INFECTION AND IMMUNITY, Apr. 1979, p. 127-131 0019-9567/79/04-0127/05$02.00/0
Vol. 24, No. 1
Endotoxin Lethality and Tolerance in Mice: Analysis with the B-Lymphocyte-Defective CBA/N Straint N. M. ZALDIVAR'* AND I. SCHER2 Department of Experimental Medicine, Naval Medical Research Institute,' and Department of Experimental Pathology and Medicine, Uniformed Services University of the Health Sciences,2 Bethesda, Maryland 20014
Received for publication 19 October 1978
Immune-defective and immunologically normal F1 mice derived from the CBA/ N strain were used to study the influence of anti-endotoxin antibody on the lethal effects of endotoxin. Immune-defective F1 male mice were unable to make specific responses to purified preparations of E. coli O111:B4 endotoxin, whereas their immunologically normal F1 female littermates made excellent responses. The ability to form antibody to lipopolysaccharide (LPS) in these F1 mice did not influence either their natural resistance to endotoxin challenge or the effects of pretreatment with sublethal amounts of endotoxin on subsequent challenge with higher normally lethal doses. Furthermore, transfer of sera with high titers of anti-LPS antibody to mice prior to challenge with LPS failed to protect. Thus, anti-LPS antibody does not appear to play a critical role in protection of immunedefective (CBA/N x DBA/2) F1 male mice to the lethal effects of endotoxin or to the protective effects of a single sublethal dose of endotoxin on subsequent endotoxin challenge. It has been known for many years that the administration of endotoxin to either experimental animals or humans induces a state of resistance to the toxic effects of subsequent doses (8, 13). The mechanisms underlying the development of early vs. late resistance (tolerance) appear to be distinct, since considerable evidence supports the view that a macrophage refractory state is important in early tolerance, whereas the appearance of anti-endotoxin antibodies mediates late tolerance (8, 11). The evidence that late tolerance is mediated by antibody comes from studies which demonstrate that it: (i) is delayed in its appearance and persists for weeks or months; (ii) is relatively specific for the endotoxin initially administered; (iii) is transferred with the immunoglobulin G and immunoglobulin M fractions of immune sera; and (iv) is accelerated in its development in a preimmunized host (8-11, 14). To study the role of protective antibodies in the lethal effects of endotoxin in mice, we have administered sublethal doses of purified preparations of bacterial lipopolysaccharide (LPS) to the F1 progeny of CBA/N mice. Male and female mice of this inbred strain and F1 male mice derived from CBA/N females are unable to form antibody to LPS (2, 24; N. M. Zaldivar and I. Scher, Abstr. Annu. Meet. Am. Soc. Microbiol.
1977, B77, p. 28), an abnormality that is the result of an X-linked B-lymphocyte defect (1, 15, 21-23). In this communication we demonstrate: (i) that immune-defective (CBA/N x DBA/2) F1 male mice are unable to form specific antibody to LPS derived from Escherichia coli O111:B4; (ii) that (CBA/N x DBA/2) F1 males and their immunologically normal F1 female littermates have similar sensitivities to endotoxin lethality; (iii) that 100 jig of E. coli LPS had the same protective effect in the subsequent challenge of F1 male or female mice with 800 ,ug of E. coli (a uniformly lethal dose in untreated mice); (iv) that the route of administration of the E. coli LPS does not change its protective effect to subsequent challenge in these mice; and (v) that serum transfer from immune animals is not protective to mice challenged with 800 ,ug of LPS. These studies demonstrate that the absence of a specific immune response to LPS does not result in an increased sensitivity to the lethal effects of endotoxin or prevent the acquisition of resistance to these effects as a result of the administration of sublethal doses of LPS in immune-defective F1 mice. MATERIALS AND METHODS Mice. (CBA/N x DBa/2) F, female and male mice,
aged 12-14 weeks, were obtained from the Division of Research Services, National Institutes of Health. Endotoxin. A Boivin preparation of E. coli endot Naval Medical Research and Development Command, Research Work Unit No. MR04102010037. toxin O111:B4 (Difco Laboratories, Detroit, Mich.) 127
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studied. To purify this preparation of E. coli endotoxin, we extracted it with hot phenol by the method of Rudbach (20). Briefly, a 10% mixture of the E. coli endotoxin and pyrogen-free sterile water was made and added to an equal volume of 88% liquid phenol. The mixture was incubated at 68°C for 30 min with constant stirring. After an overnight incubation at 4°C, the phenol and water phases were separated by centrifugation at 4°C for 60 min at 1,000 x g, and the water phases were recovered. This extraction was repeated, and the water phases from both extractions were dialyzed against running tap water at 4°C for 7 days and against three daily changes of distilled water for 3 days. After concentration to Vio of its original volume, the water phase was brought to 0.5 M with sodium acetate and the LPS was precipitated at 4°C overnight by adding 95% ethanol to a final alcohol concentration of 68%. The LPS which was isolated by centrifugation was dissolved in water, dialyzed at 4°C against distilled water, and centrifuged at 1,000 x g for 30 min, and the supernatant fluid was lyophilized. When studied by amino acid analysis, this preparation of E. coli LPS had a protein concentration of approximately 2% protein. Quantitation of antibody responses. Mice were given 100 ,ug of LPS by intravenous (i.v.) or intraperitoneal (i.p.) inoculation, and blood was collected by orbital sinus puncture at 5-day intervals from days 5 through 25. Sera were stored at -20°C before assay. A modification of the procedure of Rudbach et al. (19) was used to coat sheep erythrocytes (SRBC) with E. coli LPS. A 10-mg portion of E. coli LPS was added to 10 ml of 0.1 M phosphate buffer (pH 7.4) and placed in a boiling water bath for 150 min. A 5-ml amount of this solution was diluted to 50 ml with 0.9 M saline, and 1.25 ml of twice-washed (in saline), packed SRBC was added. This suspension was incubated at 37°C for 30 min, and the E. coli LPS-coated SRBC (LPSSRBC) were washed three times in saline and resuspended in 10 ml of saline containing 1 mg of bovine serum albumin per ml. A 100-pl portion of packed LPS-SRBC was diluted to 10 ml in bovine serum albumin saline, and 25,ul of this suspension was added to wells of a microtiter tray; 25 M1 of serial dilutions of individual sera was added to the wells, the microtiter plates were shaken, and the LPS-SRBC were allowed to settle at room temperature for 1 h. The highest titer of sera which caused agglutination of the LPS-SRBC was determined, the plates were centrifuged at 500 rpm for 5 min, and the supernatant was discarded. The LPS-SRBC were resuspended in 25 ,ul of bovine serum albumin saline by gentle agitation on a Vortex mixer, and 25 Ml of a 1: 1,000 dilution of a polyspecific goat anti-mouse immunoglobulin (Meloy Laboratories, Springfield, Va.) was added to each well to enhance the agglutination reaction. This amount of the goat anti-mouse immunoglobulin was chosen after it was shown that it produced the greatest enhancement using F1 female sera and LPS-SRBC. In the case of the F1 male sera this dilution of the goat antisera and a range of others 1:10 to 1:1,500 failed to enhance. In other experiments a 1: 1,000 dilution of the goat anti-mouse immunoglobulin optimally enhanced sera from F1 males immunized and tested against SRBC. After the anti-mouse imwas
INFECT. IMMUN. munoglobulin was added the LPS-SRBC were allowed to settle for 1 h, and the highest titer of sera at which the enhanced agglutination occurred was determined. Controls consisting of SRBC and trinitrophenol-coupled SRBC (TNP-SRBC) (16) were also tested in each experiment. No agglutination (either enhanced or unenhanced) was observed with either SRBC or TNPSRBC. Serum transfers. F. mice of either sex were bled through the tail vein 10 days after they received 100 Iug of E. coli LPS by i.p. inoculation. The sera from these bleeds were pooled and given in different amounts to groups of F1 female mice by i.v. injection 24 h before challenge with varying amounts of E. coli endotoxin.
RESULTS Groups of 30 (CBA/N x DBA/2) F1 male or female mice were given 100, 200, or 400 ,ig of E. coli endotoxin i.p., and deaths were recorded over a period of 72 h. As shown in Table 1, all mice (both males and females) that received 400 Iug of E. coli endotoxin by i.p. injection were dead within 72 h, and equivalent numbers of F1 males and F1 females died after receiving 200 ,Ig. No deaths were observed when 100 ,ug of E. coli endotoxin was administered. These data demonstrate that the X-linked B-lymphocyte defect of (CBA/N x DBA/2) F1 male mice does not influence their resistance to the lethal effects of endotoxin. The anti-LPS antibody responses of (CBA/N x DBA/2) F1 male and female mice were analyzed by immunizing with 100 ,ug of E. coli LPS, a nonlethal dose which in preliminary experiments protected F1 female mice from a subsequent challenge with 800 ,ug of E. coli endotoxin. As shown in Table 2, all F1 female mice formed antibody to this amount of LPS, with a response which reached a maximum at 10 days (mean reciprocal enhanced hemagglutination titer of >10,240). By contrast, none of 40 F1 male mice made detectable responses. Having established the pattern of anti-LPS antibody production in (CBA/N x DBA/2) F1 male and female mice, we administered 100 jIg of E. coli LPS by i.p. or i.v. inoculation to groups of F1 male and female mice, waited 10 days, and challenged them with 800 jig of E. coli endotoxin. TABLE 1. E. coli endotoxin-induced lethality in (CBA/N x DBA/2)F1 males and females No. alive/total no. at 72 h Amnt of endotoxin F1 males FF females (Wg) 100 30/30 30/30 200 6/30 5/30 400 0/30 0/30 a Mice were given E. coli endotoxin i.p., and deaths were recorded over a 72-h period.
VOL. 24, 1979
ENDOTOXIN LETHALITY
TABLE 2. Anti-E. coli O111:B4 endotoxin responses of (CBA/N x DBA/2) F, males and females to 100 pg of E. coli LPSG Geometric mean reciprocal hemagglutination Days after immunization
titer
Unenhanced
Enhanced
F, male 0 0 0 -1 0 0 0 5 0 10 0 15.8 0 20 0 15 0 67.9 0 25 aGroups of 40 mice were immunized with 100 pg of E. coli LPS i.p., and the response of individual mice was determined by a hemagglutination assay using LPS-SRBC. All sera of F. female mice induced agglutination on or after 10 days, with enhanced titers of 210,240 at day 10, 1:640 to 1:2,560 at day 15 and 1:160 to 1:1,280 at day 25. No agglutination was observed with sera from F, male mice at any day after immuF. female F, male
F, female 0 0 >10,240 1,097 403
nization.
As shown in Table 3, the protection induced by 100 jig of E. coli LPS given i.p. or i.v. against the lethal effects of E. coli endotoxin was equivalent in immune-defective F1 males and their immunologically normal F1 female littermates. The possible protective effects of sera derived from F1 female or male mice that had received 100 Mg of E. coli LPS 10 days previously were tested by giving 10, 25, 50, 100, or 500 yl of F1 male or female immune sera to F1 female mice. On the following day the F1 female mice were challenged with 800 Mug of E. coli endotoxin. The pooled enhanced anti-LPS titers of the F1 female mice that received 500 1d of immune sera 16 h previously were 1:2,560. As shown in Table 4, neither the F1 male or female sera protected F1 female mice against challenge with 800 ,g of E. coli endotoxin.
DISCUSSION Resistance to a number of the toxic effects of bacterial endotoxins can be induced in experimental animals by the administration of sublethal amounts of LPS. This phenomenon, which has been referred to as "tolerance," has been
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with the late phase of tolerance. The loss of specificity when rough mutants were used to induce late tolerance suggested that common core antigens were also capable of inducing late tolerance. However, it has not been definitively shown that anti-O-specific or common core antibodies were responsible for late tolerance, since the protective effects of passively transferred sera were not shown to be eliminated by absorption with either anti-immunoglobulins or LPS. We have utilized CBA/N mice to study the role of anti-endotoxin antibodies in the development of tolerance to the lethal effects of endotoxin. CBA/N mice have an X-linked defect in B-lymphocyte function which influences a number of their immune functions (1, 2, 15, 21-23). It has previously been shown that mice that express the CBA/N immune defect respond normally to the macrophage- and lymphocyte-activating effects of LPS and that they can mount a specific thymic-independent TNP response to TABLE 3. Influence of 100 pg of E. coli LPS on subsequent challenge of (CBA/N x DBA/2) F, male or female mice with 800 pg of E. coli endotoxina Sex of F, mice
Source and method of No.no.alive/total at 72 h
administration of LPS
Female Male
E. coli (i.p.)
38/41 38/41
Female E. coli (i.v.) 12/12 Male 16/16 a Mice were challenged by i.p. injection of 800 of E. coli endotoxin 10 days after they were inoculated with 100 pg of LPS given by iv. or i.p. injection. All of the eight F, males and eight F, females that received 800 pg of E. coli endotoxin alone were dead within 24 h.
pg
TABLE 4. Influence of (CBA/N x DBA/2) F. male or female sera given i.v. on subsequent challenge with 800 pg of E. coli endotoxin of F, female mice Sex of serum donor
Serum adminiis(1)
Female
10 25 50 100 500 10 25 50 100 500
tereda
No.no.alive/total at 72 h
0/10 0/15 0/15 0/16 0/10 Male 0/5 0/5 0/7 0/7 0/5 a Sera were derived from F, male or female mice that were immunized with 100 pg of E. coli LPS administered by i.p. inoculation 10 days previously. The reciprocal hemagglutination titer of these pooled sera were >10,240 and 0 for the females and males,
studied extensively in both humans and rabbits (8, 10-14). Early studies suggested that resistance to the toxic effects of homologous and heterologous endotoxins was the result of enhanced reticuloendothelial activity (4, 5, 9). However, subsequent passive transfer studies have demonstrated a protective role of antiserum directed against the O-specific antigens of endotoxins in endotoxin-induced toxicity (8). In addition, studies in humans have shown that increases in anti-O antibody were associated respectively.
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Boivin preparations of TNP-endotoxin (2,15,18, 21). However, recent studies have indicated that these mice are unable to respond with specific antibody to protein-depleted preparations of E. coli LPS (24; Zaldivar and Scher, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B77, p. 28). In this report, antibody formation was assayed by an enhanced hemagglutination technique. This procedure detected anti-LPS and not antiSRBC antibodies, since agglutination was not observed when preimmune sera were tested with LPS-SRBC or when high-titer anti-LPS sera were tested with SRBC. Analysis of sera derived from (CBA/N x DBA/2) F1 male mice after immunization with a purified preparation ofLPS (derived from E. coli) demonstrated that these immune-defective mice were unable to form anti-LPS antibody to this preparation. In spite of this unresponsiveness, (CBA/N x DBA/2) F1 male mice were no more susceptible to the lethal effects of E. coli endotoxin than their immunologically normal F1 female littermates. These findings are quite distinct from those observed with C3H/HeJ mice, a strain which is also unable to form anti-LPS antibody and is highly resistant to the toxic actions of LPS (25, 26). These findings are reminiscent of the contrasting mitogenic, adjuvant, and lymphocyte-macrophage-activating actions of LPS in these two strains since C3H/HeJ mice are resistant to these effects, whereas (CBA/N x DBA/2) F1 male mice are not (6, 7, 17, 18). Thus, the lethal effects of endotoxin appear to be correlated with its nonspecific biological properties, but not with its immunogenicity in C3H/HeJ and (CBA/N x DBA/2) F1 male mice. The equivalent sensitivity of (CBA/ x DBA/ 2) F1 male and female mice to the lethal effects of endotoxin allowed us to test the effects of 100 ,ug of LPS on the lethality of 800 ,ug of endotoxin in these mice. We challenged at 10 days after the administration of 100 ,ug of LPS because at this time the F1 females produced their highest antiLPS antibody titers. This protocol protected 38 out of 41 immunologically normal F1 female mice and 38 out of 41 immune-defective F1 male mice. Administration of the LPS by i.v. injection also resulted in excellent protection in both F1 males and F1 females (12 out of 12 and 16 out of 16 alive, respectively), suggesting that the protection induced by i.p. inoculation was not the result of impaired absorption or increased nonspecific phagocytosis of the endotoxin challenge dose. The possible protective effects of serum from F1 mice that received 100 ,Ag of LPS was also measured directly by assaying the ability of F1 female and male 10-day sera to confer resistance in passive transfer experiments.
INFECT. IMMUN.
These transfer experiments did not influence the toxicity of 800 ,ug of endotoxin in spite of a titer of 1:2,560 in mice that received 500,ul of F1 female sera 16 h previously. Thus, neither F1 female or male sera afforded protection. It should be emphasized that these experiments do not by themselves indicate that passively transferred sera could not under different experimental conditions induce protection, particularly in view of the fact that we utilized only one dose of endotoxin for challenge. With lower doses of endotoxin, it is entirely possible that sera from tolerant animals could be protective. However, these findings, along with the excellent protection observed in F1 male mice that had no detectable anti-LPS antibodies, strongly suggest that factors other than antibody play a critical role in the development of tolerance to the lethal effects of endotoxin and that anti-LPS antibody is not necessary for the development of resistance to an 800-,ug challenge of endotoxin in F, male mice. The ability of (CBA/N x DBA/2) F1 male mice to respond to certain of the immune effects of LPS, as noted above, may provide insights into the mechanisms involved in this phenomenon. Certainly, analysis of LPS-induced resistance in these mice, where anti-LPS antibody is not formed, will be useful as an experimental model to analyze the multiple biological effects of LPS. LITERATURE CITED 1. Amsbaugh, D. F., C. T. Hanson, B. Prescott, P. W. Stashak, 0. R. Barthold, and P. J. Baker. 1972. Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. I. Evidence that an X-linked gene plays a decisive role in determining responsiveness. J. Exp. Med. 136:931-949. 2. Amsbaugh, D. F., C. T. Hansen, B. Prescott, P. W. Stashak, R. Asofsky, and P. J. Baker. 1974. Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. II. Relationship between IgM immunoglobulin levels and the ability to give an IgM antibody response. J. Exp. Med. 139:1499-1512. 3. Beeson, P. B. 1946. Development of tolerance to typhoid bacterial pyrogen and its abolition by reticuloendothelial blockade. Proc. Soc. Exp. Biol. Med. 61:248-250. 4. Beeson, P. B. 1947. Tolerance to bacterial pyrogens. I. Factors influencing its development. J. Exp. Med. 86: 29-38. 5. Beeson, P. B. 1947. Tolerance to bacterial pyrogens. II. Role of the reticuloendothelial system. J. Exp. Med. 86: 39-44. 6. Glode, L M., A. Jacques, S. E. Mergenhagen, and D. L. Rosenstreich. 1977. Resistance of macrophages from C3H/HeJ mice to the in vitro cytotoxic effects of endotoxin. J. Immunol. 119:162-166. 7. Glode, L M., L. Scher, B. Osborne, and D. L. Rosenstreich. 1976. Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice. J. Immunol. 116:454461. 8. Greisman, S. E., and R. B. Hornick. 1976. Endotoxin tolerance, p. 43-50. In R. F. Beers, Jr. and E. G. Basset (ed.), The role of immunological factors in infectious,
VOL. 24, 1979 allergic and autoimmune disease. Raven Press, New York. 9. Greisman, S. E., H. N. Wagner, Jr., M. His, and R. B. Hornick. 1964. Mechanism of endotoxin tolerance. II. Relationship between endotoxin tolerance and reticuloendothelial system phagocytic activity in man. J. Exp. Med. 119:241-264. 10. Greisman, S. E., and E. J. Young. 1969. Mechanisms of endotoxin tolerance. VI. Transfer of the "anamnestic" tolerant response with primed spleen cells. J. Immunol. 103:1237-1241. 11. Greisman, S. E., E. J. Young, and F. A. Carozza, Jr. 1969. Mechanisms of endotoxin tolerance. V. Specificity of the early and late phases of pyrogenic tolerance. J. Immunol. 103:1223-1236. 12. Greisman, S. E., E. J. Young, and J. B. DuBuy. 1973. Mechanisms of endotoxin tolerance. VIII. Specificity of serum transfer. J. Immunol. 111:1349-1360. 13. McCabe, W. R. 1975. Humoral protection against gramnegative bacilli and endotoxins, p. 336-340. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 14. Milner, K. C. 1973. Patterns of tolerance to endotoxin. J. Infect. Dis. 128(Suppl.):237-245. 15. Mosier, D., I. Scher, and W. E. Paul. 1976. In vitro responses of CBA/N mice: spleen cells of mice with an X-linked defect that precludes immune responses to several thymus-independent antigens can respond to TNP-lipopolysaccharide. J. Immunol. 117:1363-1369. 16. Rittenberg, M. B., and K. L. Pratt. 1968. A nitrophenyl (TNP) plaque assay. Primary response of Balb/c mice to soluble and particulate immunogen. Proc. Soc. Exp. Biol. Med. 132:575-581. 17. Rosenstreich, D. L,, L. M. Glode, L. M. Wahl, A. L. Sandberg, and S. E. Mergenhagen. 1977. Analysis of the cellular defects of endotoxin-unresponsive C3H/ HeJ mice, p. 314-320. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology,
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Washington, D.C. 18. Rosenstreich, D. L., S. N. Vogel, A. Jacques, L. M. Wahl, I. Scher, and S. E. Mergenhagen. 1978. Differential endotoxin sensitivity of lymphocytes and macrophages from mice with an X-linked defect in B cell maturation. J. Immunol. 121:685-690. 19. Rudbach, J. A. 1971. Molecular immunogenicity of bacterial lipopolysaccharide antigens: establishing a quantitative system. J. Immunol. 106:993-1001. 20. Rudbach, J. A., F. I. Akiya, R. J. Elin, H. D. Hochstein, M. K. Luoma, E. C. B. Milner, K. C. Milner, and K. R. Thomas. 1976. Preparation and properties of a national reference endotoxin. J. Clin. Microbiol. 3: 21-25. 21. Scher, I., A. Ahmed, D. M. Strong, A. D. Steinberg, and W. E. Paul. 1975. X-linked B-lymphocyte defect in CBA/N mice. I. Studies of the function and composition of spleen cells. J. Exp. Med. 141:788-803. 23. Scher, I., A. D. Steinberg, A. K. Berning, and W. E. Paul. 1975. X-linked B-lymphocyte defect in CBA/N mice. II. Studies of the mechanisms underlying the immune defect. J. Exp. Med. 142:637-650. 22. Scher, I., S. 0. Sharrow, and W. E. Paul. 1976. Xlinked B-lymphocyte defect in CBA/N mice. III. Abnormal development of B-lymphocyte populations defined by their density of surface immunoglobulin. J. Exp. Med. 144:507-518. 24. Scher, I., N. M. Zaldivar, and D. E. Mosier. 1977. Blymphocyte subpopulations and endotoxin response in CBA/N mice, p. 310-313. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 25. Sultzer, B. M. 1968. Genetic control of leukocyte responses to endotoxin. Nature (London) 219:1253-1254. 26. Watson, J., K. Kelly, and M. Langen. 1977. Genetic and cellular aspects of host response to endotozin, p. 298-303. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C.
Vol. 24, No. 1
Endotoxin Lethality and Tolerance in Mice: Analysis with the B-Lymphocyte-Defective CBA/N Straint N. M. ZALDIVAR'* AND I. SCHER2 Department of Experimental Medicine, Naval Medical Research Institute,' and Department of Experimental Pathology and Medicine, Uniformed Services University of the Health Sciences,2 Bethesda, Maryland 20014
Received for publication 19 October 1978
Immune-defective and immunologically normal F1 mice derived from the CBA/ N strain were used to study the influence of anti-endotoxin antibody on the lethal effects of endotoxin. Immune-defective F1 male mice were unable to make specific responses to purified preparations of E. coli O111:B4 endotoxin, whereas their immunologically normal F1 female littermates made excellent responses. The ability to form antibody to lipopolysaccharide (LPS) in these F1 mice did not influence either their natural resistance to endotoxin challenge or the effects of pretreatment with sublethal amounts of endotoxin on subsequent challenge with higher normally lethal doses. Furthermore, transfer of sera with high titers of anti-LPS antibody to mice prior to challenge with LPS failed to protect. Thus, anti-LPS antibody does not appear to play a critical role in protection of immunedefective (CBA/N x DBA/2) F1 male mice to the lethal effects of endotoxin or to the protective effects of a single sublethal dose of endotoxin on subsequent endotoxin challenge. It has been known for many years that the administration of endotoxin to either experimental animals or humans induces a state of resistance to the toxic effects of subsequent doses (8, 13). The mechanisms underlying the development of early vs. late resistance (tolerance) appear to be distinct, since considerable evidence supports the view that a macrophage refractory state is important in early tolerance, whereas the appearance of anti-endotoxin antibodies mediates late tolerance (8, 11). The evidence that late tolerance is mediated by antibody comes from studies which demonstrate that it: (i) is delayed in its appearance and persists for weeks or months; (ii) is relatively specific for the endotoxin initially administered; (iii) is transferred with the immunoglobulin G and immunoglobulin M fractions of immune sera; and (iv) is accelerated in its development in a preimmunized host (8-11, 14). To study the role of protective antibodies in the lethal effects of endotoxin in mice, we have administered sublethal doses of purified preparations of bacterial lipopolysaccharide (LPS) to the F1 progeny of CBA/N mice. Male and female mice of this inbred strain and F1 male mice derived from CBA/N females are unable to form antibody to LPS (2, 24; N. M. Zaldivar and I. Scher, Abstr. Annu. Meet. Am. Soc. Microbiol.
1977, B77, p. 28), an abnormality that is the result of an X-linked B-lymphocyte defect (1, 15, 21-23). In this communication we demonstrate: (i) that immune-defective (CBA/N x DBA/2) F1 male mice are unable to form specific antibody to LPS derived from Escherichia coli O111:B4; (ii) that (CBA/N x DBA/2) F1 males and their immunologically normal F1 female littermates have similar sensitivities to endotoxin lethality; (iii) that 100 jig of E. coli LPS had the same protective effect in the subsequent challenge of F1 male or female mice with 800 ,ug of E. coli (a uniformly lethal dose in untreated mice); (iv) that the route of administration of the E. coli LPS does not change its protective effect to subsequent challenge in these mice; and (v) that serum transfer from immune animals is not protective to mice challenged with 800 ,ug of LPS. These studies demonstrate that the absence of a specific immune response to LPS does not result in an increased sensitivity to the lethal effects of endotoxin or prevent the acquisition of resistance to these effects as a result of the administration of sublethal doses of LPS in immune-defective F1 mice. MATERIALS AND METHODS Mice. (CBA/N x DBa/2) F, female and male mice,
aged 12-14 weeks, were obtained from the Division of Research Services, National Institutes of Health. Endotoxin. A Boivin preparation of E. coli endot Naval Medical Research and Development Command, Research Work Unit No. MR04102010037. toxin O111:B4 (Difco Laboratories, Detroit, Mich.) 127
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studied. To purify this preparation of E. coli endotoxin, we extracted it with hot phenol by the method of Rudbach (20). Briefly, a 10% mixture of the E. coli endotoxin and pyrogen-free sterile water was made and added to an equal volume of 88% liquid phenol. The mixture was incubated at 68°C for 30 min with constant stirring. After an overnight incubation at 4°C, the phenol and water phases were separated by centrifugation at 4°C for 60 min at 1,000 x g, and the water phases were recovered. This extraction was repeated, and the water phases from both extractions were dialyzed against running tap water at 4°C for 7 days and against three daily changes of distilled water for 3 days. After concentration to Vio of its original volume, the water phase was brought to 0.5 M with sodium acetate and the LPS was precipitated at 4°C overnight by adding 95% ethanol to a final alcohol concentration of 68%. The LPS which was isolated by centrifugation was dissolved in water, dialyzed at 4°C against distilled water, and centrifuged at 1,000 x g for 30 min, and the supernatant fluid was lyophilized. When studied by amino acid analysis, this preparation of E. coli LPS had a protein concentration of approximately 2% protein. Quantitation of antibody responses. Mice were given 100 ,ug of LPS by intravenous (i.v.) or intraperitoneal (i.p.) inoculation, and blood was collected by orbital sinus puncture at 5-day intervals from days 5 through 25. Sera were stored at -20°C before assay. A modification of the procedure of Rudbach et al. (19) was used to coat sheep erythrocytes (SRBC) with E. coli LPS. A 10-mg portion of E. coli LPS was added to 10 ml of 0.1 M phosphate buffer (pH 7.4) and placed in a boiling water bath for 150 min. A 5-ml amount of this solution was diluted to 50 ml with 0.9 M saline, and 1.25 ml of twice-washed (in saline), packed SRBC was added. This suspension was incubated at 37°C for 30 min, and the E. coli LPS-coated SRBC (LPSSRBC) were washed three times in saline and resuspended in 10 ml of saline containing 1 mg of bovine serum albumin per ml. A 100-pl portion of packed LPS-SRBC was diluted to 10 ml in bovine serum albumin saline, and 25,ul of this suspension was added to wells of a microtiter tray; 25 M1 of serial dilutions of individual sera was added to the wells, the microtiter plates were shaken, and the LPS-SRBC were allowed to settle at room temperature for 1 h. The highest titer of sera which caused agglutination of the LPS-SRBC was determined, the plates were centrifuged at 500 rpm for 5 min, and the supernatant was discarded. The LPS-SRBC were resuspended in 25 ,ul of bovine serum albumin saline by gentle agitation on a Vortex mixer, and 25 Ml of a 1: 1,000 dilution of a polyspecific goat anti-mouse immunoglobulin (Meloy Laboratories, Springfield, Va.) was added to each well to enhance the agglutination reaction. This amount of the goat anti-mouse immunoglobulin was chosen after it was shown that it produced the greatest enhancement using F1 female sera and LPS-SRBC. In the case of the F1 male sera this dilution of the goat antisera and a range of others 1:10 to 1:1,500 failed to enhance. In other experiments a 1: 1,000 dilution of the goat anti-mouse immunoglobulin optimally enhanced sera from F1 males immunized and tested against SRBC. After the anti-mouse imwas
INFECT. IMMUN. munoglobulin was added the LPS-SRBC were allowed to settle for 1 h, and the highest titer of sera at which the enhanced agglutination occurred was determined. Controls consisting of SRBC and trinitrophenol-coupled SRBC (TNP-SRBC) (16) were also tested in each experiment. No agglutination (either enhanced or unenhanced) was observed with either SRBC or TNPSRBC. Serum transfers. F. mice of either sex were bled through the tail vein 10 days after they received 100 Iug of E. coli LPS by i.p. inoculation. The sera from these bleeds were pooled and given in different amounts to groups of F1 female mice by i.v. injection 24 h before challenge with varying amounts of E. coli endotoxin.
RESULTS Groups of 30 (CBA/N x DBA/2) F1 male or female mice were given 100, 200, or 400 ,ig of E. coli endotoxin i.p., and deaths were recorded over a period of 72 h. As shown in Table 1, all mice (both males and females) that received 400 Iug of E. coli endotoxin by i.p. injection were dead within 72 h, and equivalent numbers of F1 males and F1 females died after receiving 200 ,Ig. No deaths were observed when 100 ,ug of E. coli endotoxin was administered. These data demonstrate that the X-linked B-lymphocyte defect of (CBA/N x DBA/2) F1 male mice does not influence their resistance to the lethal effects of endotoxin. The anti-LPS antibody responses of (CBA/N x DBA/2) F1 male and female mice were analyzed by immunizing with 100 ,ug of E. coli LPS, a nonlethal dose which in preliminary experiments protected F1 female mice from a subsequent challenge with 800 ,ug of E. coli endotoxin. As shown in Table 2, all F1 female mice formed antibody to this amount of LPS, with a response which reached a maximum at 10 days (mean reciprocal enhanced hemagglutination titer of >10,240). By contrast, none of 40 F1 male mice made detectable responses. Having established the pattern of anti-LPS antibody production in (CBA/N x DBA/2) F1 male and female mice, we administered 100 jIg of E. coli LPS by i.p. or i.v. inoculation to groups of F1 male and female mice, waited 10 days, and challenged them with 800 jig of E. coli endotoxin. TABLE 1. E. coli endotoxin-induced lethality in (CBA/N x DBA/2)F1 males and females No. alive/total no. at 72 h Amnt of endotoxin F1 males FF females (Wg) 100 30/30 30/30 200 6/30 5/30 400 0/30 0/30 a Mice were given E. coli endotoxin i.p., and deaths were recorded over a 72-h period.
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ENDOTOXIN LETHALITY
TABLE 2. Anti-E. coli O111:B4 endotoxin responses of (CBA/N x DBA/2) F, males and females to 100 pg of E. coli LPSG Geometric mean reciprocal hemagglutination Days after immunization
titer
Unenhanced
Enhanced
F, male 0 0 0 -1 0 0 0 5 0 10 0 15.8 0 20 0 15 0 67.9 0 25 aGroups of 40 mice were immunized with 100 pg of E. coli LPS i.p., and the response of individual mice was determined by a hemagglutination assay using LPS-SRBC. All sera of F. female mice induced agglutination on or after 10 days, with enhanced titers of 210,240 at day 10, 1:640 to 1:2,560 at day 15 and 1:160 to 1:1,280 at day 25. No agglutination was observed with sera from F, male mice at any day after immuF. female F, male
F, female 0 0 >10,240 1,097 403
nization.
As shown in Table 3, the protection induced by 100 jig of E. coli LPS given i.p. or i.v. against the lethal effects of E. coli endotoxin was equivalent in immune-defective F1 males and their immunologically normal F1 female littermates. The possible protective effects of sera derived from F1 female or male mice that had received 100 Mg of E. coli LPS 10 days previously were tested by giving 10, 25, 50, 100, or 500 yl of F1 male or female immune sera to F1 female mice. On the following day the F1 female mice were challenged with 800 Mug of E. coli endotoxin. The pooled enhanced anti-LPS titers of the F1 female mice that received 500 1d of immune sera 16 h previously were 1:2,560. As shown in Table 4, neither the F1 male or female sera protected F1 female mice against challenge with 800 ,g of E. coli endotoxin.
DISCUSSION Resistance to a number of the toxic effects of bacterial endotoxins can be induced in experimental animals by the administration of sublethal amounts of LPS. This phenomenon, which has been referred to as "tolerance," has been
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with the late phase of tolerance. The loss of specificity when rough mutants were used to induce late tolerance suggested that common core antigens were also capable of inducing late tolerance. However, it has not been definitively shown that anti-O-specific or common core antibodies were responsible for late tolerance, since the protective effects of passively transferred sera were not shown to be eliminated by absorption with either anti-immunoglobulins or LPS. We have utilized CBA/N mice to study the role of anti-endotoxin antibodies in the development of tolerance to the lethal effects of endotoxin. CBA/N mice have an X-linked defect in B-lymphocyte function which influences a number of their immune functions (1, 2, 15, 21-23). It has previously been shown that mice that express the CBA/N immune defect respond normally to the macrophage- and lymphocyte-activating effects of LPS and that they can mount a specific thymic-independent TNP response to TABLE 3. Influence of 100 pg of E. coli LPS on subsequent challenge of (CBA/N x DBA/2) F, male or female mice with 800 pg of E. coli endotoxina Sex of F, mice
Source and method of No.no.alive/total at 72 h
administration of LPS
Female Male
E. coli (i.p.)
38/41 38/41
Female E. coli (i.v.) 12/12 Male 16/16 a Mice were challenged by i.p. injection of 800 of E. coli endotoxin 10 days after they were inoculated with 100 pg of LPS given by iv. or i.p. injection. All of the eight F, males and eight F, females that received 800 pg of E. coli endotoxin alone were dead within 24 h.
pg
TABLE 4. Influence of (CBA/N x DBA/2) F. male or female sera given i.v. on subsequent challenge with 800 pg of E. coli endotoxin of F, female mice Sex of serum donor
Serum adminiis(1)
Female
10 25 50 100 500 10 25 50 100 500
tereda
No.no.alive/total at 72 h
0/10 0/15 0/15 0/16 0/10 Male 0/5 0/5 0/7 0/7 0/5 a Sera were derived from F, male or female mice that were immunized with 100 pg of E. coli LPS administered by i.p. inoculation 10 days previously. The reciprocal hemagglutination titer of these pooled sera were >10,240 and 0 for the females and males,
studied extensively in both humans and rabbits (8, 10-14). Early studies suggested that resistance to the toxic effects of homologous and heterologous endotoxins was the result of enhanced reticuloendothelial activity (4, 5, 9). However, subsequent passive transfer studies have demonstrated a protective role of antiserum directed against the O-specific antigens of endotoxins in endotoxin-induced toxicity (8). In addition, studies in humans have shown that increases in anti-O antibody were associated respectively.
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Boivin preparations of TNP-endotoxin (2,15,18, 21). However, recent studies have indicated that these mice are unable to respond with specific antibody to protein-depleted preparations of E. coli LPS (24; Zaldivar and Scher, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B77, p. 28). In this report, antibody formation was assayed by an enhanced hemagglutination technique. This procedure detected anti-LPS and not antiSRBC antibodies, since agglutination was not observed when preimmune sera were tested with LPS-SRBC or when high-titer anti-LPS sera were tested with SRBC. Analysis of sera derived from (CBA/N x DBA/2) F1 male mice after immunization with a purified preparation ofLPS (derived from E. coli) demonstrated that these immune-defective mice were unable to form anti-LPS antibody to this preparation. In spite of this unresponsiveness, (CBA/N x DBA/2) F1 male mice were no more susceptible to the lethal effects of E. coli endotoxin than their immunologically normal F1 female littermates. These findings are quite distinct from those observed with C3H/HeJ mice, a strain which is also unable to form anti-LPS antibody and is highly resistant to the toxic actions of LPS (25, 26). These findings are reminiscent of the contrasting mitogenic, adjuvant, and lymphocyte-macrophage-activating actions of LPS in these two strains since C3H/HeJ mice are resistant to these effects, whereas (CBA/N x DBA/2) F1 male mice are not (6, 7, 17, 18). Thus, the lethal effects of endotoxin appear to be correlated with its nonspecific biological properties, but not with its immunogenicity in C3H/HeJ and (CBA/N x DBA/2) F1 male mice. The equivalent sensitivity of (CBA/ x DBA/ 2) F1 male and female mice to the lethal effects of endotoxin allowed us to test the effects of 100 ,ug of LPS on the lethality of 800 ,ug of endotoxin in these mice. We challenged at 10 days after the administration of 100 ,ug of LPS because at this time the F1 females produced their highest antiLPS antibody titers. This protocol protected 38 out of 41 immunologically normal F1 female mice and 38 out of 41 immune-defective F1 male mice. Administration of the LPS by i.v. injection also resulted in excellent protection in both F1 males and F1 females (12 out of 12 and 16 out of 16 alive, respectively), suggesting that the protection induced by i.p. inoculation was not the result of impaired absorption or increased nonspecific phagocytosis of the endotoxin challenge dose. The possible protective effects of serum from F1 mice that received 100 ,Ag of LPS was also measured directly by assaying the ability of F1 female and male 10-day sera to confer resistance in passive transfer experiments.
INFECT. IMMUN.
These transfer experiments did not influence the toxicity of 800 ,ug of endotoxin in spite of a titer of 1:2,560 in mice that received 500,ul of F1 female sera 16 h previously. Thus, neither F1 female or male sera afforded protection. It should be emphasized that these experiments do not by themselves indicate that passively transferred sera could not under different experimental conditions induce protection, particularly in view of the fact that we utilized only one dose of endotoxin for challenge. With lower doses of endotoxin, it is entirely possible that sera from tolerant animals could be protective. However, these findings, along with the excellent protection observed in F1 male mice that had no detectable anti-LPS antibodies, strongly suggest that factors other than antibody play a critical role in the development of tolerance to the lethal effects of endotoxin and that anti-LPS antibody is not necessary for the development of resistance to an 800-,ug challenge of endotoxin in F, male mice. The ability of (CBA/N x DBA/2) F1 male mice to respond to certain of the immune effects of LPS, as noted above, may provide insights into the mechanisms involved in this phenomenon. Certainly, analysis of LPS-induced resistance in these mice, where anti-LPS antibody is not formed, will be useful as an experimental model to analyze the multiple biological effects of LPS. LITERATURE CITED 1. Amsbaugh, D. F., C. T. Hanson, B. Prescott, P. W. Stashak, 0. R. Barthold, and P. J. Baker. 1972. Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. I. Evidence that an X-linked gene plays a decisive role in determining responsiveness. J. Exp. Med. 136:931-949. 2. Amsbaugh, D. F., C. T. Hansen, B. Prescott, P. W. Stashak, R. Asofsky, and P. J. Baker. 1974. Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. II. Relationship between IgM immunoglobulin levels and the ability to give an IgM antibody response. J. Exp. Med. 139:1499-1512. 3. Beeson, P. B. 1946. Development of tolerance to typhoid bacterial pyrogen and its abolition by reticuloendothelial blockade. Proc. Soc. Exp. Biol. Med. 61:248-250. 4. Beeson, P. B. 1947. Tolerance to bacterial pyrogens. I. Factors influencing its development. J. Exp. Med. 86: 29-38. 5. Beeson, P. B. 1947. Tolerance to bacterial pyrogens. II. Role of the reticuloendothelial system. J. Exp. Med. 86: 39-44. 6. Glode, L M., A. Jacques, S. E. Mergenhagen, and D. L. Rosenstreich. 1977. Resistance of macrophages from C3H/HeJ mice to the in vitro cytotoxic effects of endotoxin. J. Immunol. 119:162-166. 7. Glode, L M., L. Scher, B. Osborne, and D. L. Rosenstreich. 1976. Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice. J. Immunol. 116:454461. 8. Greisman, S. E., and R. B. Hornick. 1976. Endotoxin tolerance, p. 43-50. In R. F. Beers, Jr. and E. G. Basset (ed.), The role of immunological factors in infectious,
VOL. 24, 1979 allergic and autoimmune disease. Raven Press, New York. 9. Greisman, S. E., H. N. Wagner, Jr., M. His, and R. B. Hornick. 1964. Mechanism of endotoxin tolerance. II. Relationship between endotoxin tolerance and reticuloendothelial system phagocytic activity in man. J. Exp. Med. 119:241-264. 10. Greisman, S. E., and E. J. Young. 1969. Mechanisms of endotoxin tolerance. VI. Transfer of the "anamnestic" tolerant response with primed spleen cells. J. Immunol. 103:1237-1241. 11. Greisman, S. E., E. J. Young, and F. A. Carozza, Jr. 1969. Mechanisms of endotoxin tolerance. V. Specificity of the early and late phases of pyrogenic tolerance. J. Immunol. 103:1223-1236. 12. Greisman, S. E., E. J. Young, and J. B. DuBuy. 1973. Mechanisms of endotoxin tolerance. VIII. Specificity of serum transfer. J. Immunol. 111:1349-1360. 13. McCabe, W. R. 1975. Humoral protection against gramnegative bacilli and endotoxins, p. 336-340. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 14. Milner, K. C. 1973. Patterns of tolerance to endotoxin. J. Infect. Dis. 128(Suppl.):237-245. 15. Mosier, D., I. Scher, and W. E. Paul. 1976. In vitro responses of CBA/N mice: spleen cells of mice with an X-linked defect that precludes immune responses to several thymus-independent antigens can respond to TNP-lipopolysaccharide. J. Immunol. 117:1363-1369. 16. Rittenberg, M. B., and K. L. Pratt. 1968. A nitrophenyl (TNP) plaque assay. Primary response of Balb/c mice to soluble and particulate immunogen. Proc. Soc. Exp. Biol. Med. 132:575-581. 17. Rosenstreich, D. L,, L. M. Glode, L. M. Wahl, A. L. Sandberg, and S. E. Mergenhagen. 1977. Analysis of the cellular defects of endotoxin-unresponsive C3H/ HeJ mice, p. 314-320. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology,
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Washington, D.C. 18. Rosenstreich, D. L., S. N. Vogel, A. Jacques, L. M. Wahl, I. Scher, and S. E. Mergenhagen. 1978. Differential endotoxin sensitivity of lymphocytes and macrophages from mice with an X-linked defect in B cell maturation. J. Immunol. 121:685-690. 19. Rudbach, J. A. 1971. Molecular immunogenicity of bacterial lipopolysaccharide antigens: establishing a quantitative system. J. Immunol. 106:993-1001. 20. Rudbach, J. A., F. I. Akiya, R. J. Elin, H. D. Hochstein, M. K. Luoma, E. C. B. Milner, K. C. Milner, and K. R. Thomas. 1976. Preparation and properties of a national reference endotoxin. J. Clin. Microbiol. 3: 21-25. 21. Scher, I., A. Ahmed, D. M. Strong, A. D. Steinberg, and W. E. Paul. 1975. X-linked B-lymphocyte defect in CBA/N mice. I. Studies of the function and composition of spleen cells. J. Exp. Med. 141:788-803. 23. Scher, I., A. D. Steinberg, A. K. Berning, and W. E. Paul. 1975. X-linked B-lymphocyte defect in CBA/N mice. II. Studies of the mechanisms underlying the immune defect. J. Exp. Med. 142:637-650. 22. Scher, I., S. 0. Sharrow, and W. E. Paul. 1976. Xlinked B-lymphocyte defect in CBA/N mice. III. Abnormal development of B-lymphocyte populations defined by their density of surface immunoglobulin. J. Exp. Med. 144:507-518. 24. Scher, I., N. M. Zaldivar, and D. E. Mosier. 1977. Blymphocyte subpopulations and endotoxin response in CBA/N mice, p. 310-313. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 25. Sultzer, B. M. 1968. Genetic control of leukocyte responses to endotoxin. Nature (London) 219:1253-1254. 26. Watson, J., K. Kelly, and M. Langen. 1977. Genetic and cellular aspects of host response to endotozin, p. 298-303. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C.
