It seems that the immunoglobulin-synthesizing cells can appear and rapidly disappear two or three successive times without the concomitant appearance of a high number of antibody-synthesizing cells. When this occurred, the level reached by both populations of cells was practically identical after either one or two injections, indicating that probably no memory cells were induced during this
time. With high doses of antigen, proliferation of both populations was noted, indicating that in this case, memory cells were induced after the first injection of antigen. It appears also that the more immunogenic an antigen (human IgG or high doses of the other antigens) the smaller was the ratio between cells synthesizing immunoglobulin without antibody function and antibody-forming cells. We were intrigued to find no great differences between the two physical forms of human IgG, one known to easily induce tolerance and the other to be a potent immunogen inducing a high immune response when injected without adjuvant. Are immunoglobulins without detectable antibody function very low affinity antibodies? From the results reported here, it is difficult to definitely eliminate this hypothesis. It was observed by Werblin et al. (Werblin, Kim, Quagliata & Siskind, 1973) and by Kim & Siskind (1974) that after immunization of rabbits and mice with DNP-bovine gammaglobulin
AIg- and Ab-synthesizing cell development emulsified in Freund's complete adjuvant, the amount of low affinity antibodies was relatively constant whatever the quantity of antigen injected and whatever the time after immunization. Furthermore, by using DNP-haemocyanin in the absence of adjuvants, Mond, Kim & Siskind (1974) found that low dose of antigen induced higher affinity antibody than a larger dose. Despite the fact that with low doses of antigen we have sometimes detected antibody-forming cells without detectable immunoglobulin-synthesizing cells, their number was always very small. On the other hand we have sometimes observed cells producing only immunoglobulin without antibody function in high amount. Furthermore, when we increased the quantity of antigen injected, both populations rose and the ratio between immunoglobulin-producing cells and antibody-synthesizing cells did not increase and sometimes even decreased, indicating either that no population is preferentially selected in the range of doses tested, or that antibody-forming cells proliferate more than immunoglobulin-producing cells when the dose of antigen is increased. If immunoglobulin without antibody function were low affinity antibody, it would be expected that cells synthesizing these molecules would preferentially proliferate when the dose of antigen injected is increased. This did not occur, and indeed sometimes the contrary result was obtained. Are our present results compatible with our previously formulated hypothesis that all or a part of the population of cells synthesizing Ig without antibody function are precursors of the antibodyforming cells (Miller, Ternynck & Avrameas, 1974, 1975; Antoine & Avrameas, 1976)? In the experiments reported here we found that the number of the immunoglobulin-synthesizing cells was strictly dependent on the amount of antigen injected, and in general, that these cells were the first to appear in substantial numbers at low doses of antigen. Also, from the results obtained with the secondary response it seems that immunoglobulin-synthesizing cells may differentiate from memory cells, unlike what happens in animals stimulated a second time with antigen in adjuvant (Antoine & Avrameas, 1976). Finally the fact that after a booster injection antibody-synthesizing cells increased more than cells producing immunoglobulin without antibody function may be explained as development of the former population by a recruitment from the latter.
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ACKNOWLEDGMENTS We should like to thank Dr P. A. Cazenave for his generous gift of hen ovalbumin and Mr Andre Chedeville for his excellent technical assistance. This work was supported by research grant A.T.P. CNRS n' 2111.
REFERENCES ANTOINE J.C. & AVRAMEAS S. (1973) Etude de la formation des anticorps anti-tyrosinase au cours de la reponse immunitaire chez le Lapin. I. ttude cellulaire. Ann. Immunol. (Inst. Pasteur), 124C, 121. ANTOINE J.C. & AVRAMEAS S. (1974) Surface immunoglobulins of rat immunocytes: quantitation and fate of cell-bound peroxidase-labelled antibody and Fab fragment. Europ. J. Immunol. 4, 468. ANTOINE J.C. & AVRAMEAS S. (1976) Correlations between immunoglobulin and antibody-synthesizing cells during primary and secondary immune responses of rats immunized with peroxidase. Immunology, 30, 537. ANTOINE J.C., AVRAMEAS S., GONATAs N.K., STIEBER A. & GONATAS J.O. (1974) Plasma membrane and internalized immunoglobulins of lymph node cells studied with conjugates of antibody or its Fab fragment with horseradish peroxidase. J. Cell Biol. 63, 12. AVRAMEAS S. & TERNYNCK T. (1971) Peroxidase-labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry, 9, 1175. CHILLER J.M., HABICHT G.S. & WEIGLE W.O. (1971) Kinetic differences in unresponsiveness of thymus and bone marrow cells. Science, 171, 813. FAHEY J.L. & TERRY E.W. (1973) Ion exchange chromatography and gel filtration. Handbook of Experimental Immunology, 2nd edn (ed. by D. M. Weir), p. 7.1. Blackwell Scientific Publications, Oxford. GONATAs N.K., ANTOINE J.C., STIEBER A. & AVRAMEAS S. (1972) Surface immunoglobulins of thymus and lymph node cells demonstrated by the peroxidase coupling technique. Lab. Invest. 26, 253. GRAHAM R. & KARNOVSKY M.S. (1966) The early stages of adsorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291. KIM Y.T. & SISKIND G.W. (1974) Studies on the control of antibody synthesis. VI. Effect of antigen dose and time after immunization on antibody affinity and heterogeneity in the mouse. Clin. exp. Immunol. 17, 329. MILLER H.R.P., TERNYCK T. & AVRAMEAS S. (1974) A comparison of specific antibody and immunoglobulinsynthesizing immunocytes in peroxidase stimulated lymph nodes. Ann. Immunol. (Inst. Pasteur), 125C, 231. MILLER H.R.P., TERNYNCK T. & AVRAMEAS S. (1975) Synthesis of antibody and immunoglobulins without detectable antibody function in cells responding to horse radish peroxidase. J. Immunol. 114, 626. MOND J., KiM Y.T. & SISKIND G.W. (1974) Studies on the
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control of antibody synthesis. V. Effect of nonspecific modification of the magnitude of the immune response on the affinity of the antibody synthesized. J. Immunol. 112, 1255. PORTER R.R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem. J. 73, 119.
SISKIND G.W. & BENACERRAF B. (1969) Cell selection by antigen in the immune response. Advanc. Immunol. 10, 1. WERBLIN T.P., KIM Y.T., QUAGLIATA F. & SISKIND G.W. (1973) Studies on the control of antibody synthesis. HII. Changes in heterogeneity of antibody affinity during the course of the immune response. Immunology, 24, 477.
