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Sera from lipopolysaccharide (LPS)-injected mice exhibit complement-dependent cytotoxicity against syngeneic and autologous spleen cells
CELLULAR
IMMUNOLOGY
32, 252-262
(1977)
Sera from Lipopolysaccharide (LPS)-fnjected Mice Exhibit Complement-Dependent Cytotoxicity against Syngeneic and Autologous Spleen Cells1 DANIELE Division
PRIMI, C. I. EDVARD SMITH, LENNART HAMMARSTR~M, GGRAN MILLER
of Immunobiology,
Karolinska Ins&&t, Wallenberglaboratoriet, 104 05 Stockholm 50, Sweden Received
September
Lilla
AND Frcskati,
24,1976
To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearanec of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.
INTRODUCTION The ability of distinguish self from nonself is a fundamental property of the immune system. The mechanism by which the self-nonself discrimination is carried out at the cellular level is not resolved as yet, but two basically different hypotheses have been advanced. In terms of the two-signal concept of lymphocyte activation (1)) each responding lymphocyte can distinguish tolerogenic (signal 1) from activating signals (signals 1 and 2). The tolerogenic signal 1 is assumed to be mediated by the interaction between antigen and immunoglobulin receptors, whereas signal 2 results from “associative recognition” of the antigen-Ig receptor complex by, e.g., antibody directed against the antigen, antigen-specific thymus-derived (T) cells, or nonspecific products either released by T cells or intrinsic to certain types of antigens (thymus-independent, TI). The basic postulate in this hypothesis is that tolerance occurs when antigens interact with Ig receptors, whereas activation requires this 1 This work was supported by grants from the European Molecular Biology Organization, the Ollie and Elof Ericsson Foundation, the Ahlen Fundation, and the Swedish Cancer Society. 252 1977by AcademicPress, in any form reserved. 9 reproduction Inc.
ISSN 0008-8749
INDUCTION
OF CYTOTOXIC
FACTORS
BY
LPS
253
interaction plus a second signal of the type outlined. In this way, immunological tolerance is the most easy event to achieve and, thus, the activation of autoreactive cells is prevented. According to a different view (2)) self-nonself discrimination is carried out at the T-cell level by so far unknown mechanisms, whereas B cells can never be tolerized to thymus-dependent antigens, including autoantigens. This “one non-specific signal” concept postulates that immunocyte triggering is caused by one signal not delivered by the antigen-Ig receptor interaction, but rather by nonclonally distributed receptors on B cells for triggering signals intrinsic to the TI antigens produced by helper T cells (2). Previous studies on experimentally induced tolerance have not resolved this discrepancy, since it has not been possible to distinguish critically between the presence of nonactivated cells and the absence of immunocompetent cells. However, by using polyclonal B-cell activators (PBAs) such studies are now possible. PBAs have the ability to activate directly antibody synthesis in resting B cells by interacting with receptors which are not the immunoglobulin receptors. It follows that even B cells with blocked Ig receptors can be activated by PBA. By use of these PBAs it could be shown that tolerance to a hapten (3, 4) or a hapten-protein conjugate (5) does not affect the B cells, which would be successfully activated to antibody synthesis against the tolerogen by lipopolysaccharide from gram-negative bacteria, a potent PBA. Since PBAs are competent to activate B cells of any immunological specificity and, thus, to reveal the total V-gene repertoire of the B cells, it should be possible to investigate whether competent B cells against autoantigens exist in the organism. Two such studies have been carried out. In one it was found that injection of LPS in mice resulted in production of antibodies against DNA (6), and, in the second, stimulation of bovine spleen cells by LPS resulted in the production of antibodies to autologous red cells, as measured in the PFC assay using autologous erythrocytes as target cells (7). To investigate this phenomenon further, we have injected LPS, at optimal doses for induction of polyclonal responses, into mice and have studied the cytotoxic activity of the sera from these animals in the presence of complement using autologous and syngeneic spleen lymphocytes as target cells. We regularly found that sera from LPS-injected mice exhibited complement dependent cytotoxicity against syngeneic target cells. The possible causes of such an effect and the implications of our findings for an understanding of self-nonself ,discrimination are discussed. MATERIALS
AND
METHODS
C3HJTif, C3H/HeJ, BlO SM, A/Sri, and A x Animals and immunizations. C57Bl/lO, matched for age and sex, were used throughout these experiments. The mice were injected iv and ip with varying doses of LPS in a volume not exceeding 0.1 ml in BSS. Mitogens. Lipopolysaccharide (LPS) from Escherichia co& 055: B5 was obtained by phenol-water extraction according to the method of Westphal et al. (8) and was provided by Dr. Holme (Department of Bacteriology, Karolinska Institutet, Stockholm).
254
PRIM1
ET
AL.
Sera. Mice were bled from the retro-orbital plexus at various times after LPS injection. The sera were separated 1 hr later by centrifugation and were stored in aliquots at - ZO”C, after inactivation at 56°C for 30 min. Reconstitution of irradiated a&n&. C3H/Tif mice were irradiated with 750 R and were left for 4 hr. The animals were injected with 100 IU of heparin reconstituted with 30 X 10’ B10.5M spleen cells. The cells were injected iv with or without 100 pg of LPS in a total volume of 0.3 ml of BSS. The mice were protected by Tetracycline (Dumocylin), 50 &ml, in the drinking water. Target cells. Spleen cell suspensions from mice were obtained by pressing the spleen through stainless steel screens. The resultant suspension was pipetted vigorously, and the remaining cellular aggregates were allowed to settle for 5 min. The cells remaining in the supernatant were washed twice in BSS before labeling with Wr. In some experiments the red blood cells were separataed from the lymphocytes by centrifuging the cells in a gradient consisting of 10, 29, and 30% bovine serum albumin (BSA) fractions. Labeling with 51Cr. One-tenth milliliter of sodium chromate (specific activity, 10 mCi/ml) was added to lo* packed spleen cells in 0.25 ml of RPM1 medium supplemented with penicillin, streptomycin (500 III/ml), L-glutamine (200 mM) (Flow Laboratories, Irvine, Scotland), and 10% fetal calf serum. The cells were suspended and incubated for 75 min at 37°C in a COz incubator (10% COz). The cells were then washed twice in BSS and were resuspended at a concentration of 5 x 10E/ml in RPM1 medium. Complement. Rabbit serum, never older than 1 week, was used as a source of complement (usually at a l/5 dilution in BSS) following absorption according to the method of Boyse et aE. (9) using a 30% suspension of thymus and spleen cells. Assay for cytotoxicity. Fifty microliters of a cell suspension, containing 5 x 1W labeled target cells, was pipetted into hemolysis tubes with 50 ~1 of serum from LPS-injected or control mice. The controls consisted of target cells with autologous serum from noninjected mice and target cells with medium alone. To measure the total releasable isotope, target cells were incubated with sodium dodecyl sulfate (SDS). The mixture was incubated for 1 hr at 37°C in a COz incubator. Following two washings in BSS, the cells were resuspended in 50 ~1 of rabbit complement (RC’) and were incubated for another hour at 37°C.. All assays were done in triplicate. After incubation, the tubes were centrifugated, and the supernatant and pellet were counted in a gamma counter. The percentage of Yr release was calculated as follows : Counts per minute in suaernatant - background (counts per minute in pellet-counts per minute in supernatant) -2 (background)’ Specific lysis was calculated with the following formula : percentage of lysis in experimental - percentage of lysis in control percentage of maximum lysis - percentage of lysis in control . Preparation of anti-mouse Ig colwnns. One milliliter of rabbit anti-mouse Ig serum (Behringwerke AG, Germany) was diluted in 10 ml of 0.1 M sodium tetraborate and was added to 1 g of previously activated Hidrazide Bio-Gel P2 beads (BioRad, Richmond, Germany). The mixture was stirred for 5 hr. After one more wash-
13.33 11.76
25.67 f3.24
26.33 f2.60
18.67 zkl.49
22.00 *2.05
8.5OC f0.50
14.00s zh2.52
10.67s ~1~0.88
11.67s f1.03
18.10 lt2.91
A/Sri
BIOSM
A x Bl0.5M
A X C57Bl/lO
C3H/Tif
16.40s f3.06
12.10s zko.90
12.33 Al.33
13.00s f2.00
15.00s zko.00
N
l/4
29.10 Al.86
23.79 fl.75
21.00 zlz3.79
30.33 fl.76
l/6
29.80 f2.12
19.23 fl.33
21.33 f3.38
31.33 fl.20
15.50 10.50
LPS
Dilution
18.20s ho.46
11.97s ho.61
11.50s tto.50
12.00s &OS8
11.67s zkO.67
N
of serum
release (%)
l/8
31.80 zk0.76
20.40 zk2.60
22.67 zk3.84
28.00 ~3.06
19.67 f2.66
LPS
of sera from LPS-Injected
P < 0.05 by the Student’s t test.
19.00s f3.06
13.07s ~1~1.29
10.67s f0.88
13.00s zk3.00
12.00 lt1.15
N
1 Dilutions
Isotope
TABLE Cells at Various
12.00 fO.OO
LPS
Release from Target
a The sera were obtained at Day 3 after injection of 100 pg of LPS. b N, untreated ; LPS, LPS-injected. c Mean f SEM of triplicate experiments; S = significant at 0.01 <
LPS
of Isotope
Nb
Mouse strain
Percentage
19.60s f0.21
12.90s &I.50
13.50 f0.50
11.67s zkl.33
11.67s f0.33
N
l/IO
Mice*
31.50 f2.15
25.95 13.15
16.50 fl.50
24.67 ~0.67
18.00 fl.OO
LPS
and Untreated
20.00 f1.86
15.65 ~1~3.85
10.50 f2.50
12.21 f0.68
14.33s fl.33
N
l/l2
24.60 h2.36
26.20 f0.20
16.67 f2.96
12.00 zk2.52
22.33 f0.88
LPS
?v)
2 2 i3 cn z
z
X
rE ;z 3
8
E z 2 u g
2.56
PRIM1
ET
AL.
ing in sodium tetraboratae, a solution made up of 1 ml of 2 M ammonium chloride and 0.5 ml of 1 M ammonium hydroxide was added to the pellet and left overnight. The anti-mouse Ig-coupled beads were packed in short columns and were washed free of ZSO-nm-absorbing material by eluting several volumes of saline. The sample was eluted with the same solution. The eluted material was concentrated at the initial volume by ultrafiltration using a 10 PM Difco ultrafiltration membrane (Amicon Corp., USA). RESULTS Sera from LPS-Injected
Mice are Cytotoxic for Syngeneic Spleen Cells
Sera from different strains of mice injected iv with 100 pg of LPS were tested 3 days after injection for complement-dependent cytotoxic activity against syngeneic spleen cells. Table 1 shows that these sera caused lysis of autologous target cells. This property was found to be complement dependent, and the strength of cytotoxicity varied with the serum dilutions used. Thus, lysis of target cells was not observed when the complement used in the assay had been inactivated or when the sera were added at low concentrations. There was also a prozone effect with concentrated sera, and the optimal serum concentration varied from strain to strain. Cytotoxicity
Can Be Elivninated by Absorption
with Target Cells
In order to investigate whether the cytotoxic effect of sera could be removed by absorption with the target cells, we used C3H/Tif mice, a high responder strain to LPS, and collected sera 3 days after injection of 100 pg of LPS. The sera were
30 25
1
0
l/2
l/4
l/6
118
l/10
l/l2
SERA DILUTIONS
FIG. 1. Complement-dependent cytotoxic response of C3h/Tif spleen cells after incubation for 1 hr at 37°C with serum from syngeneic mice obtained 3 days after injection of 0.2 ml of BSS (a) or 100 pg of LPS ( n ). The serum from the LPS injected mice was absorbedfor 30 minutes at 4°C. with l/3 volume of target cells (@). Student’s t test carried out between the groups showed that, for certain dilutions, the serum from the LPS-injected mice was significantly more cytotoxic than the control and the absorbed serum (0.01 < I’ < 0.05).
INDUCTION
OF CYTOTOXIC
l/6
l/4
l/2
FACTORS
l/8
BY
l/10
LPS
257
l/12
SERA DILUTIONS
FIG. 2. Complement-dependent cytotoxic response of CSH/Tif spleen cells after incubation with autologous serum treated for 1 hr with 100 fig of LPS (!I!) or with serum from syngeneic mice obtained 3 days after injection of 100 pg of LPS ( n ). Student’s t test showed that the latter was significantly more cytotoxic at certain dilutions (0.001 < P < 0.005).
tested against autologous spleen cells after they had been absorbed for 30 min at 4°C with a l/3 volume of packed syngeneic spleen and thymus cells. As shown in Fig. 1, absorption significantly decreased the cytotoxicity of the serum. Although this finding could be interpreted as a removal of specific antibodies, it is possible that LPS itself was responsible for the effect. Thus, LPS bound to the target cells could fix complement via the complement-bypass mechanism and thereby cause target cell lysis.
l/2
l/4
1/6
l/IO
l/12
SERUM DILUTIONS
FIG. 3. Complement-dependent cytotoxicity of C3H/HeJ spleen cells tested against serum from syngeneic mice collected at different days after injection of 0.2 ml of BSS (IJ- - -0) or 200 pg of LPS (m-m). Student’s t test between the groups showed no significant difference.
2.58
PRIM1
ET
AL.
LPS Itself Is Not Responsible for Lysis To investigate this possibility, we incubated 1 ml of normal C3H/Tif serum with 100 e of LPS at room temperature for 1 hr. The mixture was tested in the cytotoxic assay. Figure 2 shows the result of this experiment and compares it to the response of serum from mice injected with the same amount of mitogen. Since the LPS serum mixture did not give any lysis, LPS can be excluded as being responsible for the effect. To show that the cytotoxic activity of sera from LPS-injected mice was due to the PBA property of LPS, we tested sera from LPS-injected C3H/HeJ mice, which are nonresponders to LPS, at different time periods after injection of 200 pg of the mitogen. The results clearly indicate (Fig. 3) that sera from LPS-injected C3H/HeJ mice do not have any lytic effect on the target cells. This confirms that the cytotoxic effect of C3H/Tif serum is due to the polyclonal B-cell activity of LPS and again excludes the possibility that cytotoxicity is due to any other property of LPS. Kinetics of the response The kinetics of the appearance of serum-mediated cytotoxicity after LPS injection was studied and was found to peak at Days 3-4 and, thereafter, rapidly decline (Fig. 4). This is the same kinetics as shown for the polyclonal response to LPS. To determine whether the disappearance of cytotoxicity after Day 5 could be due to absorption of immunoglobulins by self antigens in viva, C3H/Tif mice were irradiated and reconstituted with 3 x 10’ spleen cells from BlO. 5M spleen lymphocytes and, thereafter, were injected with 100 g of LPS. Sera were collected at different days after injection and were tested against BlO. 5M spleen cells. The results shown in Fig. 5 demonstrate that the kinetics of cytotoxicity was similar to that shown in Fig. 4. Thus, it appears unlikely that absorption played any role in 35 30 25
1
3
2
DAYS
AFTER
LPS
4
5
6
INJECTION
FIG. 4. Complement-dependent specific lysis of CSH/Tif spleen cells after incubation serum from syngeneic mice collected at different days after injection of 200 pg of LPS.
with
INDUCTION
OF
CYTOTOXIC
FACTORS
BY
LPS
259
25
1
2
DAYS AFTER
3 LPS
4
5
6
INJECTION
FIG. 5. Complement-dependent specific lysis of C3H/Tif spleen cells after incubation with serum obtained from irradiated syngeneic mice reconstituted with spleen cells from BlO. 5M mice and injected with 100 pg of LPS.
the disappearance of antibodies against self antigens that may be the cause of a such cytotoxic effect. Probable Immunoglobulin
Nature of the Cytotoxic Factors
CBA mice were injected with 100 lug of LPS, and sera were collected 4 days later and were pooled. Samples of 1 ml were passed through anti-mouse Ig or normal columns. The eluted sera were concentrated at the initial volume and were used in the complement-dependent cytotoxic assay diluted l/4. Figure 6A shows that serum from LPS mice completely lost its complementdependent cytotoxic activity when passed through the anti-Ig column. However, no change in lytic activity was detected when serum was eluted from an uncoupled
FIG. 6. CBA mice were injected with 100 yg of LPS. Sera were collected 4 days later, pooled, and tested for complement-dependent cytotoxicity ( IJJ) against syngeneic (A) and allogeneic (B) spleen lymphocytes. The sera were also passed through anti-mouse Ig (I) and normal (a) columns before testing.
260
PRIM1
ET
AL.
column. An identical effect was found when such sera were tested against spleen lymphocytes obtained from the allogeneic strain 5M. Therefore, it seems likely that the cytotoxic factors in such sera are immunoglobulins produced by the polyclonal activation of B cells. DISCUSSION Our results show that sera from LPS-injected mice have a cytotoxic activity against syngeneic cells. The variability between different experiments that was observed could be traced mainly to the source of complement. Thus, the complement activity of the rabbit sera declined with time of storage, starting after a few days. We have tried to avoid this problem by using complement from only one rabbit and by never storing sera for more than 1 week. The appearance of cytotoxic serum factor after LPS injection could be caused principally by two mechanisms : (i) A direct or indirect effect of LPS itself or (ii) the induction of antibodies competent to react with autologous and syngeneic target cells. The results presented above exclude the possibility that LPS itself induced cytotoxicity, since injection of LPS to the nonresponder strain C3H/HeJ did not induce complement-dependent cytotoxicity, nor did the admixture of LPS to normal serum cause any cytotoxicity in the presence of complement. The possibility that LPSbound complement and that the LPS-complement complex caused cytotoxicity is excluded by the previous findings that such complexes do not have any deleterious effect on lymphocytes kept in culture over a 2-day period (10). It is possible that LPS induced formation of antibodies to LPS and that the antibody-LPS complex, possibly after having fixed complement, bound to Fc or C’3 receptors and induced cytotoxicity. This possibility is unlikely for several reasons. LPS, at the polyclonal concentrations employed in the present experiments, is capable of activating antibody synthesis in all B cells except those having specificity for the inducer, e.g., these concentrations are tolerogenic for LPS itself ( 11). Furthermore, interactions between different ligands, such as LPS, PPD, dextran sulfate, hapten-protein complexes, and Fc or Fc and C’3 receptors have never resulted in cytotoxicity (12), even though high complement concentrations were employed. It should be pointed out that LPS is not unique in its ability to cause the appearance of cytotoxic serum factors. As will be shown elsewhere, PPD and dextran sulfate also have this ability. The only known common property among these substances is that they are polyclonal B-cell activators. In particular, dextran sulfate is well studied with regard to the condition leading to appearance of anti-dextran antibodies, induction of polyclonal antibody synthesis, and interaction with C’3 and Fc receptors. It has been clearly shown (13) that, at the concentration used for induction of cytotoxic serum factors against syngeneic spleen cells, there is complete tolerance to dextran itself and interaction between dextran sulfate and Fc or c’3 or both receptors together never results in cytotoxicity. Therefore, it is unlikely that the present experiments are caused by trivial explanations of this type. The second alternative listed above appears more likely, e.g., that cytotoxity was caused by antibodies directed against membrane antigens of autologous and syngeneic spleen cells. LPS is known to induce polyclonal antibody synthesis including antibodies against the tolerogen in experimentally induced tolerance (5). In addition, LPS has been shown to cause the appearance of anti-
INDUCTION
OF CYTOTOXIC
FACTORS
BY
LPS
261
body-producing cells against autologous red cells tested in the Jerne plaque assay (7). In the latter case it is unquestionable that the cytotoxic principle was complement-fixing antibody directed against autologous red blood cells. The fact that in our experiments cytotoxic factors were completely eliminated when sera were passed through anti-mouse Ig columns is further evidence of the immunoglobulin nature of such factors. The specificity of the antibodies induced has not been studied as yet. They could be directed against any number of cell membrane antigenic determinants, including those determined by MuLV such as gp 70. It is well known that there are a number of clones of B cells which carry activity against these viral components. These cells release immunoglobulins which can fix the complement and give target cells killing (14, 15). We do not have evidence to exclude this possibility. However it has been shown that polyclonally activated T cells do not react against autologous cells (16). It is, therefore, rather unlikely that B cells and not T cells would react against virus-induced antigens on the cell membrane, especially since B cells probably would need T-cell cooperation to be activated against these thymus-dependent antigens. Another argument against the virus-induced antigens as target for the cytotoxic antibodies comes from the recent finding that supernatants from LPS-cultured human tonsil cells have a similar complement-dependent cytotoxic activity to autologous targets (17). The important finding from an immunological point of view is that the LPS-reactive B-cell subpopulation contained cells with immunoglobulin receptors directed against self-antigens, and that these cells were not tolerant but could be activated by LPS to the synthesis of immunoglobulins with specificity for self. Therefore, our findings have important implications for the understanding of the triggering mechanism of B lymphocytes. The one nonspecific signal theory (18) assumes that only one nonspecific signal activates B lymphocytes, and this signal is delivered by non-clonally distributed receptors for polyclonal B-cell activators. Therefore, PBA stimulation activates B cells to produce antibodies of all Ig specifities. Normally, there is no production of autoantibodies because B cells cannot be activated by thymus-dependent antigens, since thymus-dependent antigens require cooperation between T and B cells for induction of B-cell immune responses. Since self-reactive T cells do not exist (17), the self-reactive B cells are normally kept in a resting state. The findings that LPS induced the formation of cytotoxic factors, probably immunoglobulins, capable of reacting with autologous and syngeneic cells, are in contrast to the two-signal hypothesis (1) stating that contact between antigen and immunoglobulin receptors leads to an irreversible state of immunological paralysis, while our results are in agreement with the above one nonspecific signal theory. It seems likely that activation of autoantibody synthesis could occur in infections, since most PBAs known are of bacterial or parasitic origin. A large number of parasites and bacteria have already been demonstrated to have PBA properties, such as M. Tuberculosis, M. Leprae, Malaria, Mycoplasma (19-21), etc., and it is also known that the diseases caused by these agents are accompanied by more or less generalized autoantibody synthesis. Selective autoimmunity, directed against a particular autoantigen probably requires a different explanation, since selectively itself renders it unlikely that PBAs are responsible for activation.
262
PRIM1
ET AL.
In conclusion, our findings support the concept that self-reactive B cells are normally present in untreated animals, and that these cells, as well as B cells of all other Ig specifities, can be activated by different PBAs into synthesis of their Ig specifities, thus excluding that contact between Ig receptors and autoantigens causes a tolerogenic signal. REFERENCES 1. 2. 3. 4. 5.
Bretscher, P. A., and Cohn, ‘M. S., Science 169, 1042, 1970. Coutinho, A., and Mijller, G., ScaBd. .I. Immunol. 4, 99, 1975. Aldo-Benson, M., and Borel, Y., J. Zmmzlnol. 112, 1793, 1974. Gronowicz, E., and Coutinho, A., Ew-. J. Immunol. 5,413, 1975. Mijller, G., Gronowicz, E., Persson, U., Coutinho, A., Moller, E., Hammarstrom, L., and Smith, C. I., J. Es-p. Med. 143, 1429, 1976. 6. Gilbert, J., Four&, P. H., Lambert, P. H., and Miescher, P. A. J. E., J. Exp. Med. 140, 1189, 1974. 7. Hammarstriim, L., Smith, E., Primi, D., and Moller, G., Nature (London) 263, 60, 1976. 8. Westphal, O., Luderitz, O., and Bister, Nafzlrforscher. Teil. B 7b, 148, 1952. 9. Boyse, E. A., Hubbard, L., Stockert, E., and Lamm, M. E., Transplantation 10,446, 1970. 10. Moller, G., SjBberg, O., and Andersson, J. Eur. J. Immunol. 2, 586, 1972. 11. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 4, 349, 1972. 12. Mijller, G., and Coutinho, A., J. Exp. Med. 141, 647, 1975. 13. Coutinho, A., and Moller, G., Advan. Immunol. 21, 114, 1975. 14. Ihle, J. N., Hanna, M. G., Roberson, L. E., and Kenney, F. T. J. Exp. Med. 136, 1568, 1974. 1.5. Ihle, J. N., Yurconic, M., Jr., and Hanna, M. G., Jr., J. Exp. Med. 138, 194, 1973. 16. Primi, D., Smith, C. I., Hammarstrom, L., Lundquist, P., and Moller, G., Clin. Exp. Zmmunol., in press, 1977. 17. Waterfield, J. D., Waterfield, E. M., and Moller, G., Cell. Immwaol. 17, 392, 1975. 18. Coutinho, A., and MGller, G., Stand. J. Zmmunol. 3, 133, 1974. 19. Sultzer, B. M., and Nilsson, B. S., Nature New Biol. 240, 198, 1972. 20. Greenwood, B. M., and Rosalind, M. V., Natwe (London) 257, 592, 1975. 21. Biberfeld, G., and Gronowicz, E., Nature (London) 261,238, 1976.
IMMUNOLOGY
32, 252-262
(1977)
Sera from Lipopolysaccharide (LPS)-fnjected Mice Exhibit Complement-Dependent Cytotoxicity against Syngeneic and Autologous Spleen Cells1 DANIELE Division
PRIMI, C. I. EDVARD SMITH, LENNART HAMMARSTR~M, GGRAN MILLER
of Immunobiology,
Karolinska Ins&&t, Wallenberglaboratoriet, 104 05 Stockholm 50, Sweden Received
September
Lilla
AND Frcskati,
24,1976
To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearanec of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.
INTRODUCTION The ability of distinguish self from nonself is a fundamental property of the immune system. The mechanism by which the self-nonself discrimination is carried out at the cellular level is not resolved as yet, but two basically different hypotheses have been advanced. In terms of the two-signal concept of lymphocyte activation (1)) each responding lymphocyte can distinguish tolerogenic (signal 1) from activating signals (signals 1 and 2). The tolerogenic signal 1 is assumed to be mediated by the interaction between antigen and immunoglobulin receptors, whereas signal 2 results from “associative recognition” of the antigen-Ig receptor complex by, e.g., antibody directed against the antigen, antigen-specific thymus-derived (T) cells, or nonspecific products either released by T cells or intrinsic to certain types of antigens (thymus-independent, TI). The basic postulate in this hypothesis is that tolerance occurs when antigens interact with Ig receptors, whereas activation requires this 1 This work was supported by grants from the European Molecular Biology Organization, the Ollie and Elof Ericsson Foundation, the Ahlen Fundation, and the Swedish Cancer Society. 252 1977by AcademicPress, in any form reserved. 9 reproduction Inc.
ISSN 0008-8749
INDUCTION
OF CYTOTOXIC
FACTORS
BY
LPS
253
interaction plus a second signal of the type outlined. In this way, immunological tolerance is the most easy event to achieve and, thus, the activation of autoreactive cells is prevented. According to a different view (2)) self-nonself discrimination is carried out at the T-cell level by so far unknown mechanisms, whereas B cells can never be tolerized to thymus-dependent antigens, including autoantigens. This “one non-specific signal” concept postulates that immunocyte triggering is caused by one signal not delivered by the antigen-Ig receptor interaction, but rather by nonclonally distributed receptors on B cells for triggering signals intrinsic to the TI antigens produced by helper T cells (2). Previous studies on experimentally induced tolerance have not resolved this discrepancy, since it has not been possible to distinguish critically between the presence of nonactivated cells and the absence of immunocompetent cells. However, by using polyclonal B-cell activators (PBAs) such studies are now possible. PBAs have the ability to activate directly antibody synthesis in resting B cells by interacting with receptors which are not the immunoglobulin receptors. It follows that even B cells with blocked Ig receptors can be activated by PBA. By use of these PBAs it could be shown that tolerance to a hapten (3, 4) or a hapten-protein conjugate (5) does not affect the B cells, which would be successfully activated to antibody synthesis against the tolerogen by lipopolysaccharide from gram-negative bacteria, a potent PBA. Since PBAs are competent to activate B cells of any immunological specificity and, thus, to reveal the total V-gene repertoire of the B cells, it should be possible to investigate whether competent B cells against autoantigens exist in the organism. Two such studies have been carried out. In one it was found that injection of LPS in mice resulted in production of antibodies against DNA (6), and, in the second, stimulation of bovine spleen cells by LPS resulted in the production of antibodies to autologous red cells, as measured in the PFC assay using autologous erythrocytes as target cells (7). To investigate this phenomenon further, we have injected LPS, at optimal doses for induction of polyclonal responses, into mice and have studied the cytotoxic activity of the sera from these animals in the presence of complement using autologous and syngeneic spleen lymphocytes as target cells. We regularly found that sera from LPS-injected mice exhibited complement dependent cytotoxicity against syngeneic target cells. The possible causes of such an effect and the implications of our findings for an understanding of self-nonself ,discrimination are discussed. MATERIALS
AND
METHODS
C3HJTif, C3H/HeJ, BlO SM, A/Sri, and A x Animals and immunizations. C57Bl/lO, matched for age and sex, were used throughout these experiments. The mice were injected iv and ip with varying doses of LPS in a volume not exceeding 0.1 ml in BSS. Mitogens. Lipopolysaccharide (LPS) from Escherichia co& 055: B5 was obtained by phenol-water extraction according to the method of Westphal et al. (8) and was provided by Dr. Holme (Department of Bacteriology, Karolinska Institutet, Stockholm).
254
PRIM1
ET
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Sera. Mice were bled from the retro-orbital plexus at various times after LPS injection. The sera were separated 1 hr later by centrifugation and were stored in aliquots at - ZO”C, after inactivation at 56°C for 30 min. Reconstitution of irradiated a&n&. C3H/Tif mice were irradiated with 750 R and were left for 4 hr. The animals were injected with 100 IU of heparin reconstituted with 30 X 10’ B10.5M spleen cells. The cells were injected iv with or without 100 pg of LPS in a total volume of 0.3 ml of BSS. The mice were protected by Tetracycline (Dumocylin), 50 &ml, in the drinking water. Target cells. Spleen cell suspensions from mice were obtained by pressing the spleen through stainless steel screens. The resultant suspension was pipetted vigorously, and the remaining cellular aggregates were allowed to settle for 5 min. The cells remaining in the supernatant were washed twice in BSS before labeling with Wr. In some experiments the red blood cells were separataed from the lymphocytes by centrifuging the cells in a gradient consisting of 10, 29, and 30% bovine serum albumin (BSA) fractions. Labeling with 51Cr. One-tenth milliliter of sodium chromate (specific activity, 10 mCi/ml) was added to lo* packed spleen cells in 0.25 ml of RPM1 medium supplemented with penicillin, streptomycin (500 III/ml), L-glutamine (200 mM) (Flow Laboratories, Irvine, Scotland), and 10% fetal calf serum. The cells were suspended and incubated for 75 min at 37°C in a COz incubator (10% COz). The cells were then washed twice in BSS and were resuspended at a concentration of 5 x 10E/ml in RPM1 medium. Complement. Rabbit serum, never older than 1 week, was used as a source of complement (usually at a l/5 dilution in BSS) following absorption according to the method of Boyse et aE. (9) using a 30% suspension of thymus and spleen cells. Assay for cytotoxicity. Fifty microliters of a cell suspension, containing 5 x 1W labeled target cells, was pipetted into hemolysis tubes with 50 ~1 of serum from LPS-injected or control mice. The controls consisted of target cells with autologous serum from noninjected mice and target cells with medium alone. To measure the total releasable isotope, target cells were incubated with sodium dodecyl sulfate (SDS). The mixture was incubated for 1 hr at 37°C in a COz incubator. Following two washings in BSS, the cells were resuspended in 50 ~1 of rabbit complement (RC’) and were incubated for another hour at 37°C.. All assays were done in triplicate. After incubation, the tubes were centrifugated, and the supernatant and pellet were counted in a gamma counter. The percentage of Yr release was calculated as follows : Counts per minute in suaernatant - background (counts per minute in pellet-counts per minute in supernatant) -2 (background)’ Specific lysis was calculated with the following formula : percentage of lysis in experimental - percentage of lysis in control percentage of maximum lysis - percentage of lysis in control . Preparation of anti-mouse Ig colwnns. One milliliter of rabbit anti-mouse Ig serum (Behringwerke AG, Germany) was diluted in 10 ml of 0.1 M sodium tetraborate and was added to 1 g of previously activated Hidrazide Bio-Gel P2 beads (BioRad, Richmond, Germany). The mixture was stirred for 5 hr. After one more wash-
13.33 11.76
25.67 f3.24
26.33 f2.60
18.67 zkl.49
22.00 *2.05
8.5OC f0.50
14.00s zh2.52
10.67s ~1~0.88
11.67s f1.03
18.10 lt2.91
A/Sri
BIOSM
A x Bl0.5M
A X C57Bl/lO
C3H/Tif
16.40s f3.06
12.10s zko.90
12.33 Al.33
13.00s f2.00
15.00s zko.00
N
l/4
29.10 Al.86
23.79 fl.75
21.00 zlz3.79
30.33 fl.76
l/6
29.80 f2.12
19.23 fl.33
21.33 f3.38
31.33 fl.20
15.50 10.50
LPS
Dilution
18.20s ho.46
11.97s ho.61
11.50s tto.50
12.00s &OS8
11.67s zkO.67
N
of serum
release (%)
l/8
31.80 zk0.76
20.40 zk2.60
22.67 zk3.84
28.00 ~3.06
19.67 f2.66
LPS
of sera from LPS-Injected
P < 0.05 by the Student’s t test.
19.00s f3.06
13.07s ~1~1.29
10.67s f0.88
13.00s zk3.00
12.00 lt1.15
N
1 Dilutions
Isotope
TABLE Cells at Various
12.00 fO.OO
LPS
Release from Target
a The sera were obtained at Day 3 after injection of 100 pg of LPS. b N, untreated ; LPS, LPS-injected. c Mean f SEM of triplicate experiments; S = significant at 0.01 <
LPS
of Isotope
Nb
Mouse strain
Percentage
19.60s f0.21
12.90s &I.50
13.50 f0.50
11.67s zkl.33
11.67s f0.33
N
l/IO
Mice*
31.50 f2.15
25.95 13.15
16.50 fl.50
24.67 ~0.67
18.00 fl.OO
LPS
and Untreated
20.00 f1.86
15.65 ~1~3.85
10.50 f2.50
12.21 f0.68
14.33s fl.33
N
l/l2
24.60 h2.36
26.20 f0.20
16.67 f2.96
12.00 zk2.52
22.33 f0.88
LPS
?v)
2 2 i3 cn z
z
X
rE ;z 3
8
E z 2 u g
2.56
PRIM1
ET
AL.
ing in sodium tetraboratae, a solution made up of 1 ml of 2 M ammonium chloride and 0.5 ml of 1 M ammonium hydroxide was added to the pellet and left overnight. The anti-mouse Ig-coupled beads were packed in short columns and were washed free of ZSO-nm-absorbing material by eluting several volumes of saline. The sample was eluted with the same solution. The eluted material was concentrated at the initial volume by ultrafiltration using a 10 PM Difco ultrafiltration membrane (Amicon Corp., USA). RESULTS Sera from LPS-Injected
Mice are Cytotoxic for Syngeneic Spleen Cells
Sera from different strains of mice injected iv with 100 pg of LPS were tested 3 days after injection for complement-dependent cytotoxic activity against syngeneic spleen cells. Table 1 shows that these sera caused lysis of autologous target cells. This property was found to be complement dependent, and the strength of cytotoxicity varied with the serum dilutions used. Thus, lysis of target cells was not observed when the complement used in the assay had been inactivated or when the sera were added at low concentrations. There was also a prozone effect with concentrated sera, and the optimal serum concentration varied from strain to strain. Cytotoxicity
Can Be Elivninated by Absorption
with Target Cells
In order to investigate whether the cytotoxic effect of sera could be removed by absorption with the target cells, we used C3H/Tif mice, a high responder strain to LPS, and collected sera 3 days after injection of 100 pg of LPS. The sera were
30 25
1
0
l/2
l/4
l/6
118
l/10
l/l2
SERA DILUTIONS
FIG. 1. Complement-dependent cytotoxic response of C3h/Tif spleen cells after incubation for 1 hr at 37°C with serum from syngeneic mice obtained 3 days after injection of 0.2 ml of BSS (a) or 100 pg of LPS ( n ). The serum from the LPS injected mice was absorbedfor 30 minutes at 4°C. with l/3 volume of target cells (@). Student’s t test carried out between the groups showed that, for certain dilutions, the serum from the LPS-injected mice was significantly more cytotoxic than the control and the absorbed serum (0.01 < I’ < 0.05).
INDUCTION
OF CYTOTOXIC
l/6
l/4
l/2
FACTORS
l/8
BY
l/10
LPS
257
l/12
SERA DILUTIONS
FIG. 2. Complement-dependent cytotoxic response of CSH/Tif spleen cells after incubation with autologous serum treated for 1 hr with 100 fig of LPS (!I!) or with serum from syngeneic mice obtained 3 days after injection of 100 pg of LPS ( n ). Student’s t test showed that the latter was significantly more cytotoxic at certain dilutions (0.001 < P < 0.005).
tested against autologous spleen cells after they had been absorbed for 30 min at 4°C with a l/3 volume of packed syngeneic spleen and thymus cells. As shown in Fig. 1, absorption significantly decreased the cytotoxicity of the serum. Although this finding could be interpreted as a removal of specific antibodies, it is possible that LPS itself was responsible for the effect. Thus, LPS bound to the target cells could fix complement via the complement-bypass mechanism and thereby cause target cell lysis.
l/2
l/4
1/6
l/IO
l/12
SERUM DILUTIONS
FIG. 3. Complement-dependent cytotoxicity of C3H/HeJ spleen cells tested against serum from syngeneic mice collected at different days after injection of 0.2 ml of BSS (IJ- - -0) or 200 pg of LPS (m-m). Student’s t test between the groups showed no significant difference.
2.58
PRIM1
ET
AL.
LPS Itself Is Not Responsible for Lysis To investigate this possibility, we incubated 1 ml of normal C3H/Tif serum with 100 e of LPS at room temperature for 1 hr. The mixture was tested in the cytotoxic assay. Figure 2 shows the result of this experiment and compares it to the response of serum from mice injected with the same amount of mitogen. Since the LPS serum mixture did not give any lysis, LPS can be excluded as being responsible for the effect. To show that the cytotoxic activity of sera from LPS-injected mice was due to the PBA property of LPS, we tested sera from LPS-injected C3H/HeJ mice, which are nonresponders to LPS, at different time periods after injection of 200 pg of the mitogen. The results clearly indicate (Fig. 3) that sera from LPS-injected C3H/HeJ mice do not have any lytic effect on the target cells. This confirms that the cytotoxic effect of C3H/Tif serum is due to the polyclonal B-cell activity of LPS and again excludes the possibility that cytotoxicity is due to any other property of LPS. Kinetics of the response The kinetics of the appearance of serum-mediated cytotoxicity after LPS injection was studied and was found to peak at Days 3-4 and, thereafter, rapidly decline (Fig. 4). This is the same kinetics as shown for the polyclonal response to LPS. To determine whether the disappearance of cytotoxicity after Day 5 could be due to absorption of immunoglobulins by self antigens in viva, C3H/Tif mice were irradiated and reconstituted with 3 x 10’ spleen cells from BlO. 5M spleen lymphocytes and, thereafter, were injected with 100 g of LPS. Sera were collected at different days after injection and were tested against BlO. 5M spleen cells. The results shown in Fig. 5 demonstrate that the kinetics of cytotoxicity was similar to that shown in Fig. 4. Thus, it appears unlikely that absorption played any role in 35 30 25
1
3
2
DAYS
AFTER
LPS
4
5
6
INJECTION
FIG. 4. Complement-dependent specific lysis of CSH/Tif spleen cells after incubation serum from syngeneic mice collected at different days after injection of 200 pg of LPS.
with
INDUCTION
OF
CYTOTOXIC
FACTORS
BY
LPS
259
25
1
2
DAYS AFTER
3 LPS
4
5
6
INJECTION
FIG. 5. Complement-dependent specific lysis of C3H/Tif spleen cells after incubation with serum obtained from irradiated syngeneic mice reconstituted with spleen cells from BlO. 5M mice and injected with 100 pg of LPS.
the disappearance of antibodies against self antigens that may be the cause of a such cytotoxic effect. Probable Immunoglobulin
Nature of the Cytotoxic Factors
CBA mice were injected with 100 lug of LPS, and sera were collected 4 days later and were pooled. Samples of 1 ml were passed through anti-mouse Ig or normal columns. The eluted sera were concentrated at the initial volume and were used in the complement-dependent cytotoxic assay diluted l/4. Figure 6A shows that serum from LPS mice completely lost its complementdependent cytotoxic activity when passed through the anti-Ig column. However, no change in lytic activity was detected when serum was eluted from an uncoupled
FIG. 6. CBA mice were injected with 100 yg of LPS. Sera were collected 4 days later, pooled, and tested for complement-dependent cytotoxicity ( IJJ) against syngeneic (A) and allogeneic (B) spleen lymphocytes. The sera were also passed through anti-mouse Ig (I) and normal (a) columns before testing.
260
PRIM1
ET
AL.
column. An identical effect was found when such sera were tested against spleen lymphocytes obtained from the allogeneic strain 5M. Therefore, it seems likely that the cytotoxic factors in such sera are immunoglobulins produced by the polyclonal activation of B cells. DISCUSSION Our results show that sera from LPS-injected mice have a cytotoxic activity against syngeneic cells. The variability between different experiments that was observed could be traced mainly to the source of complement. Thus, the complement activity of the rabbit sera declined with time of storage, starting after a few days. We have tried to avoid this problem by using complement from only one rabbit and by never storing sera for more than 1 week. The appearance of cytotoxic serum factor after LPS injection could be caused principally by two mechanisms : (i) A direct or indirect effect of LPS itself or (ii) the induction of antibodies competent to react with autologous and syngeneic target cells. The results presented above exclude the possibility that LPS itself induced cytotoxicity, since injection of LPS to the nonresponder strain C3H/HeJ did not induce complement-dependent cytotoxicity, nor did the admixture of LPS to normal serum cause any cytotoxicity in the presence of complement. The possibility that LPSbound complement and that the LPS-complement complex caused cytotoxicity is excluded by the previous findings that such complexes do not have any deleterious effect on lymphocytes kept in culture over a 2-day period (10). It is possible that LPS induced formation of antibodies to LPS and that the antibody-LPS complex, possibly after having fixed complement, bound to Fc or C’3 receptors and induced cytotoxicity. This possibility is unlikely for several reasons. LPS, at the polyclonal concentrations employed in the present experiments, is capable of activating antibody synthesis in all B cells except those having specificity for the inducer, e.g., these concentrations are tolerogenic for LPS itself ( 11). Furthermore, interactions between different ligands, such as LPS, PPD, dextran sulfate, hapten-protein complexes, and Fc or Fc and C’3 receptors have never resulted in cytotoxicity (12), even though high complement concentrations were employed. It should be pointed out that LPS is not unique in its ability to cause the appearance of cytotoxic serum factors. As will be shown elsewhere, PPD and dextran sulfate also have this ability. The only known common property among these substances is that they are polyclonal B-cell activators. In particular, dextran sulfate is well studied with regard to the condition leading to appearance of anti-dextran antibodies, induction of polyclonal antibody synthesis, and interaction with C’3 and Fc receptors. It has been clearly shown (13) that, at the concentration used for induction of cytotoxic serum factors against syngeneic spleen cells, there is complete tolerance to dextran itself and interaction between dextran sulfate and Fc or c’3 or both receptors together never results in cytotoxicity. Therefore, it is unlikely that the present experiments are caused by trivial explanations of this type. The second alternative listed above appears more likely, e.g., that cytotoxity was caused by antibodies directed against membrane antigens of autologous and syngeneic spleen cells. LPS is known to induce polyclonal antibody synthesis including antibodies against the tolerogen in experimentally induced tolerance (5). In addition, LPS has been shown to cause the appearance of anti-
INDUCTION
OF CYTOTOXIC
FACTORS
BY
LPS
261
body-producing cells against autologous red cells tested in the Jerne plaque assay (7). In the latter case it is unquestionable that the cytotoxic principle was complement-fixing antibody directed against autologous red blood cells. The fact that in our experiments cytotoxic factors were completely eliminated when sera were passed through anti-mouse Ig columns is further evidence of the immunoglobulin nature of such factors. The specificity of the antibodies induced has not been studied as yet. They could be directed against any number of cell membrane antigenic determinants, including those determined by MuLV such as gp 70. It is well known that there are a number of clones of B cells which carry activity against these viral components. These cells release immunoglobulins which can fix the complement and give target cells killing (14, 15). We do not have evidence to exclude this possibility. However it has been shown that polyclonally activated T cells do not react against autologous cells (16). It is, therefore, rather unlikely that B cells and not T cells would react against virus-induced antigens on the cell membrane, especially since B cells probably would need T-cell cooperation to be activated against these thymus-dependent antigens. Another argument against the virus-induced antigens as target for the cytotoxic antibodies comes from the recent finding that supernatants from LPS-cultured human tonsil cells have a similar complement-dependent cytotoxic activity to autologous targets (17). The important finding from an immunological point of view is that the LPS-reactive B-cell subpopulation contained cells with immunoglobulin receptors directed against self-antigens, and that these cells were not tolerant but could be activated by LPS to the synthesis of immunoglobulins with specificity for self. Therefore, our findings have important implications for the understanding of the triggering mechanism of B lymphocytes. The one nonspecific signal theory (18) assumes that only one nonspecific signal activates B lymphocytes, and this signal is delivered by non-clonally distributed receptors for polyclonal B-cell activators. Therefore, PBA stimulation activates B cells to produce antibodies of all Ig specifities. Normally, there is no production of autoantibodies because B cells cannot be activated by thymus-dependent antigens, since thymus-dependent antigens require cooperation between T and B cells for induction of B-cell immune responses. Since self-reactive T cells do not exist (17), the self-reactive B cells are normally kept in a resting state. The findings that LPS induced the formation of cytotoxic factors, probably immunoglobulins, capable of reacting with autologous and syngeneic cells, are in contrast to the two-signal hypothesis (1) stating that contact between antigen and immunoglobulin receptors leads to an irreversible state of immunological paralysis, while our results are in agreement with the above one nonspecific signal theory. It seems likely that activation of autoantibody synthesis could occur in infections, since most PBAs known are of bacterial or parasitic origin. A large number of parasites and bacteria have already been demonstrated to have PBA properties, such as M. Tuberculosis, M. Leprae, Malaria, Mycoplasma (19-21), etc., and it is also known that the diseases caused by these agents are accompanied by more or less generalized autoantibody synthesis. Selective autoimmunity, directed against a particular autoantigen probably requires a different explanation, since selectively itself renders it unlikely that PBAs are responsible for activation.
262
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ET AL.
In conclusion, our findings support the concept that self-reactive B cells are normally present in untreated animals, and that these cells, as well as B cells of all other Ig specifities, can be activated by different PBAs into synthesis of their Ig specifities, thus excluding that contact between Ig receptors and autoantigens causes a tolerogenic signal. REFERENCES 1. 2. 3. 4. 5.
Bretscher, P. A., and Cohn, ‘M. S., Science 169, 1042, 1970. Coutinho, A., and Mijller, G., ScaBd. .I. Immunol. 4, 99, 1975. Aldo-Benson, M., and Borel, Y., J. Zmmzlnol. 112, 1793, 1974. Gronowicz, E., and Coutinho, A., Ew-. J. Immunol. 5,413, 1975. Mijller, G., Gronowicz, E., Persson, U., Coutinho, A., Moller, E., Hammarstrom, L., and Smith, C. I., J. Es-p. Med. 143, 1429, 1976. 6. Gilbert, J., Four&, P. H., Lambert, P. H., and Miescher, P. A. J. E., J. Exp. Med. 140, 1189, 1974. 7. Hammarstriim, L., Smith, E., Primi, D., and Moller, G., Nature (London) 263, 60, 1976. 8. Westphal, O., Luderitz, O., and Bister, Nafzlrforscher. Teil. B 7b, 148, 1952. 9. Boyse, E. A., Hubbard, L., Stockert, E., and Lamm, M. E., Transplantation 10,446, 1970. 10. Moller, G., SjBberg, O., and Andersson, J. Eur. J. Immunol. 2, 586, 1972. 11. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 4, 349, 1972. 12. Mijller, G., and Coutinho, A., J. Exp. Med. 141, 647, 1975. 13. Coutinho, A., and Moller, G., Advan. Immunol. 21, 114, 1975. 14. Ihle, J. N., Hanna, M. G., Roberson, L. E., and Kenney, F. T. J. Exp. Med. 136, 1568, 1974. 1.5. Ihle, J. N., Yurconic, M., Jr., and Hanna, M. G., Jr., J. Exp. Med. 138, 194, 1973. 16. Primi, D., Smith, C. I., Hammarstrom, L., Lundquist, P., and Moller, G., Clin. Exp. Zmmunol., in press, 1977. 17. Waterfield, J. D., Waterfield, E. M., and Moller, G., Cell. Immwaol. 17, 392, 1975. 18. Coutinho, A., and MGller, G., Stand. J. Zmmunol. 3, 133, 1974. 19. Sultzer, B. M., and Nilsson, B. S., Nature New Biol. 240, 198, 1972. 20. Greenwood, B. M., and Rosalind, M. V., Natwe (London) 257, 592, 1975. 21. Biberfeld, G., and Gronowicz, E., Nature (London) 261,238, 1976.