INFECTION AND IMMUNITY, May 1977, p. 513-517 Copyright © 1977 American Society for Microbiology
Vol. 16, No. 2 Printed in U.S.A.
Immunological Responses of Mice to Lipopolysaccharide: Lack of Secondary Responsiveness by C3H/HeJ Mice JON A. RUDBACH* AND NORMAN D. REED Department of Microbiology, University of Montana, Missoula, Montana 59812,* and Department of Microbiology, Montana State University, Bozeman, Montana 59715
Received for publication 11 November 1976
Mice of the C3H/HeJ strain, which were unresponsive to the biological effects of bacterial lipopolysaccharide (LPS), could not be induced to make specific secondary immunological responses to LPS; they responded to two doses of LPS with a primary response. This lack of secondary responsiveness by C3H/HeJ mice was due to a defect in a single, autosomal, dominant gene. Thus, further evidence was provided that an intact second immunological signal and responsiveness thereto were required to trigger secondary antibody responses in primed animals. In a continuing effort to discover the cellular reactions with antigen that will result in the generation of immunological memory and trigger secondary antibody responses, we have studied as a model system the antibody responses of mice to lipopolysaccharide (LPS) antigens. It has been shown that nanogram amounts of LPS sensitized mice for secondary responses (9). Secondary responses to LPS could be produced in congenitally athymic (nude) mice (8); this observation suggested that thymus-derived cells (T-cells) were not required for immunological memory to LPS. More recently it has been shown, with naturally occurring bacterial antigens (10, 20) and with chemically modified LPS preparations (21), that mice could be sensitized specifically for secondary responses with polysaccharide antigens that contained only the immunodeterminant groups, i.e., only the antigenic signal. However, intact LPS containing both the second (toxic, mitogenic lipid A) and the antigenic signal was required to trigger specific secondary responses in primed mice. Therefore, it appeared that both the antigenic and the second signals played necessary but distinct roles in the generation of immunological responses. The antigenic signal appeared to be sufficient to elicit primary responses and to prime immunologically virgin animals. However, both the antigenic and the second signals were required to trigger secondary responses. The present study was designed to test the accuracy of the conclusions on requisites for triggering secondary responses in bone marrow-derived cells. In these experiments the antigen was always LPS that possessed both the antigenic and the second signals, but the geno-
types of the mice used were varied; some mice could respond only to the antigenic signal, and others could respond to both the antigenic and
the second signals. MATERIALS AND METHODS Antigen. The LPS used in these studies was extracted by the phenol-water procedure from Escherichia coli 0113 (Braude strain). Its preparation and properties have been described (11). Animals. Mice of the RML stock were obtained from the Rocky Mountain Laboratory, Hamilton, Mont. BALB/c mice were raised in our colonies at Missoula and Bozeman. Because the RML and BALB/c mice gave similar immunological responses to LPS, data from them were pooled and are reported under the heading of "white mice." The C3H/ HeJ mice were purchased from the Jackson Laboratory, Bar Harbor, Me. C3H/HeJ mice, studied early by Sultzer (16-18) and more recently by others (3, 14, 15, 19, 23), have a defect that can be traced to an inability of their cells to be stimulated by the toxicologically active, lipid A portion of the LPS molecule (1, 7, 13, 15). To obtain F1 generation mice, we crossed C3H/ HeJ females with white males. F1 males and females were intercrossed to produce the F2 generation. F1 males were mated with both C3H/HeJ females and white females to produce backcross generation mice. Because preliminary experiments indicated that responsiveness to LPS was not a sex-linked trait, young adult mice of both sexes were used interchangeably in experiments. All mice were allowed mouse chow and water ad libitum and were maintained under conditions approved by the American Association for Accreditation of Laboratory Animal Care. Immunizations and assays. Mice were injected intravenously on day 0 and/or day 21 with LPS or whole, heat-killed E. coli cells contained in 0.2 ml of phosphate-buffered saline. At the times cited (4 or 513
514
INPFECT . IMMUN .
RUDBACH AND REED
25 days after the last injection), the mice were anesthesized with ether and bled, and the spleens were removed and processed for enumeration of direct LPS-specific plaque-forming cells (PFC). Sera were titrated by passive hemagglutination (HA), and the PFC responses were determined by methods described previously (5, 9), wherein LPS-coated sheep erythrocytes (SRBC) (6) were the indicator cells. In one series of experiments, mice were immunized
with SRBC, and the immunological responses were assessed by using noncoated SRBC. In these experiments (Table 3), both direct and facilitated PFC were enumerated; plaques were facilitated with an optimal dilution of rabbit anti-mouse gamma globulin.
RESULTS
When immune responses were quantified 4 days after a single injection of 1 or 10 jig of LPS, both white and C3H/HeJ mice made primary immune responses; however, as observed by others (23), the primary responses in C3H/HeJ mice were smaller (Table 1, groups B and E compared with groups G and J). A marked difference between the immunological capacities of C3H/HeJ and white mice was noted when attempts were made to induce secondary responses to LPS. Mice were injected on day 0 with 1 jig of LPS and on day 21 with a second injection of 1 or 10 jig of LPS, and immune responses were measured on day 25. White mice gave typical secondary responses (Table 1, groups F and I). In marked contrast, mice of the C3H/HeJ strain could not be induced to give secondary responses to LPS (Table 1, groups A and D); they responded in a quantitatively similar fashion to both one and two injections of LPS.
In an attempt to overcome the inability of C3H/HeJ mice to produce a secondary response to LPS, the LPS was administered in a more immunogenic form, as whole bacterial cells (Table 2). One or two doses of whole E. coli cells elicited similar PFC responses in C3H/HeJ mice, whereas white mice again made good secondary responses. Next to be shown was whether the inability of C3H/HeJ mice to give secondary responses was limited to the T-cell-independent antigen LPS or whether it was a general deficiency in the immune response of C3H/HeJ mice. This was tested by injecting C3H/HeJ and white mice with one or two doses of SRBC. White mice and C3H/HeJ mice both mustered strong secondary antibody responses to SRBC (Table 3). Thus, it appeared that a specific defect was present in C3H/HeJ mice that prevented them from responding secondarily to LPS. Lastly, it was of interest to determine the nature of inheritance of the capacity of mice to give secondary responses to LPS. By appropriate mating, F,, F2, and backcross generations were obtained. These mice were tested for their ability to make secondary responses to LPS. In prior experiments, no C3H/HeJ mouse had responded to two doses of LPS with as many as 3,000 PFC per spleen or an HA titer of more than 64; no white mouse had responded secondarily to LPS with as few as 3,000 PFC per spleen or an HA titer of less than 128. Therefore, we considered, after two injections of LPS, responses of less than 3,000 PFC per spleen and HA titers of 64 or less to be primary-level responses; responses greater than 3,000 PFC per spleen and HA titers of 128 or more were con-
TABLE 1. Immunological responses of C3H/HeJ and white mice to injections of LPS
Injections (,ug) Group
C3H/HeJ A B C D E
No. of mice
23 17 18 6 6
PFC/spleena
Day O
Day 21
1 0 1 1 0
1 1 0 10 10
962 1,013 268
1,736
2,698
232 206 50 11 858
HAb 11 7 8 16 8
White 2,170 1 1 F 23 42,840-- 7,397 86 11,740 ± 2,093 1 0 17 G 41 210 1,093 0 1 H 18 512 79,060 ± 21,480 I 10 1 6 256 408 11,330 ± 10 J 6 0 aArithmetic mean LPS-specific PFC per spleen + standard error of the mean; PFC assay was done on day 25. b Geometric mean passive HA titer; mice were bled on day 25.
IMMUNOLOGICAL RESPONSES TO LPS
VOL. 16, 1977
sidered to be secondary-level responses. Preliminary experiments had established that the ability to make secondary responses to LPS was not a sex-linked trait. Therefore, we calculated the number of mice expected to make primary and secondary-level responses, assuming that the capacity to respond in a heightened fashion to the second injection of LPS was inherited as a single, autosomal, dominant gene. In two experiments carried out in separate laboratories (Table 4) the numbers of primary- and secondary-level responders observed correlated very closely with the numbers expected. It appeared that our assumption was correct. The ability of mice to make secondary responses to LPS was due to the activity of a single, autosomal, dominant gene. DISCUSSION The interpretations of data derived from prior experiments (10, 20, 21), which were designed to elucidate the signaling mechanisms in primary and secondary antibody responses, may be summarized as follows. (i) Unprimed bone marrow-derived cells can be stimulated to produce a primary antibody response and can TABLE 2. Immunological responses of C3HIHeJ and white mice to injections of whole E. coli cells No.
of
Type of mouse
mice
C3H/HeJ
2 4
Injectionsa Day Day 0
21
WC PBS
WC WC
PFC/spleen PCsle 25,160 25,680
be sensitized for a secondary response by the antigenic signal alone, i.e., by native protoplasmic polysaccharide (20) or alkaline detoxified LPS (21). (ii) The triggering of a secondary antibody response in a primed animal requires both the antigenic and the second signal. In the case of LPS, the second signal probably resides in the lipid A portion of the molecule (20). In the present study mice of the C3H/HeJ strain, which are unresponsive to the toxicologically active, lipid A portion of the LPS molecule, made primary immunological responses to LPS or whole E. coli cells but failed to make secondary responses; they responded to two doses of LPS or whole E. coli cells with a primary-level response. The results of the genetic analysis of the capacity to produce a secondary response to LPS suggested that responsiveness was determined by a single, autosomal, dominant gene. A similar pattern of inheritance of the ability to make mitogenic responses to LPS has been proposed (23). Thus, more data were provided that support the concept that the second signal and the mitogenic signal are identical. From the data presented here, a third conclusion may be added to the two mentioned previously: to respond in a secondary fashion, primed mice must have the genetic capacity to respond to the second signal. C3H/HeJ mice failed to respond to lipid A, the second signal in the immune response to the T-cell-independent antigen LPS, and they were, therfore, incapable of making secondary responses to LPS. This inability to make a secondary response to LPS was
WC WC 310,900 WC PBS 40,010 a Phosphate-buffered saline (PBS) or 109 whole E. coli cells (WC) were injected intravenously. b See footnote a, Table 1. White
2 2
515
not due to a general
anergy
in the immuno-
logical systems of C3H/HeJ mice because they could make strong secondary responses to the T-cell-dependent antigen SRBC. In the latter response, the second signal is not lipid A but rather some product of stimulated T-cells (2, 4, 12, 22). Apparently, the inability of C3H/HeJ
TABLE 3. Immunological responses of C3H/HeJ and white mice to injections of SRBC Injectionsa
Type of mouse
No. of mice
C3H/HeJ
9 10 8
White
10 10 8
Day 0 SRBC PBS SRBC
Day 21 SRBC SRBC PBS
PFC/spleenb
HAc
452,080 ± 139,000 42,500 ± 13,040 6,681 + 2,843
1,634 127 181
SRBC SRBC 686,000 + 209,000 4,720 PBS SRBC 128 138,100 ± 40,370 SRBC 216 PBS 8,531 + 2,531 a Phosphate-buffered saline (PBS) or approximately 10" SRBC were injected intravenously. b Arithmetic mean of total (direct and facilitated) SRBC-specific PFC per spleen + standard error of the mean; PFC assay done on day 25. c Geometric mean HA titer; mice were bled on day 25.
516
INFECT. IMMUN.
RUDBACH AND REED
TABLE 4. Secondary immunological responses to LPS in the parental strains and in the F1, F2, and backcross generationsa Type of mouse Expt 1 C3H/HeJ White F, F, x white F, x C3H/HeJ
F2 Expt 2 C3H/HeJ White
F, F, x white
F2
No. of mice
No. of primary- and secondary-level responses" Observed Expectedc 10
20
10
20
11 12 13 15 14 42
11 0 0 0 7 10.5
0 11 12 0 0 13 0 15 6 7 31.5 11
0 12 13 15 8 31
16 107 24 94 72
16 0 0 0 18
16 4 0 1 15
0 103 24 93 57
0 107 24 94 54
All mice were injected with 1 Ag of LPS on days 0 and 21; on day 25, the mice were tested for immunological responses to LPS. b Passive HA titers of 64 or less were considered primarylevel responses (10), and passive hemagglutination titers of 128 or more were considered secondary-level responses (2°). Identical distributions of mice were obtained if the number of splenic PFC was determined: 3,000 PFC or less indicated a primary-level response; more than 3,000 PFC indicated a secondary-level response. c Expected frequency was calculated assuming that responsiveness to LPS is determined by one dominant, autosomal gene. a
mice to respond to the second, toxic signal of LPS was due to an inability of their cells or to a lack of a membrane component on their cells that could react with the lipid A region of the LPS molecule (24). Inasmuch as memory cells could be triggered by a T-cell-dependent antigen, this would indicate that the receptors for the T-cell product and the receptors for the lipid A were different. ACKNOWLEDGMENTS This study was supported by Public Health Service research grant Al 10384 and Research Career Development Award AI 70208 (to N.D.R.) from the National Institute of Allergy and Infectious Diseases. We thank M. K. Luoma and Pat Healow for their expert technical assistance. LITERATURE CITED 1. Andersson, J., F. Mechers, C. Galanos, and 0. Luderitz. 1973. The mitogenic effect of lipopolysaccharide on bone marrow-derived mouse lymphocytes. Lipid A as the mitogenic part of the molecule. J. Exp. Med. 137:943-953. 2. Armerding, D., and D. H. Katz. 1974. Activation of T and B lymphocytes in vitro. II. Biological and biochemical properties of an allogeneic effect factor (AEF) active in triggering specific B lymphocytes. J. Exp. Med. 140:19-37. 3. Coutinho, A., E. Gronowicz, and B. M. Sultzer. 1975.
Genetic control of B-cell responses. I. Selective unresponsiveness to lipopolysaccharide. Scand. J. Immunol. 4:139-143. 4. Gorczynski, R. M., R. G. Miller, and R. A. Phillips. 1973. Reconstitution of T cell-depleted spleen cell populations by factors derived from T cells. I. Conditions for the production of active T cell supernatants. J. Immunol. 110:968-983. 5. Manning, J. K., N. D. Reed, and J. W. Jutila. 1972.
Antibody response to Escherichia coli lipopolysaccha-
ride and type III pneumococcal polysaccharide by congenitally thymusless (nude) mice. J. Immunol. 108:1470-1472. 6. Neter, E., 0. Westphal, 0. Luderitz, E. A. Gorzynski, and E. Eichenberger. 1956. Studies of enterobacterial lipopolysaccharides. Effects of heat and chemicals on erythrocyte-modifying, antigenic toxic and pyrogenic properties. J. Immunol. 76:377-385. 7. Peavy, D. L., J. W. Shands, Jr., W. H. Adler, and R. T. Smith. 1973. Mitogenicity of bacterial endotoxins: characterization of the mitogenic principle. J. Immunol. 111:352-357. 8. Reed, N. D., J. K. Manning, and J. A. Rudbach. 1973. Immunologic responses of mice to lipopolysaccharide
from Escherichia coli. J. Infect. Dis. 128:S70-S74.
9. Rudbach, J. A. 1971. Molecular immunogenicity of bacterial lipopolysaccharide antigens: establishing a quantitative system. J. Immunol. 106:993-1001. 10. Rudbach, J. A. 1976. Immunogenicity of lipopolysaccharides, p. 29-41. In R. F. Beers, Jr., and E. G. Bassett (ed.), The role of immunological factors in infectious, allergic, and autoimmune processes. Raven Press, New York. 11. 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. 12. Sjoberg, O., J. Andersson, and G. Moller. 1972. Reconstitution of antibody response in vitro of T cell-deprived spleen cells by supernatants from spleen cell cultures. J. Immunol. 109:1379-1385. 13. Skidmore, B. J., J. M. Chiller, D. C. Morrison, and W. 0. Weigle. 1975. Immunologic properties of bacterial lipopolysaccharide (LPS): correlation between the mitogenic, adjuvant, and immunogenic activities. J. Immunol. 114:770-775. 14. Skidmore, B. J., J. M. Chiller, W. 0. Weigle, R. Riblet, and J. Watson. 1976. Immunologic properties of bacterial lipopolysaccharide (LPS). III. Genetic linkage between the in vitro mitogenic and in vivo adjuvant properties of LPS. J. Exp. Med. 143:143-150. 15. Skidmore, B. J., D. C. Morrison, J. M. Chiller, and W. 0. Weigle. 1975. Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS. J. Exp. Med. 142:1488-1508. 16. Sultzer, B. M. 1968. Genetic control of leucocytic responses to endotoxin. Nature (London) 219:1253-1254. 17. Sultzer, B. M. 1969. Genetic factors in leucocyte responses to endotoxin: further studies in mice. J. Immunol. 103:32-38. 18. Sultzer, B. M. 1972. Genetic control ofhost responses to endotoxin. Infect. Immun. 5:107-113. 19. Talcott, J. A., D. B. Ness, and F. C. Grumet. 1975. Adjuvant effect of LPS on in vivo antibody response: genetic defect in C3H/HeJ mice. Immunogenetics 2:507-515. 20. Von Eschen, K. B., and J. A. Rudbach. 1974. Immunological responses of mice to native protoplasmic polysaccharide and lipopolysaccharide. Functional separation of the two signals required to stimulate a sec-
VOL. 16, 1977 ondary antibody response. J. Exp. Med. 140:16041614. 21. Von Eschen, K. B., and J. A. Rudbach. 1976. Antibody responses of mice to alkaline detoxified lipopolysaccharide. J. Immunol. 116:8-11. 22. Watson, J. 1973. The role of humoral factors in the initiation of in vitro primary immune responses. Ifl. Characterization of factors that replace thymus-derived cells. J. Immunol. 111:1301-1313. 23. Watson, J., and R. Riblet. 1974. Genetic control of
IMMUNOLOGICAL RESPONSES TO LPS
517
responses to bacterial lipopolysaccharides in mice. I. Evidence for' a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J. Exp. Med. 140:1147-1161. 24. Watson, J., and R. Riblet. 1975. Genetic control of responses to bacterial lipopolysaccharides in mice. H. A gene that influences a membrane component involved in the activation of bone marrow-derived lymphocytes by lipopolysaccharides. J. Immunol. 114:1462-1468.
Vol. 16, No. 2 Printed in U.S.A.
Immunological Responses of Mice to Lipopolysaccharide: Lack of Secondary Responsiveness by C3H/HeJ Mice JON A. RUDBACH* AND NORMAN D. REED Department of Microbiology, University of Montana, Missoula, Montana 59812,* and Department of Microbiology, Montana State University, Bozeman, Montana 59715
Received for publication 11 November 1976
Mice of the C3H/HeJ strain, which were unresponsive to the biological effects of bacterial lipopolysaccharide (LPS), could not be induced to make specific secondary immunological responses to LPS; they responded to two doses of LPS with a primary response. This lack of secondary responsiveness by C3H/HeJ mice was due to a defect in a single, autosomal, dominant gene. Thus, further evidence was provided that an intact second immunological signal and responsiveness thereto were required to trigger secondary antibody responses in primed animals. In a continuing effort to discover the cellular reactions with antigen that will result in the generation of immunological memory and trigger secondary antibody responses, we have studied as a model system the antibody responses of mice to lipopolysaccharide (LPS) antigens. It has been shown that nanogram amounts of LPS sensitized mice for secondary responses (9). Secondary responses to LPS could be produced in congenitally athymic (nude) mice (8); this observation suggested that thymus-derived cells (T-cells) were not required for immunological memory to LPS. More recently it has been shown, with naturally occurring bacterial antigens (10, 20) and with chemically modified LPS preparations (21), that mice could be sensitized specifically for secondary responses with polysaccharide antigens that contained only the immunodeterminant groups, i.e., only the antigenic signal. However, intact LPS containing both the second (toxic, mitogenic lipid A) and the antigenic signal was required to trigger specific secondary responses in primed mice. Therefore, it appeared that both the antigenic and the second signals played necessary but distinct roles in the generation of immunological responses. The antigenic signal appeared to be sufficient to elicit primary responses and to prime immunologically virgin animals. However, both the antigenic and the second signals were required to trigger secondary responses. The present study was designed to test the accuracy of the conclusions on requisites for triggering secondary responses in bone marrow-derived cells. In these experiments the antigen was always LPS that possessed both the antigenic and the second signals, but the geno-
types of the mice used were varied; some mice could respond only to the antigenic signal, and others could respond to both the antigenic and
the second signals. MATERIALS AND METHODS Antigen. The LPS used in these studies was extracted by the phenol-water procedure from Escherichia coli 0113 (Braude strain). Its preparation and properties have been described (11). Animals. Mice of the RML stock were obtained from the Rocky Mountain Laboratory, Hamilton, Mont. BALB/c mice were raised in our colonies at Missoula and Bozeman. Because the RML and BALB/c mice gave similar immunological responses to LPS, data from them were pooled and are reported under the heading of "white mice." The C3H/ HeJ mice were purchased from the Jackson Laboratory, Bar Harbor, Me. C3H/HeJ mice, studied early by Sultzer (16-18) and more recently by others (3, 14, 15, 19, 23), have a defect that can be traced to an inability of their cells to be stimulated by the toxicologically active, lipid A portion of the LPS molecule (1, 7, 13, 15). To obtain F1 generation mice, we crossed C3H/ HeJ females with white males. F1 males and females were intercrossed to produce the F2 generation. F1 males were mated with both C3H/HeJ females and white females to produce backcross generation mice. Because preliminary experiments indicated that responsiveness to LPS was not a sex-linked trait, young adult mice of both sexes were used interchangeably in experiments. All mice were allowed mouse chow and water ad libitum and were maintained under conditions approved by the American Association for Accreditation of Laboratory Animal Care. Immunizations and assays. Mice were injected intravenously on day 0 and/or day 21 with LPS or whole, heat-killed E. coli cells contained in 0.2 ml of phosphate-buffered saline. At the times cited (4 or 513
514
INPFECT . IMMUN .
RUDBACH AND REED
25 days after the last injection), the mice were anesthesized with ether and bled, and the spleens were removed and processed for enumeration of direct LPS-specific plaque-forming cells (PFC). Sera were titrated by passive hemagglutination (HA), and the PFC responses were determined by methods described previously (5, 9), wherein LPS-coated sheep erythrocytes (SRBC) (6) were the indicator cells. In one series of experiments, mice were immunized
with SRBC, and the immunological responses were assessed by using noncoated SRBC. In these experiments (Table 3), both direct and facilitated PFC were enumerated; plaques were facilitated with an optimal dilution of rabbit anti-mouse gamma globulin.
RESULTS
When immune responses were quantified 4 days after a single injection of 1 or 10 jig of LPS, both white and C3H/HeJ mice made primary immune responses; however, as observed by others (23), the primary responses in C3H/HeJ mice were smaller (Table 1, groups B and E compared with groups G and J). A marked difference between the immunological capacities of C3H/HeJ and white mice was noted when attempts were made to induce secondary responses to LPS. Mice were injected on day 0 with 1 jig of LPS and on day 21 with a second injection of 1 or 10 jig of LPS, and immune responses were measured on day 25. White mice gave typical secondary responses (Table 1, groups F and I). In marked contrast, mice of the C3H/HeJ strain could not be induced to give secondary responses to LPS (Table 1, groups A and D); they responded in a quantitatively similar fashion to both one and two injections of LPS.
In an attempt to overcome the inability of C3H/HeJ mice to produce a secondary response to LPS, the LPS was administered in a more immunogenic form, as whole bacterial cells (Table 2). One or two doses of whole E. coli cells elicited similar PFC responses in C3H/HeJ mice, whereas white mice again made good secondary responses. Next to be shown was whether the inability of C3H/HeJ mice to give secondary responses was limited to the T-cell-independent antigen LPS or whether it was a general deficiency in the immune response of C3H/HeJ mice. This was tested by injecting C3H/HeJ and white mice with one or two doses of SRBC. White mice and C3H/HeJ mice both mustered strong secondary antibody responses to SRBC (Table 3). Thus, it appeared that a specific defect was present in C3H/HeJ mice that prevented them from responding secondarily to LPS. Lastly, it was of interest to determine the nature of inheritance of the capacity of mice to give secondary responses to LPS. By appropriate mating, F,, F2, and backcross generations were obtained. These mice were tested for their ability to make secondary responses to LPS. In prior experiments, no C3H/HeJ mouse had responded to two doses of LPS with as many as 3,000 PFC per spleen or an HA titer of more than 64; no white mouse had responded secondarily to LPS with as few as 3,000 PFC per spleen or an HA titer of less than 128. Therefore, we considered, after two injections of LPS, responses of less than 3,000 PFC per spleen and HA titers of 64 or less to be primary-level responses; responses greater than 3,000 PFC per spleen and HA titers of 128 or more were con-
TABLE 1. Immunological responses of C3H/HeJ and white mice to injections of LPS
Injections (,ug) Group
C3H/HeJ A B C D E
No. of mice
23 17 18 6 6
PFC/spleena
Day O
Day 21
1 0 1 1 0
1 1 0 10 10
962 1,013 268
1,736
2,698
232 206 50 11 858
HAb 11 7 8 16 8
White 2,170 1 1 F 23 42,840-- 7,397 86 11,740 ± 2,093 1 0 17 G 41 210 1,093 0 1 H 18 512 79,060 ± 21,480 I 10 1 6 256 408 11,330 ± 10 J 6 0 aArithmetic mean LPS-specific PFC per spleen + standard error of the mean; PFC assay was done on day 25. b Geometric mean passive HA titer; mice were bled on day 25.
IMMUNOLOGICAL RESPONSES TO LPS
VOL. 16, 1977
sidered to be secondary-level responses. Preliminary experiments had established that the ability to make secondary responses to LPS was not a sex-linked trait. Therefore, we calculated the number of mice expected to make primary and secondary-level responses, assuming that the capacity to respond in a heightened fashion to the second injection of LPS was inherited as a single, autosomal, dominant gene. In two experiments carried out in separate laboratories (Table 4) the numbers of primary- and secondary-level responders observed correlated very closely with the numbers expected. It appeared that our assumption was correct. The ability of mice to make secondary responses to LPS was due to the activity of a single, autosomal, dominant gene. DISCUSSION The interpretations of data derived from prior experiments (10, 20, 21), which were designed to elucidate the signaling mechanisms in primary and secondary antibody responses, may be summarized as follows. (i) Unprimed bone marrow-derived cells can be stimulated to produce a primary antibody response and can TABLE 2. Immunological responses of C3HIHeJ and white mice to injections of whole E. coli cells No.
of
Type of mouse
mice
C3H/HeJ
2 4
Injectionsa Day Day 0
21
WC PBS
WC WC
PFC/spleen PCsle 25,160 25,680
be sensitized for a secondary response by the antigenic signal alone, i.e., by native protoplasmic polysaccharide (20) or alkaline detoxified LPS (21). (ii) The triggering of a secondary antibody response in a primed animal requires both the antigenic and the second signal. In the case of LPS, the second signal probably resides in the lipid A portion of the molecule (20). In the present study mice of the C3H/HeJ strain, which are unresponsive to the toxicologically active, lipid A portion of the LPS molecule, made primary immunological responses to LPS or whole E. coli cells but failed to make secondary responses; they responded to two doses of LPS or whole E. coli cells with a primary-level response. The results of the genetic analysis of the capacity to produce a secondary response to LPS suggested that responsiveness was determined by a single, autosomal, dominant gene. A similar pattern of inheritance of the ability to make mitogenic responses to LPS has been proposed (23). Thus, more data were provided that support the concept that the second signal and the mitogenic signal are identical. From the data presented here, a third conclusion may be added to the two mentioned previously: to respond in a secondary fashion, primed mice must have the genetic capacity to respond to the second signal. C3H/HeJ mice failed to respond to lipid A, the second signal in the immune response to the T-cell-independent antigen LPS, and they were, therfore, incapable of making secondary responses to LPS. This inability to make a secondary response to LPS was
WC WC 310,900 WC PBS 40,010 a Phosphate-buffered saline (PBS) or 109 whole E. coli cells (WC) were injected intravenously. b See footnote a, Table 1. White
2 2
515
not due to a general
anergy
in the immuno-
logical systems of C3H/HeJ mice because they could make strong secondary responses to the T-cell-dependent antigen SRBC. In the latter response, the second signal is not lipid A but rather some product of stimulated T-cells (2, 4, 12, 22). Apparently, the inability of C3H/HeJ
TABLE 3. Immunological responses of C3H/HeJ and white mice to injections of SRBC Injectionsa
Type of mouse
No. of mice
C3H/HeJ
9 10 8
White
10 10 8
Day 0 SRBC PBS SRBC
Day 21 SRBC SRBC PBS
PFC/spleenb
HAc
452,080 ± 139,000 42,500 ± 13,040 6,681 + 2,843
1,634 127 181
SRBC SRBC 686,000 + 209,000 4,720 PBS SRBC 128 138,100 ± 40,370 SRBC 216 PBS 8,531 + 2,531 a Phosphate-buffered saline (PBS) or approximately 10" SRBC were injected intravenously. b Arithmetic mean of total (direct and facilitated) SRBC-specific PFC per spleen + standard error of the mean; PFC assay done on day 25. c Geometric mean HA titer; mice were bled on day 25.
516
INFECT. IMMUN.
RUDBACH AND REED
TABLE 4. Secondary immunological responses to LPS in the parental strains and in the F1, F2, and backcross generationsa Type of mouse Expt 1 C3H/HeJ White F, F, x white F, x C3H/HeJ
F2 Expt 2 C3H/HeJ White
F, F, x white
F2
No. of mice
No. of primary- and secondary-level responses" Observed Expectedc 10
20
10
20
11 12 13 15 14 42
11 0 0 0 7 10.5
0 11 12 0 0 13 0 15 6 7 31.5 11
0 12 13 15 8 31
16 107 24 94 72
16 0 0 0 18
16 4 0 1 15
0 103 24 93 57
0 107 24 94 54
All mice were injected with 1 Ag of LPS on days 0 and 21; on day 25, the mice were tested for immunological responses to LPS. b Passive HA titers of 64 or less were considered primarylevel responses (10), and passive hemagglutination titers of 128 or more were considered secondary-level responses (2°). Identical distributions of mice were obtained if the number of splenic PFC was determined: 3,000 PFC or less indicated a primary-level response; more than 3,000 PFC indicated a secondary-level response. c Expected frequency was calculated assuming that responsiveness to LPS is determined by one dominant, autosomal gene. a
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