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Binding of radioiodinated bovine serum albumin to lymphoid cells of specifically primed or immunized mice in vitro
Life 3ciencee Vol. 7, Part II, pp . 377 -382, 1988. Printed in Great Britain.
Pergamon Press
BINDING OF RADIOIODINATED HOYINE SERUM ALH[TMIN Ta LYMPHOID CELLS OF SPECIFICALLY PRIMED OR IMMJNIZED MICE IN VITRO D. Naor and D. Sulitaeanu Department of Immunology, Hebrew University - Hadassah Medical School Jerusalem,
Israel
(Received 12 December 1987 ; in final form 28 January 1988) The mechanism determining the specificity of antibody induction is still unlmovn. Hurnet~s suggestion (1) that the specificity is due to antigen reacting with specific cell receptors on the lymphoid cells has gained vide acceptance, as it is compatible with a variety of ezperimental findings . However, the existence of these receptors has remained unproven . In a previous communication (2) we described the binding of a small amount of highly radioiodinated Bovine Serum Albumin (1z5I-HSA) to normal mouse spleen cells . Although far from cancluaive, this work could be taken ea tangible evidence that specific cell receptors might be real . We decided, therefore,
to test by our technique two
additional postulates of the clonal selection theory, namely that : e) the primed animals have an increased number of cells carrying the specific receptors and b) the tolerant animals have a reduced number of such cells . For comparison purposes, we studied also the antigen binding ability of cells from immunised animals . The results described in this preliminary report negate unequivocally the first postulate : affinity of primed cells for antigen vas no different from that of normal cells . On the other hand, a considerable proportion of the immune cells were capable of binding antigen. Materials and Methods The technique used in this work has been described in the previous publication (2) . Briefly, suspensions of mouse spleen or popliteal lymph node cells were incubated with
125
I-HSA (9 x 10 5 - 1 x 106 cpm, appr . 0.003 - 0.01, ug,
377
BII~ING OF H3A
378
Vol. 7, No. 8
specific activity 50 - 100 uC/ug) in the presence of 4~ normal mouse serum . The incubation was carried out at +4~, to minimize uptake by pinocytosis . The non bound radioactivity was removed by washing the cells three times with physiological salt solution (Saline G, 3) containing 1 .8~ polyvinyl pyrrolidone and 10 units/ml heparin . The radioactivity left in the tubes was measured in a Scintillation counter . Cell smears were then prepared, dried quickly and dipped in Ilford K 5 nuclear emulsion for autoradiography . The autoradiographs were exposed for 30 - 137 days, developed and stained lightly (10 min .) with Giemsa . The distribution of grain counts was determined by surveying 2000 - 5000 cells . Autoradiographs made by placing on slides known amounts of labeled antigen revealed that 1 silver grain corresponded to 50 - 150 molecules of 125 I-HSA . Mice were immunized by foot-pad injections of crystalline BSA incorporated in incomplete Freund~s adjuvant . The immune state of the animals was assayed one day before removing the cells for antigen binding experiments . A rapid immune elimination test was developed for this purpose, performed as fcllows : lug non labeled BSA mixed with labeled BSA (appr . 2 x 10 5 cpm) was injected into the tail vein . Blood samples were taken from the retroorbital plexus at one minute and siz hours after injection and their radioactivity was determined . Thirty to forty percent of the initial radioactivity, depending on the size of the animal, remained in the blood of normal mice at six hrs . The corresponding value for the blood of immune mice was 20~ or less (down to 5~) " With this technique, a clear cut indication of the presence of antibodies in the blood was obtained when 3 ug of rabbit anti BSA were injected intramuscularly 1g rss before the assay. Results Preliminary experiments showed extensive binding of 125 I - HSA by cells of immunized mice . It was therefore important in the studies with primed cells to select an immunization schedule which would ensure adequate priming (as mani fested by a strong response on second stimulation) without inducing detectable antibody formation . The procedure finally devised is shown in Table 1 : mice we^e
HIIdDING OF HSA
Vol . 7, No . 8
injected with 0.1
ug
379
HSA in incomplete ~eund~s adjuvant . After 118 days some
of the primed mice, together with control, untreated animals, received a booster TABLE 1 Distribution of Grain Counts in nymph Node Cells of Normal, Primed and Immunised Mice hg antigen injected to cell donors on days 0
0+118
0.1
Immune statè of cell donors at time of experiment
No . of cells containing grain counts as follows
(Day 0+12Q)
0-3
Q-6
7-10
11-15
16 or more
Total
1o
Stimulated twice and producing antibody
1926
25
18
8
23
2000
10
Stimulated once (recently), not producing antibôdy
1996
2
1
-
1
2000
0.1
-
Stimulated once PRIMED
1995
4
-
1
-
2000
-
-
Normal
1981
11
5
-
3
2000
injection of 10 ~g HSA. Antigen binding assays were performed 6 days later, at which time the boostered animals were strongly positive by the immune elimination test, while the other animals were negative . The distribution of grain counts in popliteal node cells incubated with antigen ie shown in Table 1 ; as in previously reported experiments (2) most cells of normal mice bound no radioactivity whatsoever (less than 3 grains) . A few normal cells were moderately labeled and only 3 of these cells (out of 2000) contained larger amounts of radioactivity (more than 16 grains) . The primed cells did not appear. tô bind the radioiodinated HSA more effectively than the normal cells . The cells of animals receiving the second &SA dose only (10 fig) were no different from the primed cells . On the other hand, many of the cells from the boostered mice were strongly labeled {23 out of 2000), thus providing additional evidence that priming had been effective .
380
HII~iDIIdG OF H3A
Vol . 7, No. 8
In order to dray valid conclusions from these experiments it was essential to know to what extent circulating antibody passively bound to the lymphoid cells might contribute to the observed binding of antigen. An estimate of this contribution was obtained by injecting to normal animals mouse anti BSA (10 ug per mouse) and performing the binding assays 7_!~ hra later. With the eaception of e slight increase in the number of labeled macrophages, no differences were seen between cells of passively immunised and of normal animals . This is in agreement with the known property of cytophilic antibody to attach to macrophages but not to lymphocytes (Q,5) . We concluded therefore that labeling of immune lymphoid cells was due to antibody actually produced by these cells . Several observations relating to the labeled cells of the boostered mice are worth reporting: s) Plasma cells were only a small minority among the labeled cells . Many of the radioactive cells were "naked nuclei" (Fig . 1) - a cell type rarely mentioned in the literature (6) . Identical looking nuclei were sometimes seen surrounded by a small amount of pale cytoplasm . b) Many strongly labeled macrophages were found . In similar experiments xith peritoneal eaudate cells of immune mice, a large proportion of the macrophages xere also labeled. It seems most likely that the macrophages carrier cytophilic antibody bound in vivo .
FIG. 1 labeled "~k d nucleus" found among immune popliteal node cells incubated wit , ~SI-BSA in vitro . The unlabeled cell is also a "naked nucleus" . Exposition time for autoradiography was 105 days .
Vol . 7, No. 8
BII~IIdG OF HSA
381
Discussion The work described here has shown that cells of primed mice (Y cells of Sercara and Coons, 7) are no different from normal cells as regards ability to bind antigen on their éurface . The results do not support therefore the widely held belife (1~,g,9,10) that the efficiency of the secondary response is due to the increased ability of Y cells ~to capture antigen through an antibody-like configuration on the cell surface. The possibility still remains that Y cells might be able to concentrate antigen by an active metabolic process. Our experiments provide additional confirmation for the well known property of immune cells to bind specific antigen (11 j, The affinity of antigen for these cells is quite strong, ns no amount of washing nppeara capable of dissociating the complez. To account for this fact, ane must assume that the antibody is tightly bound to the cell membrane . How this is achieved is not known. Qie might speculate that the antibody molecule is a structural component of the immune cell membrane . ~periments are in progress designed to compare the binding of 12 5I-HSA to cells of normal and specifically tolerant mice . Preliminary results indicate that cells of tolerant mice are totally unable to bind antigen . Spa The ability of mouse popliteal lymph node cells to bind redioiodinated BSA "in vitro" w~s studied by radioautograp}~p . Cells of mice primed by n single injection of 0.1 Fig HSA in incomplete F~eund's adjuvant bound BSA to the same ex tent as cells of normal mice . The increased efficiency of the secondary response cannot be therefore explained in terms of the classical clonal selection theory. Large numbers of cells from imm,inised mice bound the label, some of them quite strongly . Many of the labeled immune cells were of the "naked nucleus" type and only a few of them were typical plasma cells . References 1, M. HURNET, The Clonal Selection Theo Press, Cambridge 1959 .
of Ac uired Immunit , p. 67, University
382
2.
BII~ING OF BSA
D . NAOR and D . SULITZEANU, Nature 2 ~,
Vol . 7, No . 8
687 (1967) .
3 . R .A . HAtß~fLSTROM and R.D . STONER, J. Lab. Clin . Med.
62, 985 (1963) .
4.
L . HULLIGER and E . SORKIN, lnmninol .
Q, 391 (1965) "
5.
A . HERKEN and B . BENACERRAF, J. Exp. Med . 1~, 11q
6.
D .B . McMILLSN and V.E . ENGELHERT, Canad. J. Zool .
7.
E .SERCARZ and A.H . COONS, Mechanisms of Immunoloeical Tolerance, M. Hasek, A. Lengerova end M. Vojtiskova, eds ., p . 73, Czech . Acad . Sci ., Prague (1962)
8.
A :M . PAPPEMHEIMER, M. SCHARFF and J .W . UHR, In "Mechanisms of i~", J.H . Shaffer, ed ., p. 417, Little, Brown, Boston 1959 .
ersensitiv-
9.
R .W . WISSLER, F .W . FITCH and M.F . LA VIA, Ann . N .Y . Acad . Sci .
88, 134 (1960) .
(1966) .
~, 613 (1960) .
10 .E .E . SERCARZ and V.S . HYERS, J. Immunol.
28, 836 (1967) .
11 .0 . MAKELA and G .J .V . NOSSAL, J . Immunol.
87, 4L,7 (1961) .
.
Pergamon Press
BINDING OF RADIOIODINATED HOYINE SERUM ALH[TMIN Ta LYMPHOID CELLS OF SPECIFICALLY PRIMED OR IMMJNIZED MICE IN VITRO D. Naor and D. Sulitaeanu Department of Immunology, Hebrew University - Hadassah Medical School Jerusalem,
Israel
(Received 12 December 1987 ; in final form 28 January 1988) The mechanism determining the specificity of antibody induction is still unlmovn. Hurnet~s suggestion (1) that the specificity is due to antigen reacting with specific cell receptors on the lymphoid cells has gained vide acceptance, as it is compatible with a variety of ezperimental findings . However, the existence of these receptors has remained unproven . In a previous communication (2) we described the binding of a small amount of highly radioiodinated Bovine Serum Albumin (1z5I-HSA) to normal mouse spleen cells . Although far from cancluaive, this work could be taken ea tangible evidence that specific cell receptors might be real . We decided, therefore,
to test by our technique two
additional postulates of the clonal selection theory, namely that : e) the primed animals have an increased number of cells carrying the specific receptors and b) the tolerant animals have a reduced number of such cells . For comparison purposes, we studied also the antigen binding ability of cells from immunised animals . The results described in this preliminary report negate unequivocally the first postulate : affinity of primed cells for antigen vas no different from that of normal cells . On the other hand, a considerable proportion of the immune cells were capable of binding antigen. Materials and Methods The technique used in this work has been described in the previous publication (2) . Briefly, suspensions of mouse spleen or popliteal lymph node cells were incubated with
125
I-HSA (9 x 10 5 - 1 x 106 cpm, appr . 0.003 - 0.01, ug,
377
BII~ING OF H3A
378
Vol. 7, No. 8
specific activity 50 - 100 uC/ug) in the presence of 4~ normal mouse serum . The incubation was carried out at +4~, to minimize uptake by pinocytosis . The non bound radioactivity was removed by washing the cells three times with physiological salt solution (Saline G, 3) containing 1 .8~ polyvinyl pyrrolidone and 10 units/ml heparin . The radioactivity left in the tubes was measured in a Scintillation counter . Cell smears were then prepared, dried quickly and dipped in Ilford K 5 nuclear emulsion for autoradiography . The autoradiographs were exposed for 30 - 137 days, developed and stained lightly (10 min .) with Giemsa . The distribution of grain counts was determined by surveying 2000 - 5000 cells . Autoradiographs made by placing on slides known amounts of labeled antigen revealed that 1 silver grain corresponded to 50 - 150 molecules of 125 I-HSA . Mice were immunized by foot-pad injections of crystalline BSA incorporated in incomplete Freund~s adjuvant . The immune state of the animals was assayed one day before removing the cells for antigen binding experiments . A rapid immune elimination test was developed for this purpose, performed as fcllows : lug non labeled BSA mixed with labeled BSA (appr . 2 x 10 5 cpm) was injected into the tail vein . Blood samples were taken from the retroorbital plexus at one minute and siz hours after injection and their radioactivity was determined . Thirty to forty percent of the initial radioactivity, depending on the size of the animal, remained in the blood of normal mice at six hrs . The corresponding value for the blood of immune mice was 20~ or less (down to 5~) " With this technique, a clear cut indication of the presence of antibodies in the blood was obtained when 3 ug of rabbit anti BSA were injected intramuscularly 1g rss before the assay. Results Preliminary experiments showed extensive binding of 125 I - HSA by cells of immunized mice . It was therefore important in the studies with primed cells to select an immunization schedule which would ensure adequate priming (as mani fested by a strong response on second stimulation) without inducing detectable antibody formation . The procedure finally devised is shown in Table 1 : mice we^e
HIIdDING OF HSA
Vol . 7, No . 8
injected with 0.1
ug
379
HSA in incomplete ~eund~s adjuvant . After 118 days some
of the primed mice, together with control, untreated animals, received a booster TABLE 1 Distribution of Grain Counts in nymph Node Cells of Normal, Primed and Immunised Mice hg antigen injected to cell donors on days 0
0+118
0.1
Immune statè of cell donors at time of experiment
No . of cells containing grain counts as follows
(Day 0+12Q)
0-3
Q-6
7-10
11-15
16 or more
Total
1o
Stimulated twice and producing antibody
1926
25
18
8
23
2000
10
Stimulated once (recently), not producing antibôdy
1996
2
1
-
1
2000
0.1
-
Stimulated once PRIMED
1995
4
-
1
-
2000
-
-
Normal
1981
11
5
-
3
2000
injection of 10 ~g HSA. Antigen binding assays were performed 6 days later, at which time the boostered animals were strongly positive by the immune elimination test, while the other animals were negative . The distribution of grain counts in popliteal node cells incubated with antigen ie shown in Table 1 ; as in previously reported experiments (2) most cells of normal mice bound no radioactivity whatsoever (less than 3 grains) . A few normal cells were moderately labeled and only 3 of these cells (out of 2000) contained larger amounts of radioactivity (more than 16 grains) . The primed cells did not appear. tô bind the radioiodinated HSA more effectively than the normal cells . The cells of animals receiving the second &SA dose only (10 fig) were no different from the primed cells . On the other hand, many of the cells from the boostered mice were strongly labeled {23 out of 2000), thus providing additional evidence that priming had been effective .
380
HII~iDIIdG OF H3A
Vol . 7, No. 8
In order to dray valid conclusions from these experiments it was essential to know to what extent circulating antibody passively bound to the lymphoid cells might contribute to the observed binding of antigen. An estimate of this contribution was obtained by injecting to normal animals mouse anti BSA (10 ug per mouse) and performing the binding assays 7_!~ hra later. With the eaception of e slight increase in the number of labeled macrophages, no differences were seen between cells of passively immunised and of normal animals . This is in agreement with the known property of cytophilic antibody to attach to macrophages but not to lymphocytes (Q,5) . We concluded therefore that labeling of immune lymphoid cells was due to antibody actually produced by these cells . Several observations relating to the labeled cells of the boostered mice are worth reporting: s) Plasma cells were only a small minority among the labeled cells . Many of the radioactive cells were "naked nuclei" (Fig . 1) - a cell type rarely mentioned in the literature (6) . Identical looking nuclei were sometimes seen surrounded by a small amount of pale cytoplasm . b) Many strongly labeled macrophages were found . In similar experiments xith peritoneal eaudate cells of immune mice, a large proportion of the macrophages xere also labeled. It seems most likely that the macrophages carrier cytophilic antibody bound in vivo .
FIG. 1 labeled "~k d nucleus" found among immune popliteal node cells incubated wit , ~SI-BSA in vitro . The unlabeled cell is also a "naked nucleus" . Exposition time for autoradiography was 105 days .
Vol . 7, No. 8
BII~IIdG OF HSA
381
Discussion The work described here has shown that cells of primed mice (Y cells of Sercara and Coons, 7) are no different from normal cells as regards ability to bind antigen on their éurface . The results do not support therefore the widely held belife (1~,g,9,10) that the efficiency of the secondary response is due to the increased ability of Y cells ~to capture antigen through an antibody-like configuration on the cell surface. The possibility still remains that Y cells might be able to concentrate antigen by an active metabolic process. Our experiments provide additional confirmation for the well known property of immune cells to bind specific antigen (11 j, The affinity of antigen for these cells is quite strong, ns no amount of washing nppeara capable of dissociating the complez. To account for this fact, ane must assume that the antibody is tightly bound to the cell membrane . How this is achieved is not known. Qie might speculate that the antibody molecule is a structural component of the immune cell membrane . ~periments are in progress designed to compare the binding of 12 5I-HSA to cells of normal and specifically tolerant mice . Preliminary results indicate that cells of tolerant mice are totally unable to bind antigen . Spa The ability of mouse popliteal lymph node cells to bind redioiodinated BSA "in vitro" w~s studied by radioautograp}~p . Cells of mice primed by n single injection of 0.1 Fig HSA in incomplete F~eund's adjuvant bound BSA to the same ex tent as cells of normal mice . The increased efficiency of the secondary response cannot be therefore explained in terms of the classical clonal selection theory. Large numbers of cells from imm,inised mice bound the label, some of them quite strongly . Many of the labeled immune cells were of the "naked nucleus" type and only a few of them were typical plasma cells . References 1, M. HURNET, The Clonal Selection Theo Press, Cambridge 1959 .
of Ac uired Immunit , p. 67, University
382
2.
BII~ING OF BSA
D . NAOR and D . SULITZEANU, Nature 2 ~,
Vol . 7, No . 8
687 (1967) .
3 . R .A . HAtß~fLSTROM and R.D . STONER, J. Lab. Clin . Med.
62, 985 (1963) .
4.
L . HULLIGER and E . SORKIN, lnmninol .
Q, 391 (1965) "
5.
A . HERKEN and B . BENACERRAF, J. Exp. Med . 1~, 11q
6.
D .B . McMILLSN and V.E . ENGELHERT, Canad. J. Zool .
7.
E .SERCARZ and A.H . COONS, Mechanisms of Immunoloeical Tolerance, M. Hasek, A. Lengerova end M. Vojtiskova, eds ., p . 73, Czech . Acad . Sci ., Prague (1962)
8.
A :M . PAPPEMHEIMER, M. SCHARFF and J .W . UHR, In "Mechanisms of i~", J.H . Shaffer, ed ., p. 417, Little, Brown, Boston 1959 .
ersensitiv-
9.
R .W . WISSLER, F .W . FITCH and M.F . LA VIA, Ann . N .Y . Acad . Sci .
88, 134 (1960) .
(1966) .
~, 613 (1960) .
10 .E .E . SERCARZ and V.S . HYERS, J. Immunol.
28, 836 (1967) .
11 .0 . MAKELA and G .J .V . NOSSAL, J . Immunol.
87, 4L,7 (1961) .
.