Immunology 1977 33 81
The appearance of non-specific antibody-forming cells in the efferent lymph draining antigen-stimulated single lymph nodes
D. C. POSKITT, H. FROST, R. N. P. CAHILL & Z. TRNKA Basel Institute for Immunology, Grenzacherstrasse 487, Basel, Switzerland
Received 18 October 1976; acceptedfor publication 25 November 1976
Summary. Immunization of single lymph nodes with various antigens led to the appearance of cells in the efferent lymph that secreted antibody specific for the antigen which induced their formation and for a number of unrelated, non-crossreacting antigens. Immunization of single lymph nodes with mitogens led to the appearance of cells secreting antibodies specific for an even greater number of antigens, including one (TNP) that in all probability is not present in the animals' natural environment. When the node was primed with one antigen, a subsequent challenge with an unrelated antigen 12 weeks later led to the appearance of greater numbers of cells containing and secreting antibody against the previously experienced antigen, than was the case in unprimed lymph nodes. These findings indicate that the immune response to antigen provokes the maturation of lymphocytes of specificities unrelated to that of the injected immunogen. Such a mechanism may be important in maintaining immunological memory. Mitogens may directly activate lymphocytes into maturation and expression as antibody-secreting cells, whereas antigens appear to act indirectly. Correspondence: Dr D.C. Poskitt, Basel Institute for Immunology, Grenzacherstrasse 487, Postfach 4005, Basel 5, Switzerland. F
81
INTRODUCTION Many investigators have demonstrated that following the injection of an antigen, antibodies specific for the antigen and immunoglobulins without apparent specificity are synthesized during the course of the ensuing immune response (Boyd & Bernhard, 1937; Pappenheimer, Lundgren & Williams, 1940; Urbain Vansanten, 1970; De Vos Cloetens, Minsart-Baleriaux & Urbain Vansanten, 1971; Loor, 1971; Antoine & Avrameas, 1973; Miller, Avrameas & Ternynck, 1973; Miller, Ternynck & Avrameas, 1974; Moticka, 1974; Pachmann, Killander & Wigzell, 1974; Miller, Ternynck & Avrameas, 1975). Within this population of immunoglobulins, antibodies with specificities for previously experienced cross-reactive antigens have been detected; this led to the postulate of Original Antigenic Sin (Francis, 1953; Fazekas de St. Groth & Webster,
1966a, b). Further, antibodies have been demonstrated during the immune response which do not cross-react with the antigen which induced their formation (Freund, 1953; Jarret & Stewart, 1972; Nakashima & Kato, 1974; Moticka, 1975). The specificity of these antibodies appears to be directed against antigens previously experienced by the host (Weil & Felix, 1916; Bieling, 1919; Moticka, 1975; Shimada & Silverstein, 1975).
D. C. Poskitt et al.
82
We have investigated the synthesis and secretion of both specific antibody and 'non-specific' immunoglobulins by cells appearing in the efferent lymph during the course of immune responses in sheep popliteal lymph nodes. Using the in situ popliteal lymph node preparation originally described by Hall & Morris (1962) the immune response can be confined to the one lymph node and the temporal sequence of an immune response may be followed in the efferent lymph in the absence of any systemic immune reaction. Cellular and humoral events occurring within the lymph node are reflected in the efferent lymph (Frost, Braun, Poskitt, Cahill & Trnka, 1976). The appearance of cells containing and/or secreting specific and 'nonspecific' antibodies can be followed and their total numbers established at the end of the immune response. Previous reports on this subject have examined immune responses by measuring antibody titres or by looking for antibody and immunoglobulin synthesizing cells (Miller, Ternynck & Avrameas, 1974; 1975; Pachmann, Killander & Wigzell, 1974; Antoine & Avrameas 1976) in lymph nodes or spleen. Others have shown the development of 'non-specific' PFC in responses to mitogens in vitro (Andersson, Melchers, Galanos & Luderitz, 1973; Melchers & Andersson, 1974) and in vivo (Gronowicz & Coutinho, 1974). This report examines the appearance of non-specific antibody-forming and antibody-secreting cells in the efferent lymph draining antigen-stimulated single lymph nodes.
Lymph collection and cell counting Lymph was collected in sterile plastic bottles each containing 1 ml of a solution of sterile physiological saline containing 500 U-USP of Heparin (liquemin, Roche) and 500 iu each of penicillin and streptomycin (Flow Laboratories). All collections were made at room temperature. Cells were counted in a Model Fn Coulter counter equipped with a 100 u aperture, and expressed as cell output per hour. Antigens and mitogens All antigens and mitogens were suspended or dissolved in 3 ml of phosphate-buffered saline (PBS) prior to injection. Injections were given s.c. in the drainage area of the popliteal lymph node.
Red blood cells. Horse, rabbit and chicken red blood cells from randomly bred animals were used. The cells were washed thrice with PBS and a standard dose of 2 x 109 red cells was injected. Rabbit anti-SRBC antibody. Rabbit anti-SRBC antiserum No. B5221 /11 was kindly supplied by Dr A. Kelus of this Institute. Two millilitres of nonheat inactivated antiserum was added to 1 ml of packed and washed autologous SRBC and the suspension incubated for 1 h at 370 with constant stirring. The SRBC lysate was centrifuged and the membrane pellet washed three times with PBS prior to injection.
Allogeneic lymphocytes. Allogeneic lymphocytes
MATERIALS AND METHODS
were obtained from the efferent lymph of second party sheep. 3 x 108 allogeneic lymphocytes washed thrice in Eagle's (MEM) containing 10%4 of recipient lymph plasma were injected.
Animals Sheep of either sex, aged between 8 months and 2 years, were obtained from commercial flocks of 'black face' and 'white alpine' sheep. Experimental sheep were kept in metabolism cages and were given free access to hay, water and salt lick.
Bovine serum albumin. One millilitre of packed autologous SRBC coated with BSA according to the method of Sweet and Welborn (1971), to improve the immunogenicity of BSA, were injected.
Surgical procedures The lymphatic vessel efferent to the popliteal lymph node was cannulated according to the technique described by Hall & Morris (1962). Animals were allowed a 2-day post-operative recovery period prior to the injection of antigen or mitogen.
Influenza virus. Human influenza virus of the MEL strain (AO/Melbourne/1/35) was kindly supplied by Dr S. Fazekas de St. Groth of this Institute. The virus was injected at a dose of 160 pg of virus, equivalent to 4 x 1011 physical particles. The stock antigen consisted of a concentrate of purified, non-infective particles suspended in saline with 0 04% NaN3 as a preservative.
Specific and 'non-specific' antibody secretion
Streptococcal vaccine (Group A variant and Group C). Strain A486 variant, M negative and C74 vaccines originating from the collection of Dr R.C. Lancefield, The Rockefeller University, New York, were kindly supplied by Dr D.G. Braun of this Institute. One hundred microlitres of vaccine, each containing 25 4ug of Group A variant or 25 ug of group C carbohydrate was injected. Horseradish peroxidase (HRP). Sheep were primed by injecting 10 mg of HRP (Sigma grade VI) emulsified in 1 ml of Freund's complete adjuvant. To induce secondary responses 2-5 mg of HRP in 2 5 ml of PBS was injected 4 weeks after priming. Pokeweed mitogen (PWM). Two hundred micrograms PWM (Grand Island Biological Co.) was injected. Concanavalin A (Con A). Five hundred micrograms Con A (Miles) three times recrystallized, was injected.
Lipopolysaccharide (LPS). Five hundred micrograms LPS B (S. minnesota, Difco) was injected. Purified protein derivitive (PPD). 100 pug of PPD (lot 280 Weybridge) was injected s.c. in 1 ml of saline in animals which had been primed 1-2 months previously with two to three times the human dose of BCG (Berna, Berne). The detection of intracellular antibody to horseradish peroxidase Cells containing antibody specific for HRP were demonstrated by a histochemical technique which involved the reaction of the enzyme with its substrate in the presence of 3,3'-diaminobenzidine (DAB) or carbazole-H202 reagent. The methods of Avrameas & Lespinats (1967), and Avrameas (1970) were used. The detection of immunoglobulin-containing cells in HRP responses Cells containing immunoglobulins were detected using a 1 in 10 dilution of rabbit anti-sheep immunoglobulin antiserum (conjugated to HRP). Cells containing a reddish-brown precipitate after staining with carbazole-H202 reagent were assessed as positive cells containing immunoglobulin.
Cells incubated with normal rabbit immunoglobulin
83
conjugated to HRP and stained with carbazole-H202 reagent were used as controls. The number of immunoglobulin-containing cells was assessed by subtracting the number of positively stained cells observed in the controls from those observed in cell smears incubated with rabbit antisheep immunoglobulin. The number of 'non-specific' immunoglobulincontaining cells was calculated by subtracting the number of antibody-containing cells from the number of immunoglobulin-containing cells. Preparation of target cells for the plaque-forming cell assay CRBC, HRBC, RRBC, SRBC and coated SRBC were washed three times in Eagle's MEM. One millilitre of packed red cells was resuspended in Eagle's MEM to a total volume of 6 ml for use in the assay system. Washed SRBC were coated with BSA according to the method of Sweet & Welborn (1971). The method used to coat SRBC with TNP was based on that first described by Rittenberg & Pratt (1969), 2 ml of packed SRBC were suspended in 20 ml of cacodylate buffer (pH 6 9) containing 4 mg/ml TNP. The O-stearoyl-polysaccharide of Streptococcus group A variant and Streptococcus group C cell walls were used to coat SRBC according to the method of Read & Braun (1974). PFC assay Efferent lymph cells were washed and resuspended to a concentration of 106 or 107 cells/ml in Eagle's MEM. Packed uncoated or coated red cells were suspended in Eagle's MEM (1:5 v/v). Rabbit serum absorbed with the target erythrocytes was used as a source of complement. 0-5 ml of the lymphoid cell suspension was mixed with 0-1 ml of the target red cell suspension and 0 05 ml rabbit serum. The reaction mixture was thoroughly mixed and 0 05 ml immediately transferred to Cunningham slide chambers (Cunningham & Szenberg, 1968). The slide chambers were sealed with Vaseline and incubated at 370 for 30 min. Plaques were counted at forty-fold magnification. The PFC output per hour was recorded and the total number of plaques that appeared in the lymph during the course of an immune response was also calculated. Only direct PFC assays were performed. The total blast cell number represents all blast cells collected in the efferent lymph from the time that antigen was injected until PFC were no longer detectable.
D. C. Poskitt et
84
RESULTS Antibody specificity of PFC following antigenic stimulation Primary immune responses to CRBC and secondary responses to HRP were studied to determine the relative numbers of non-specific immunoglobulincontaining cells, specific antibody-containing cells and antibody-secreting cells (PFC) that appear in the efferent lymph during the course of an immune response.
In the response to 2 x 109 CRBC a total of 2-1 x lO0 cells appeared in the efferent lymph over a period of 220 hrs; 2 3 x 109 were assessed as blast cells. Of the 2 1 x 1010 cells collected, 11 7% contained immunoglobulins which had no detectable specificity for the inducing antigen, 0O9% contained specific antibody and only 0 3% secreted antibody specific for CRBC. During the course of the secondary response to HRP a total of 2-75 x 1010 cells appeared in the efferent lymph over a period of 150 h; 7-17 x 109 cells were assessed as blast cells. Of the 2 75 x 1010 cells collected, 7 % contained immunoglobulins which had no detectable specificity against the inducing antigen whilst 1133% contained antibody specific for HRP. These results indicate that many of the lymphocytes and blast cells which appear in the efferent lymph during the course of an immune response to antigen, contain immunoglobulins unable to react with the specific antigen. Furthermore, of those cells containing specific antibody only 30% were PFC. Thus, in the following experiments where the cells
al.
that appeared in the efferent lymph of popliteal lymph nodes responding to a primary antigenic stimulus were of necessity assayed for their ability to secrete antibodies against the priming antigen as well as against a panel of antigens with which the animal had no known previous experience, the PFC assay will considerably underestimate the number of cells containing specific or non-specific antibodies (Table 1). It is emphasized that cells from the efferent lymph of nonstimulated popliteal lymph nodes gave no PFC against any of the antigens tested. The primary response to allogeneic lymphocytes, xenogeneic erythrocytes, proteins, bacterial and viral antigens gave rise to cells secreting specific antibody and antibodies with specificities for some or all of the antigens selected in our panel. In certain cases cross-reactivity could account for some of the apparent non-specificity of the response, for example the Strep.Av carbohydrate and Strep. group C carbohydrate are cross-reactive. It is unlikely, however, that the whole panel of antigens chosen to screen immune responses for nonspecific components, was cross-reactive with the immunizing antigens.
Fig. la shows the changes that occurred within the efferent lymph following the injection of 3 x 108 allogeneic lymphocytes. The response was vigorous in terms of total and blast cell output, yet only a relatively small number of cells secreting antibody against chicken and horse red blood cells were detected, and PFC against the other panel antigens were not found (Table 1). The response to CRBC (Fig. lb) gave rise to cells secreting antibodies against the specific antigen and four other antigens
Table 1. Primary immune response to antigen
Antigen HRBC (2x 109) RRBC (2 x 109) CRBC (2x 109) Strep.Av. vaccine (100).) Strep.C. vaccine (1002.) BSA Influenza virus (160,ug)
Allogeneic lymphocytes (3 x 108) Rabbit anti-SRBC Ig
Non-specific PFC x 10-6 Total blast Specific PFC x 10-6 HRBC RRBC CRBC Strep.Av Strep.C cells x 10-9 12-3 2-8 56 2-4 1-2 2-1 3-4 24 104
305 1-2 46-8 13-7 1.1 0 -
0 09 1-49 0-02 011 0 93 042
-
0d13 7 91
-
0
0-25 0-04
0 0 0 0 001 0 0
-
0 07 0-02 0-01
0 05 0-02 0 3-27 04 0-23
-
0-08 0 03 0-16 7-28
0 35 0 003 0 0
0.1 2-27 0 0
-
BSA
TNP
0 0 0 005 0 0
0 0 0 0 0 0 001 0 0
-
001 0 0
The total number of blast cells and PFC that appeared in the efferent lymph of popliteal lymph nodes stimulated with antigens.
Specific and 'non-specific' antibody secretion (a) 6 So
=D
4 r
f
w
*
2
I 4 3 2
, It CL
Q
0
P.
x1
II
0 24 48 72 96 120 144168 192 216 240 Hours after s.c. injection of allogeneic lymphocytes (b)
_0. 0
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0
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0 x
IF 10A 8 6 4 2
j
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-A-
A
24 48 72 96 120144168 192 216 240264 Hours after s c. injection of CRBC (c)
0
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14 12
-
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8 620 4 2-
24 48 72 96 120 144168192218240 Hours after sc. injection of Streptococcus group C vaccine Figure 1. Primary immune response to (a) 3 x 108 0
allogeneic lymphocytes; (b) 2x 109 CRBC; (c) l00A Strep.C vaccine. The upper section of each figure shows changes in cell output in the lymph efferent to the popliteal node. The lower sections of (a) and (c) show the appearance of specific and non-specific PFC in the efferent lymph. The centre
85
from the panel (Table 1). The vigour of the immune response in terms of the increase in cell output did not appear to be directly related to the ability of an antigen to trigger cells secreting 'non-specific' antibodies. The cellular response to Strep.C vaccine (Fig. Ic) was feeble in terms of cell output and yet gave rise to sub-populations of cells secreting antibodies against three out of the six 'non-specific' antigens (Table 1). The number of cells which secrete antibody against any of the panel antigens is very small when compared to the number of cells secreting antibody against the priming antigen (Table 1). However, a more extensive panel of antigens might reveal cells secreting antibodies against many other antigens. Nearly 100% of specific and 'non-specific' antibodysecreting cells were blast cells. Thus, it is conceivable that the total number of cells containing and/or secreting both specific and 'non-specific' antibodies might exceed the total number of blast cells which appear in the efferent lymph. This is indeed suggested by the results obtained in the secondary response to horseradish peroxidase where virtually all the blast cells contained immunoglobulin and where 7 % of all cells contained 'non-specific' immunoglobulin and 11-3 % contained specific antibody.
Antibody specificity of PFC and AFC following antigenic stimulation of lymph nodes previously primed with an unrelated antigen The cells producing 'non-specific' antibodies might be memory cells or at least the progeny of cells which have been clonally expanded during some previous encounter with their specific antigen. In the next series of experiments the influence of previous antigenic experience on the number of non-specifically evoked cells was explored. Sheep were immunized with one antigen 12 weeks prior to immunizing with a second unrelated antigen in order to determine whether the response to the first antigen could be recalled. In all cases, PFC specific for the first antigen were no longer detectable in the efferent lymph 12 weeks after the section of (b) shows the appearance of non-specific PFC and the lower section shows the appearance of specific PFC in the efferent lymph. Total lymphocytes, (--*); blast cells, (0-0); HRBC/PFC, (A-A); CRBC/PFC (A-A); Strep.Av/PFC, (0-n); Strep.C/PFC, (O-O). In the response to 2x 109 CRBC (b), PFC specific for BSA and Strep.C also appeared in the efferent lymph but are omitted from this figure for simplicity (see Table 1).
D. C. Poskitt et aJ.
86
Table 2. The induction of anamnestic responses by unrelated immunogenic stimuli
Antigen 1
PFCx 10-6 Total blast cells x 10-9 HRBC RRBC CRBC Strep.Av Strep.C BSA TNP
Antigen 2
Strep.C vaccine (100).)
Allogeneic lymphocytes (3 x 108) Allogeneic lymphocytes (3 x 108) HRBC (2x 109)
CRBC (2 x 109) Strep.C -vaccine (100).)
3-4
0-02
0
0 09
0 05
0 07
0
0
4-6
0-03
0
0-87
0
0
0
0
59
73-7
0
0 37
0-23
0-17
0
0
The first antigen was injected into the drainage area of a popliteal lymph node 12 weeks prior to the second antigen. The total number of blast cells and PFC that appeared in the efferent lymph during the course of the immune response to the second antigen are shown. (a)
,0
2 0-0
10, 0-0
10.0,0
0-0-0-0/
o
0-,O-m-e-t74rL-
4
V~~~~~~~~~~
L-
-r
-yr
A
2
HI 24 48 72 96 120 144168 192 216 240 Hours after s.c. injection of allogeneic lymphocytes (b)
0
g
r
1'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0
L-
co
3 2 It -
.
.
I. .
./
A
0 24 48 72 96 120144168 192216 240 Hours after s c injection of allogeneic lymphocytes 8 6 4 2
initial immunization. The results of these experiments are summarized in Table 2. The number of cells secreting antibody against the first antigen was always higher than that of animals which had no experience of the first antigen (Tables 1 and 2). In one animal in which the popliteal lymph node had been immunized with Strep.C vaccine 12 weeks prior to challenge with allogeneic lymphocytes (Fig. 2a), PFC against Strep.Av and Strep.C carbohydrates were found whereas the contralateral lymph node which had not been immunized with Strep.C vaccine did not give rise to PFC against these antigens upon challenge with allogeneic lymphocytes. In a further experiment the number of PFC against CRBC was increased when the animal had experienced CRBC 12 weeks prior to challenge with allogeneic lymphocytes (Fig. 2b and Table 2).
(c)
/1
Figure 2. The induction of anamnestic responses by unrelated immunogenic stimuli. (a) Primary immune response to 3 x 108 allogeneic lymphocytes in a popliteal lymph node that had been primed with Strep. group C vaccine 12 weeks previously. (b) Primary immune response to 3 x 108 allogeneic lymphocytes in a popliteal lymph node that had been primed with 2x I09 CRBC 12 weeks previously. (c) Primary immune response to 2 x 109 CRBC in a popliteal lymph node that had been primed with 10 mg of HRP in Freund's complete adjuvant 12 weeks previously. The upper section of each figure shows changes in cell output in the lymph efferent to the popliteal node. The lower section of (a) and (b) and the centre section of (c) shows the appearance of PFC in the efferent lymph. The lower section of (C) shows the appearance of cells containing antibody specific for HRP. Total lymphocytes, (--*); blast cells, (o-a); HRBC/PFC, (A-A); CRBC/ PFC, (A-A); Strep.Av/PFC, (L-LI); Strep.C/PFC,
(0-c); HRP/AFC, (*-*). 0
24 48 72 96 120 144
Hours after
sc
injection of CRBC
During the course of the response to 3 x 108 allogeneic lymphocytes (a) PFC specific for HRBC also appeared in the efferent lymph but are omitted from the figure for simplicity (see Table 2).
Specific and 'non-specific' antibody secretion 6 F (a) 5 4
x _r
I
A3
./,-*
-
Similar results were obtained when Strep.C vaccine injected 12 weeks prior to HRBC. Whilst these experiments demonstrate an increase in the number of cells secreting antibody against a previously encountered and non-crossreactive antigen, they give no information as to the number of cells containing antibody specific for that antigen. To determine if there is an increase in the number of AFC we injected 2 x 109 CRBC into the drainage area of a popliteal lymph node that had been primed 12 weeks previously with HRP in Freund's complete adjuvant. Fig. 2c shows the results of this experiment. There was an increase in HRP specific AFC with a maximum output in the lymph just prior to the maximum output of PFC specific for the second antigen, CRBC.
-\/
was
/0
0-
/X
1
=2 (I
0
8 7 x
6
6
& 4 0
3
'tL O2 0
3
24 48 72 96 120144168 Hours after s.c. injection of LPS r (b) e.
/ 0"0
Antibody specificity of PFC following stimulation with mitogens Mitogens such as LPS (Andersson et al., 1973) or PPD (Melchers et al., 1974) induce in vitro the maturation of B lymphocytes to PFC secreting 'polyclonal immunoglobulins'. Therefore, mitogens, by virtue of their reported non-specific action upon lymphocytes, should give rise to cells secreting antibodies with specificities directed against a large number of antigens. The selectivity of mitogens for T and B cells well documented for other species has not been extensively examined in sheep. Investigations in this laboratory (manuscript in preparation) indicate that PWM and PPD activate both T and B cells whilst LPS activates only B cells and Con A only T cells. Table 3 and Fig. 3a and b show the numbers and the appearance of 'non-specific' PFC after stimulation with Con A, PWM, PPD and LPS. It can be seen that the spectrum of 'non-specific' PFC is
.\/x-
o
\
5
BA
4
3
0r OL
3 2 l
87
1
24 48 72 96 120 144 168 Hours after sc injection of PWM Figure 3. Responses to mitogens. The response of the popliteal lymph node to (a) LPS and (b) PWM. 0
The upper section of each figure shows the changes in cell output in the lymph efferent to the popliteal node. The
lower section shows the appearance of 'non-specific' PFC in the efferent lymph. Total cells, (0-0); blast cells, (0-a); HRBC/PFC, (A-A); CRBC/PFC, (A-A); Strep.Av/PFC, (0-E); TNP/PFC, (-). PFC with specificity for Strep.Av, Strep.C and LPS (A) and Strep.C (B) also appeared in the efferent lymph but are omitted for simplicity (see Table 3).
Table 3. Responses to mitogens
PFCx 10-6
Mitogen Con A (500 /ug) PPD (100 pg) PWM (100 jug) LPS (500jug)
Total blast cells x 10- 9 97 12-8 4-4 11-2
HRBC
RRBC
CRBC
Strep.Av
Strep.C
BSA
TNP
0-16 7-36 0 73 2-38
0 0-07 0 0
0 19 4-85
0 002 0-47 0-07 0-32
0-002 0 34 005 0-58
0 0-21 0 0
0 008 0-02 0 3-28
1P77 1-22
The total number of blast cells and PFC that appeared in the efferent lymph of popliteal lymph nodes stimulated with mitogens.
D. C. Poskitt et al.
88
broader after mitogenic stimulation, with the exception of influenza virus and that the 'nonspecific' PFC appear in the efferent lymph at a much earlier time than in the response to antigens. DISCUSSION When lymph nodes were stimulated with antigens or mitogens, cells appeared in the efferent lymph that synthesized and secreted antibodies specific for the immunizing antigen and a number of non-crossreacting antigens. Lymphocytes from the efferent lymph of non-stimulated lymph nodes never gave 'background' PFC against any of the panel antigens. This corresponds to our previous findings (Poskitt, 1974) and also those of Hay (1970). Lymphocytes can enter lymph nodes by two routes, from the blood via the post-capillary venules and from afferent lymphatics which they enter from the tissue spaces after leaving the blood stream via peripheral arteriovenous loops. The popliteal lymph node is the most distal node in a chain of lymph nodes leading to the thoracic duct and as such receives no lymph or cellular components from other lymph nodes. In response to antigens and mitogens, lymphocytes are recruited from the blood into the lymph node, the mechanism that controls and regulates this migration are unknown. During this recruitment phase antibody-secreting cell precursors are recruited into the node. In our experiments, 'non-specific' PFC in antigen responses appeared later than they did in mitogen responses and were restricted to the period in which specific PFC were present. The time of appearance of 'non-specific' PFC after mitogenic stimulation in our experiments is similar to that reported by Gronowicz & Coutinho (1974). Thus, the 'nonspecific' PFC that emerge in the efferent lymph of mitogen-stimulated lymph nodes appear to be the result of direct activation of antibody-secreting cell precursors by mitogen. The kinetics of the appearance of 'non-specific' PFC after stimulation with antigen suggest that antigens might act indirectly by way of specific cell interactions giving rise to factors which non-specifically activate bystander B lymphocytes which have no specificity for the antigen. Such factors may act within the lymph node to activate preferentially the specific antibody-secreting cell precursors in the presence of the specific antigen and to a more limited extent, bystander antibody-
secreting cell precursors of other specificities in the absence of their specific antigen. Another characteristic of the 'non-specific' PFC clones was their limited size which was several orders lower than that of the specific PFC clone. The numbers were higher in the efferent lymph from nodes with prior experience of the test antigen suggesting that memory cells might be the candidates for cells synthesizing and secreting 'non-specific' antibodies although no distinction can be made between residential and recirculating memory. This question is being further examined. The results of these studies indicate that the microenvironment within a lymph node responding to antigen induces not only the maturation of specific antigen-reactive lymphocytes but also the maturation of lymphocytes of specificities unrelated to that of the injected antigen. Such a mechanism may aid the maintenance of immunological memory by the non-specific activation of memory cells, each immune response boosting the pool of memory cells preferentially to the inducing and marginally to unrelated and non-cross-reacting antigens. If this is the case, then areas of lymphoid tissue which are continually exposed to antigenic insult, such as the gut, maybe important in maintaining immunological memory.
ACKNOWLEDGMENTS The authors would like to thank Mr H. J. Hoch for expert technical assistance. REFEREN CES ANDERSSON J., MELCHERS F., GALANOS C. & LUDERITZ 0. (1973) The mitogenic effect of lipopolysaccharide on bone marrow derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. exp. Med. 137, 943. ANTOINE J.C. & AVRAMEAS S. (1973) Etude de la formation des anticorps anti-tryosinase au cours de la reponse immunitaire chez le lapin. Ann. Immunol. (Inst. Pasteur), 124C, 121. 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. AVRAMEAS S. (1970) Immunoenzyme techniques: Enzymes as markers for the localisation of antigens and antibodies Int. rev. Cytol. 27, 349. AVRAMEAS S. & LESPINATs G. (1967) Detection d'anticorps dans des cellules immunocompetentes d'animaux immunises avec des enzymes. Compt. Rend. Acad. Sci. (Paris). 265, 302.
Specific and 'non-specific' antibody secretion BIELING R. (1919) Untersuchungen uber die veranderte Agglutininbildung, mit Ruhrbacillen vorbehandelter Kaninchen. Ztschr. Immun. 28, 246. BOYD W.C. & BERNARD H. (1937) Quantitative changes in antibodies and globulin fractions in sera ofrabbits injected with several antigens. J. Immunol. 33, 111. CUNNINGHAM A.J. & SZENBERG A. (1968) Further improvements in the plaque technique for detecting single antibody forming cells. Immunology, 14, 599. DE Vos CLOETENS C., MINsART-BALERIAUX V. & URBAIN VANSANTEN G. (1971) Possible relationships between antibody and non-specific immunoglobulin simultaneously induced after antigenic stimulation. Immunology, 20, 955. FAZEKAS DE ST. GROTH S. & WEBSTER R.G. (1966a) Disquisitions on original antigenic sin. l. Evidence in man. J. exp. Med. 124, 331. FAZEKAS DE ST. GROTH & WEBSTER R.G. (1966b) Disquisitions on original antigenic sin. 2. Proof in lower creatures. J. exp. Med. 124, 347. FRANCIS T., JR (1953) Influenza: The newe acquayantance. Ann. int. Med. 39, 203. FREUND J. (1953) The response of immunised animals to specific and non-specific stimuli. The Nature and Significance of the Antibody Response. (ed. by A.M. Pappenheimer, Jr), p. 46. Columbia University Press, New York. FROST H., BRAUN D.G., POSKITr D., CAHILL R.N.P. & TRNKA Z. (1976) Antipolysaccharide antibodies of restricted heterogeneity secreted by a single lymph node. J. exp. Med. 143, 707. GRONOWIcz E. & COUTINHO A. (1974) Selective triggering of B cell subpopulations by mitogens. Europ. J. Immunol. 4, 771. HALL J.G. & MORRIS B. (1962) The output of cells in lymph from the popliteal node of sheep Quart. J. exp. Physiol. 47, 360. HAY J.B. (1971) The role of fixed and migratory cells in immunological reactions. Ph.D. thesis. Australian National University. JARRET E.E.E. & STEWART D.C. (1972) Potentiation of rat
reagenic (IgE) antibody by helminth infection. Simultaneous potentiation of separate reagins. Immunology, 23, 749. LOOR F. (1971) On the existence of heterospecific antibodies in sera from rabbits immunised against tobacco mosaic virus determinants. Immunology, 21, 557. MELCHERS F. & ANDERSSON J. (1974) The kinetics of proliferation and maturation of mitogen activated bone marrow-derived lymphocytes. Europ. J. Immunol. 4, 687. MILLER H.R.P., AVRAMEAS S. & TERNYNCK T. (1973) Intracellular distribution of antibody in immunocytes
89
responding to primary challenge with horseradish peroxidase. Amer. J. Path. 71, 239. MILLER H.R.P., TERNYNCK T. & AVRAMEAS S. (1974) A comparison of specific antibody and immunoglobulin synthesising 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 formation in cells responding to horseradish peroxidase. J. Immunol. 114, 626. MOTICKA E.J. (1974) The non-specific stimulation of immunoglobulin secretion following specific stimulation of the immune system. Immunology, 27, 401. MOTICKA E.J. (1975) Cellular basis and nature of the polyclonal hyperimmunoglobulinemia induced by antigenic challenge. Cell. Immunol. 19, 32. NAKASHIMA I. & KATO N. (1974) Non-specific stimulation of immunoglobulin synthesis in mice by capsular polysaccharide of Klebsiella pneumoniae. Immunology, 27, 179. PACHMANN K., KILLANDER D. & WIGZELL H. (1974) Increase in intracellular immunoglobulins in the majority of splenic lymphoid cells after primary immunisation. Europ. J. Immunol. 4, 138. PAPPENHEIMER A.M., LUNDGREN H.P. & WILLIAMS J.W. (1940) Studies on the molecular weight of diphtheria toxin, antitoxin on their reaction product. J. exp. Med. 71, 247. POSKirr D.C. (1974) Studies on the regulation of the immune response. Ph.D. thesis, Australian National University. READ S.E. & BRAUN D.G. (1974) In vitro antibody response of primed rabbit peripheral blood lymphocytes to group A variant Streptococcal polysaccharide. Europ. J. Immunol. 4, 422. RITTENBERG M.B. & PRATT C. (1969) Anti-trinitrophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particulate immunogen. Proc. Soc. exp. Biol. (N. Y.), 132, 575. SHIMADA K. & SILVERSTEIN A.M. (1975) Induction of booster antibody formation without specific antigenic drive. Cell Immunol. 18, 484. SWEET G.H. & WELBORN F.L. (1971) Use of chromium chloride as the coupling agent in a modified plaqueassay, cells producing anti-protein antibody. J. Immunol. 106, 1407. URBAIN VANSANTEN G. (1970) Concomitant synthesis in separate cells, of non-reactive immunoglobulins and specific antibodies after immunization with tobacco mosaic virus. Immunology, 19, 783. WEIL E. & FELIX A. (1916) Ueber die Beziehungen der Gruber Widal'schen Reaktion zum Fleckfieber. Wien. K/in. Wschr. 29, 974.
The appearance of non-specific antibody-forming cells in the efferent lymph draining antigen-stimulated single lymph nodes
D. C. POSKITT, H. FROST, R. N. P. CAHILL & Z. TRNKA Basel Institute for Immunology, Grenzacherstrasse 487, Basel, Switzerland
Received 18 October 1976; acceptedfor publication 25 November 1976
Summary. Immunization of single lymph nodes with various antigens led to the appearance of cells in the efferent lymph that secreted antibody specific for the antigen which induced their formation and for a number of unrelated, non-crossreacting antigens. Immunization of single lymph nodes with mitogens led to the appearance of cells secreting antibodies specific for an even greater number of antigens, including one (TNP) that in all probability is not present in the animals' natural environment. When the node was primed with one antigen, a subsequent challenge with an unrelated antigen 12 weeks later led to the appearance of greater numbers of cells containing and secreting antibody against the previously experienced antigen, than was the case in unprimed lymph nodes. These findings indicate that the immune response to antigen provokes the maturation of lymphocytes of specificities unrelated to that of the injected immunogen. Such a mechanism may be important in maintaining immunological memory. Mitogens may directly activate lymphocytes into maturation and expression as antibody-secreting cells, whereas antigens appear to act indirectly. Correspondence: Dr D.C. Poskitt, Basel Institute for Immunology, Grenzacherstrasse 487, Postfach 4005, Basel 5, Switzerland. F
81
INTRODUCTION Many investigators have demonstrated that following the injection of an antigen, antibodies specific for the antigen and immunoglobulins without apparent specificity are synthesized during the course of the ensuing immune response (Boyd & Bernhard, 1937; Pappenheimer, Lundgren & Williams, 1940; Urbain Vansanten, 1970; De Vos Cloetens, Minsart-Baleriaux & Urbain Vansanten, 1971; Loor, 1971; Antoine & Avrameas, 1973; Miller, Avrameas & Ternynck, 1973; Miller, Ternynck & Avrameas, 1974; Moticka, 1974; Pachmann, Killander & Wigzell, 1974; Miller, Ternynck & Avrameas, 1975). Within this population of immunoglobulins, antibodies with specificities for previously experienced cross-reactive antigens have been detected; this led to the postulate of Original Antigenic Sin (Francis, 1953; Fazekas de St. Groth & Webster,
1966a, b). Further, antibodies have been demonstrated during the immune response which do not cross-react with the antigen which induced their formation (Freund, 1953; Jarret & Stewart, 1972; Nakashima & Kato, 1974; Moticka, 1975). The specificity of these antibodies appears to be directed against antigens previously experienced by the host (Weil & Felix, 1916; Bieling, 1919; Moticka, 1975; Shimada & Silverstein, 1975).
D. C. Poskitt et al.
82
We have investigated the synthesis and secretion of both specific antibody and 'non-specific' immunoglobulins by cells appearing in the efferent lymph during the course of immune responses in sheep popliteal lymph nodes. Using the in situ popliteal lymph node preparation originally described by Hall & Morris (1962) the immune response can be confined to the one lymph node and the temporal sequence of an immune response may be followed in the efferent lymph in the absence of any systemic immune reaction. Cellular and humoral events occurring within the lymph node are reflected in the efferent lymph (Frost, Braun, Poskitt, Cahill & Trnka, 1976). The appearance of cells containing and/or secreting specific and 'nonspecific' antibodies can be followed and their total numbers established at the end of the immune response. Previous reports on this subject have examined immune responses by measuring antibody titres or by looking for antibody and immunoglobulin synthesizing cells (Miller, Ternynck & Avrameas, 1974; 1975; Pachmann, Killander & Wigzell, 1974; Antoine & Avrameas 1976) in lymph nodes or spleen. Others have shown the development of 'non-specific' PFC in responses to mitogens in vitro (Andersson, Melchers, Galanos & Luderitz, 1973; Melchers & Andersson, 1974) and in vivo (Gronowicz & Coutinho, 1974). This report examines the appearance of non-specific antibody-forming and antibody-secreting cells in the efferent lymph draining antigen-stimulated single lymph nodes.
Lymph collection and cell counting Lymph was collected in sterile plastic bottles each containing 1 ml of a solution of sterile physiological saline containing 500 U-USP of Heparin (liquemin, Roche) and 500 iu each of penicillin and streptomycin (Flow Laboratories). All collections were made at room temperature. Cells were counted in a Model Fn Coulter counter equipped with a 100 u aperture, and expressed as cell output per hour. Antigens and mitogens All antigens and mitogens were suspended or dissolved in 3 ml of phosphate-buffered saline (PBS) prior to injection. Injections were given s.c. in the drainage area of the popliteal lymph node.
Red blood cells. Horse, rabbit and chicken red blood cells from randomly bred animals were used. The cells were washed thrice with PBS and a standard dose of 2 x 109 red cells was injected. Rabbit anti-SRBC antibody. Rabbit anti-SRBC antiserum No. B5221 /11 was kindly supplied by Dr A. Kelus of this Institute. Two millilitres of nonheat inactivated antiserum was added to 1 ml of packed and washed autologous SRBC and the suspension incubated for 1 h at 370 with constant stirring. The SRBC lysate was centrifuged and the membrane pellet washed three times with PBS prior to injection.
Allogeneic lymphocytes. Allogeneic lymphocytes
MATERIALS AND METHODS
were obtained from the efferent lymph of second party sheep. 3 x 108 allogeneic lymphocytes washed thrice in Eagle's (MEM) containing 10%4 of recipient lymph plasma were injected.
Animals Sheep of either sex, aged between 8 months and 2 years, were obtained from commercial flocks of 'black face' and 'white alpine' sheep. Experimental sheep were kept in metabolism cages and were given free access to hay, water and salt lick.
Bovine serum albumin. One millilitre of packed autologous SRBC coated with BSA according to the method of Sweet and Welborn (1971), to improve the immunogenicity of BSA, were injected.
Surgical procedures The lymphatic vessel efferent to the popliteal lymph node was cannulated according to the technique described by Hall & Morris (1962). Animals were allowed a 2-day post-operative recovery period prior to the injection of antigen or mitogen.
Influenza virus. Human influenza virus of the MEL strain (AO/Melbourne/1/35) was kindly supplied by Dr S. Fazekas de St. Groth of this Institute. The virus was injected at a dose of 160 pg of virus, equivalent to 4 x 1011 physical particles. The stock antigen consisted of a concentrate of purified, non-infective particles suspended in saline with 0 04% NaN3 as a preservative.
Specific and 'non-specific' antibody secretion
Streptococcal vaccine (Group A variant and Group C). Strain A486 variant, M negative and C74 vaccines originating from the collection of Dr R.C. Lancefield, The Rockefeller University, New York, were kindly supplied by Dr D.G. Braun of this Institute. One hundred microlitres of vaccine, each containing 25 4ug of Group A variant or 25 ug of group C carbohydrate was injected. Horseradish peroxidase (HRP). Sheep were primed by injecting 10 mg of HRP (Sigma grade VI) emulsified in 1 ml of Freund's complete adjuvant. To induce secondary responses 2-5 mg of HRP in 2 5 ml of PBS was injected 4 weeks after priming. Pokeweed mitogen (PWM). Two hundred micrograms PWM (Grand Island Biological Co.) was injected. Concanavalin A (Con A). Five hundred micrograms Con A (Miles) three times recrystallized, was injected.
Lipopolysaccharide (LPS). Five hundred micrograms LPS B (S. minnesota, Difco) was injected. Purified protein derivitive (PPD). 100 pug of PPD (lot 280 Weybridge) was injected s.c. in 1 ml of saline in animals which had been primed 1-2 months previously with two to three times the human dose of BCG (Berna, Berne). The detection of intracellular antibody to horseradish peroxidase Cells containing antibody specific for HRP were demonstrated by a histochemical technique which involved the reaction of the enzyme with its substrate in the presence of 3,3'-diaminobenzidine (DAB) or carbazole-H202 reagent. The methods of Avrameas & Lespinats (1967), and Avrameas (1970) were used. The detection of immunoglobulin-containing cells in HRP responses Cells containing immunoglobulins were detected using a 1 in 10 dilution of rabbit anti-sheep immunoglobulin antiserum (conjugated to HRP). Cells containing a reddish-brown precipitate after staining with carbazole-H202 reagent were assessed as positive cells containing immunoglobulin.
Cells incubated with normal rabbit immunoglobulin
83
conjugated to HRP and stained with carbazole-H202 reagent were used as controls. The number of immunoglobulin-containing cells was assessed by subtracting the number of positively stained cells observed in the controls from those observed in cell smears incubated with rabbit antisheep immunoglobulin. The number of 'non-specific' immunoglobulincontaining cells was calculated by subtracting the number of antibody-containing cells from the number of immunoglobulin-containing cells. Preparation of target cells for the plaque-forming cell assay CRBC, HRBC, RRBC, SRBC and coated SRBC were washed three times in Eagle's MEM. One millilitre of packed red cells was resuspended in Eagle's MEM to a total volume of 6 ml for use in the assay system. Washed SRBC were coated with BSA according to the method of Sweet & Welborn (1971). The method used to coat SRBC with TNP was based on that first described by Rittenberg & Pratt (1969), 2 ml of packed SRBC were suspended in 20 ml of cacodylate buffer (pH 6 9) containing 4 mg/ml TNP. The O-stearoyl-polysaccharide of Streptococcus group A variant and Streptococcus group C cell walls were used to coat SRBC according to the method of Read & Braun (1974). PFC assay Efferent lymph cells were washed and resuspended to a concentration of 106 or 107 cells/ml in Eagle's MEM. Packed uncoated or coated red cells were suspended in Eagle's MEM (1:5 v/v). Rabbit serum absorbed with the target erythrocytes was used as a source of complement. 0-5 ml of the lymphoid cell suspension was mixed with 0-1 ml of the target red cell suspension and 0 05 ml rabbit serum. The reaction mixture was thoroughly mixed and 0 05 ml immediately transferred to Cunningham slide chambers (Cunningham & Szenberg, 1968). The slide chambers were sealed with Vaseline and incubated at 370 for 30 min. Plaques were counted at forty-fold magnification. The PFC output per hour was recorded and the total number of plaques that appeared in the lymph during the course of an immune response was also calculated. Only direct PFC assays were performed. The total blast cell number represents all blast cells collected in the efferent lymph from the time that antigen was injected until PFC were no longer detectable.
D. C. Poskitt et
84
RESULTS Antibody specificity of PFC following antigenic stimulation Primary immune responses to CRBC and secondary responses to HRP were studied to determine the relative numbers of non-specific immunoglobulincontaining cells, specific antibody-containing cells and antibody-secreting cells (PFC) that appear in the efferent lymph during the course of an immune response.
In the response to 2 x 109 CRBC a total of 2-1 x lO0 cells appeared in the efferent lymph over a period of 220 hrs; 2 3 x 109 were assessed as blast cells. Of the 2 1 x 1010 cells collected, 11 7% contained immunoglobulins which had no detectable specificity for the inducing antigen, 0O9% contained specific antibody and only 0 3% secreted antibody specific for CRBC. During the course of the secondary response to HRP a total of 2-75 x 1010 cells appeared in the efferent lymph over a period of 150 h; 7-17 x 109 cells were assessed as blast cells. Of the 2 75 x 1010 cells collected, 7 % contained immunoglobulins which had no detectable specificity against the inducing antigen whilst 1133% contained antibody specific for HRP. These results indicate that many of the lymphocytes and blast cells which appear in the efferent lymph during the course of an immune response to antigen, contain immunoglobulins unable to react with the specific antigen. Furthermore, of those cells containing specific antibody only 30% were PFC. Thus, in the following experiments where the cells
al.
that appeared in the efferent lymph of popliteal lymph nodes responding to a primary antigenic stimulus were of necessity assayed for their ability to secrete antibodies against the priming antigen as well as against a panel of antigens with which the animal had no known previous experience, the PFC assay will considerably underestimate the number of cells containing specific or non-specific antibodies (Table 1). It is emphasized that cells from the efferent lymph of nonstimulated popliteal lymph nodes gave no PFC against any of the antigens tested. The primary response to allogeneic lymphocytes, xenogeneic erythrocytes, proteins, bacterial and viral antigens gave rise to cells secreting specific antibody and antibodies with specificities for some or all of the antigens selected in our panel. In certain cases cross-reactivity could account for some of the apparent non-specificity of the response, for example the Strep.Av carbohydrate and Strep. group C carbohydrate are cross-reactive. It is unlikely, however, that the whole panel of antigens chosen to screen immune responses for nonspecific components, was cross-reactive with the immunizing antigens.
Fig. la shows the changes that occurred within the efferent lymph following the injection of 3 x 108 allogeneic lymphocytes. The response was vigorous in terms of total and blast cell output, yet only a relatively small number of cells secreting antibody against chicken and horse red blood cells were detected, and PFC against the other panel antigens were not found (Table 1). The response to CRBC (Fig. lb) gave rise to cells secreting antibodies against the specific antigen and four other antigens
Table 1. Primary immune response to antigen
Antigen HRBC (2x 109) RRBC (2 x 109) CRBC (2x 109) Strep.Av. vaccine (100).) Strep.C. vaccine (1002.) BSA Influenza virus (160,ug)
Allogeneic lymphocytes (3 x 108) Rabbit anti-SRBC Ig
Non-specific PFC x 10-6 Total blast Specific PFC x 10-6 HRBC RRBC CRBC Strep.Av Strep.C cells x 10-9 12-3 2-8 56 2-4 1-2 2-1 3-4 24 104
305 1-2 46-8 13-7 1.1 0 -
0 09 1-49 0-02 011 0 93 042
-
0d13 7 91
-
0
0-25 0-04
0 0 0 0 001 0 0
-
0 07 0-02 0-01
0 05 0-02 0 3-27 04 0-23
-
0-08 0 03 0-16 7-28
0 35 0 003 0 0
0.1 2-27 0 0
-
BSA
TNP
0 0 0 005 0 0
0 0 0 0 0 0 001 0 0
-
001 0 0
The total number of blast cells and PFC that appeared in the efferent lymph of popliteal lymph nodes stimulated with antigens.
Specific and 'non-specific' antibody secretion (a) 6 So
=D
4 r
f
w
*
2
I 4 3 2
, It CL
Q
0
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0 24 48 72 96 120 144168 192 216 240 Hours after s.c. injection of allogeneic lymphocytes (b)
_0. 0
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0,/
0
0,
0 x
IF 10A 8 6 4 2
j
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24 48 72 96 120144168 192 216 240264 Hours after s c. injection of CRBC (c)
0
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-
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)?'
8 620 4 2-
24 48 72 96 120 144168192218240 Hours after sc. injection of Streptococcus group C vaccine Figure 1. Primary immune response to (a) 3 x 108 0
allogeneic lymphocytes; (b) 2x 109 CRBC; (c) l00A Strep.C vaccine. The upper section of each figure shows changes in cell output in the lymph efferent to the popliteal node. The lower sections of (a) and (c) show the appearance of specific and non-specific PFC in the efferent lymph. The centre
85
from the panel (Table 1). The vigour of the immune response in terms of the increase in cell output did not appear to be directly related to the ability of an antigen to trigger cells secreting 'non-specific' antibodies. The cellular response to Strep.C vaccine (Fig. Ic) was feeble in terms of cell output and yet gave rise to sub-populations of cells secreting antibodies against three out of the six 'non-specific' antigens (Table 1). The number of cells which secrete antibody against any of the panel antigens is very small when compared to the number of cells secreting antibody against the priming antigen (Table 1). However, a more extensive panel of antigens might reveal cells secreting antibodies against many other antigens. Nearly 100% of specific and 'non-specific' antibodysecreting cells were blast cells. Thus, it is conceivable that the total number of cells containing and/or secreting both specific and 'non-specific' antibodies might exceed the total number of blast cells which appear in the efferent lymph. This is indeed suggested by the results obtained in the secondary response to horseradish peroxidase where virtually all the blast cells contained immunoglobulin and where 7 % of all cells contained 'non-specific' immunoglobulin and 11-3 % contained specific antibody.
Antibody specificity of PFC and AFC following antigenic stimulation of lymph nodes previously primed with an unrelated antigen The cells producing 'non-specific' antibodies might be memory cells or at least the progeny of cells which have been clonally expanded during some previous encounter with their specific antigen. In the next series of experiments the influence of previous antigenic experience on the number of non-specifically evoked cells was explored. Sheep were immunized with one antigen 12 weeks prior to immunizing with a second unrelated antigen in order to determine whether the response to the first antigen could be recalled. In all cases, PFC specific for the first antigen were no longer detectable in the efferent lymph 12 weeks after the section of (b) shows the appearance of non-specific PFC and the lower section shows the appearance of specific PFC in the efferent lymph. Total lymphocytes, (--*); blast cells, (0-0); HRBC/PFC, (A-A); CRBC/PFC (A-A); Strep.Av/PFC, (0-n); Strep.C/PFC, (O-O). In the response to 2x 109 CRBC (b), PFC specific for BSA and Strep.C also appeared in the efferent lymph but are omitted from this figure for simplicity (see Table 1).
D. C. Poskitt et aJ.
86
Table 2. The induction of anamnestic responses by unrelated immunogenic stimuli
Antigen 1
PFCx 10-6 Total blast cells x 10-9 HRBC RRBC CRBC Strep.Av Strep.C BSA TNP
Antigen 2
Strep.C vaccine (100).)
Allogeneic lymphocytes (3 x 108) Allogeneic lymphocytes (3 x 108) HRBC (2x 109)
CRBC (2 x 109) Strep.C -vaccine (100).)
3-4
0-02
0
0 09
0 05
0 07
0
0
4-6
0-03
0
0-87
0
0
0
0
59
73-7
0
0 37
0-23
0-17
0
0
The first antigen was injected into the drainage area of a popliteal lymph node 12 weeks prior to the second antigen. The total number of blast cells and PFC that appeared in the efferent lymph during the course of the immune response to the second antigen are shown. (a)
,0
2 0-0
10, 0-0
10.0,0
0-0-0-0/
o
0-,O-m-e-t74rL-
4
V~~~~~~~~~~
L-
-r
-yr
A
2
HI 24 48 72 96 120 144168 192 216 240 Hours after s.c. injection of allogeneic lymphocytes (b)
0
g
r
1'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0
L-
co
3 2 It -
.
.
I. .
./
A
0 24 48 72 96 120144168 192216 240 Hours after s c injection of allogeneic lymphocytes 8 6 4 2
initial immunization. The results of these experiments are summarized in Table 2. The number of cells secreting antibody against the first antigen was always higher than that of animals which had no experience of the first antigen (Tables 1 and 2). In one animal in which the popliteal lymph node had been immunized with Strep.C vaccine 12 weeks prior to challenge with allogeneic lymphocytes (Fig. 2a), PFC against Strep.Av and Strep.C carbohydrates were found whereas the contralateral lymph node which had not been immunized with Strep.C vaccine did not give rise to PFC against these antigens upon challenge with allogeneic lymphocytes. In a further experiment the number of PFC against CRBC was increased when the animal had experienced CRBC 12 weeks prior to challenge with allogeneic lymphocytes (Fig. 2b and Table 2).
(c)
/1
Figure 2. The induction of anamnestic responses by unrelated immunogenic stimuli. (a) Primary immune response to 3 x 108 allogeneic lymphocytes in a popliteal lymph node that had been primed with Strep. group C vaccine 12 weeks previously. (b) Primary immune response to 3 x 108 allogeneic lymphocytes in a popliteal lymph node that had been primed with 2x I09 CRBC 12 weeks previously. (c) Primary immune response to 2 x 109 CRBC in a popliteal lymph node that had been primed with 10 mg of HRP in Freund's complete adjuvant 12 weeks previously. The upper section of each figure shows changes in cell output in the lymph efferent to the popliteal node. The lower section of (a) and (b) and the centre section of (c) shows the appearance of PFC in the efferent lymph. The lower section of (C) shows the appearance of cells containing antibody specific for HRP. Total lymphocytes, (--*); blast cells, (o-a); HRBC/PFC, (A-A); CRBC/ PFC, (A-A); Strep.Av/PFC, (L-LI); Strep.C/PFC,
(0-c); HRP/AFC, (*-*). 0
24 48 72 96 120 144
Hours after
sc
injection of CRBC
During the course of the response to 3 x 108 allogeneic lymphocytes (a) PFC specific for HRBC also appeared in the efferent lymph but are omitted from the figure for simplicity (see Table 2).
Specific and 'non-specific' antibody secretion 6 F (a) 5 4
x _r
I
A3
./,-*
-
Similar results were obtained when Strep.C vaccine injected 12 weeks prior to HRBC. Whilst these experiments demonstrate an increase in the number of cells secreting antibody against a previously encountered and non-crossreactive antigen, they give no information as to the number of cells containing antibody specific for that antigen. To determine if there is an increase in the number of AFC we injected 2 x 109 CRBC into the drainage area of a popliteal lymph node that had been primed 12 weeks previously with HRP in Freund's complete adjuvant. Fig. 2c shows the results of this experiment. There was an increase in HRP specific AFC with a maximum output in the lymph just prior to the maximum output of PFC specific for the second antigen, CRBC.
-\/
was
/0
0-
/X
1
=2 (I
0
8 7 x
6
6
& 4 0
3
'tL O2 0
3
24 48 72 96 120144168 Hours after s.c. injection of LPS r (b) e.
/ 0"0
Antibody specificity of PFC following stimulation with mitogens Mitogens such as LPS (Andersson et al., 1973) or PPD (Melchers et al., 1974) induce in vitro the maturation of B lymphocytes to PFC secreting 'polyclonal immunoglobulins'. Therefore, mitogens, by virtue of their reported non-specific action upon lymphocytes, should give rise to cells secreting antibodies with specificities directed against a large number of antigens. The selectivity of mitogens for T and B cells well documented for other species has not been extensively examined in sheep. Investigations in this laboratory (manuscript in preparation) indicate that PWM and PPD activate both T and B cells whilst LPS activates only B cells and Con A only T cells. Table 3 and Fig. 3a and b show the numbers and the appearance of 'non-specific' PFC after stimulation with Con A, PWM, PPD and LPS. It can be seen that the spectrum of 'non-specific' PFC is
.\/x-
o
\
5
BA
4
3
0r OL
3 2 l
87
1
24 48 72 96 120 144 168 Hours after sc injection of PWM Figure 3. Responses to mitogens. The response of the popliteal lymph node to (a) LPS and (b) PWM. 0
The upper section of each figure shows the changes in cell output in the lymph efferent to the popliteal node. The
lower section shows the appearance of 'non-specific' PFC in the efferent lymph. Total cells, (0-0); blast cells, (0-a); HRBC/PFC, (A-A); CRBC/PFC, (A-A); Strep.Av/PFC, (0-E); TNP/PFC, (-). PFC with specificity for Strep.Av, Strep.C and LPS (A) and Strep.C (B) also appeared in the efferent lymph but are omitted for simplicity (see Table 3).
Table 3. Responses to mitogens
PFCx 10-6
Mitogen Con A (500 /ug) PPD (100 pg) PWM (100 jug) LPS (500jug)
Total blast cells x 10- 9 97 12-8 4-4 11-2
HRBC
RRBC
CRBC
Strep.Av
Strep.C
BSA
TNP
0-16 7-36 0 73 2-38
0 0-07 0 0
0 19 4-85
0 002 0-47 0-07 0-32
0-002 0 34 005 0-58
0 0-21 0 0
0 008 0-02 0 3-28
1P77 1-22
The total number of blast cells and PFC that appeared in the efferent lymph of popliteal lymph nodes stimulated with mitogens.
D. C. Poskitt et al.
88
broader after mitogenic stimulation, with the exception of influenza virus and that the 'nonspecific' PFC appear in the efferent lymph at a much earlier time than in the response to antigens. DISCUSSION When lymph nodes were stimulated with antigens or mitogens, cells appeared in the efferent lymph that synthesized and secreted antibodies specific for the immunizing antigen and a number of non-crossreacting antigens. Lymphocytes from the efferent lymph of non-stimulated lymph nodes never gave 'background' PFC against any of the panel antigens. This corresponds to our previous findings (Poskitt, 1974) and also those of Hay (1970). Lymphocytes can enter lymph nodes by two routes, from the blood via the post-capillary venules and from afferent lymphatics which they enter from the tissue spaces after leaving the blood stream via peripheral arteriovenous loops. The popliteal lymph node is the most distal node in a chain of lymph nodes leading to the thoracic duct and as such receives no lymph or cellular components from other lymph nodes. In response to antigens and mitogens, lymphocytes are recruited from the blood into the lymph node, the mechanism that controls and regulates this migration are unknown. During this recruitment phase antibody-secreting cell precursors are recruited into the node. In our experiments, 'non-specific' PFC in antigen responses appeared later than they did in mitogen responses and were restricted to the period in which specific PFC were present. The time of appearance of 'non-specific' PFC after mitogenic stimulation in our experiments is similar to that reported by Gronowicz & Coutinho (1974). Thus, the 'nonspecific' PFC that emerge in the efferent lymph of mitogen-stimulated lymph nodes appear to be the result of direct activation of antibody-secreting cell precursors by mitogen. The kinetics of the appearance of 'non-specific' PFC after stimulation with antigen suggest that antigens might act indirectly by way of specific cell interactions giving rise to factors which non-specifically activate bystander B lymphocytes which have no specificity for the antigen. Such factors may act within the lymph node to activate preferentially the specific antibody-secreting cell precursors in the presence of the specific antigen and to a more limited extent, bystander antibody-
secreting cell precursors of other specificities in the absence of their specific antigen. Another characteristic of the 'non-specific' PFC clones was their limited size which was several orders lower than that of the specific PFC clone. The numbers were higher in the efferent lymph from nodes with prior experience of the test antigen suggesting that memory cells might be the candidates for cells synthesizing and secreting 'non-specific' antibodies although no distinction can be made between residential and recirculating memory. This question is being further examined. The results of these studies indicate that the microenvironment within a lymph node responding to antigen induces not only the maturation of specific antigen-reactive lymphocytes but also the maturation of lymphocytes of specificities unrelated to that of the injected antigen. Such a mechanism may aid the maintenance of immunological memory by the non-specific activation of memory cells, each immune response boosting the pool of memory cells preferentially to the inducing and marginally to unrelated and non-cross-reacting antigens. If this is the case, then areas of lymphoid tissue which are continually exposed to antigenic insult, such as the gut, maybe important in maintaining immunological memory.
ACKNOWLEDGMENTS The authors would like to thank Mr H. J. Hoch for expert technical assistance. REFEREN CES ANDERSSON J., MELCHERS F., GALANOS C. & LUDERITZ 0. (1973) The mitogenic effect of lipopolysaccharide on bone marrow derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. exp. Med. 137, 943. ANTOINE J.C. & AVRAMEAS S. (1973) Etude de la formation des anticorps anti-tryosinase au cours de la reponse immunitaire chez le lapin. Ann. Immunol. (Inst. Pasteur), 124C, 121. 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. AVRAMEAS S. (1970) Immunoenzyme techniques: Enzymes as markers for the localisation of antigens and antibodies Int. rev. Cytol. 27, 349. AVRAMEAS S. & LESPINATs G. (1967) Detection d'anticorps dans des cellules immunocompetentes d'animaux immunises avec des enzymes. Compt. Rend. Acad. Sci. (Paris). 265, 302.
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