Immunology 1977 33 629
Antibody response to Escherichia coil 0 antigens and influence of the protein moiety of the endotoxin S. AHLSTEDT * & L. LINDHOL M Department of Immunology, Institute of Medical Microbiology, University of Gdteborg, Goteborg, Sweden
Received 23 December 1976; acceptedfor publication 9 March 1977
Summary. Exposure of mice to different serotypes of
A secondary response to E. coli 06 bacteria was also noted in agreement with previous results. It was found that this immune response could be reduced drastically by injecting primed thymocytes, simultaneously with the second injection.
E. coli bacteria, either 04 or 06, resulted in an enhanced indirect IgG-PFC response to the alternate bacteria. This effect seemed to be mediated by a protein connected with the endotoxin structure. This protein moiety had some weak adjuvant activities and increased the antibody response against sheep red blood cell about two-fold. This effect was not likely to be due to any contamination with the B-cell mitogen lipid A, a constituent of the endotoxin from which the protein was isolated. In addition, experiments were performed in which irradiated spleen cells (0-400 R) from mice injected with E. coli 04 bacteria were transferred to irradiated (800 R) recipients together with E. coil 06 bacteria. Decreasing numbers of antibody forming cells with increasing irradiation dose were found. The parallel experiment employing E. coli 06 bacteria for both primary and secondary antigen injections revealed an increased immune response for an irradiation dose of 50 R, showing that suppressor cells are more irradiation sensitive than the other cells involved in this immune response, but that the effect of such cells is possibly overcome by the influence of the protein residue isolated from endotoxin.
INTRODUCTION The adjuvant effect of the endotoxins on the antibody response to protein antigens is well established (Johnson, Gainer & Landy, 1956). In contrast, endotoxin has been claimed not to enhance antibody formation against other endotoxins (Neter, 1969). Previously it was shown, however, that patients with recurrent urinary tract infections formed more precipitating IgG antibodies than patients with their first infection in spite of varying infecting strains of Escherichia coil bacteria (Hanson, Holmgren, Jodal & Winberg, 1969). This observation of enhanced IgG antibody production has been confirmed in experimental systems using mice and rabbits (e.g. Ahlstedt & Holmgren, 1975; Ahlstedt & Kaijser, 1975). The mechanism for the altered antibody response described has not been determined. The effects are not due to serologically cross-reactive 0 antigens nor to shared core structures nor to the so-called common antigen in the cell wall (Ahlstedt & Holmgren, 1975). However, an immunologically identical protein structure linked to the endotoxin has been
* Present address: Astra Lakemedel AB, Research and Development Laboratories, SodertUlje, Sweden. Correspondence: Dr Staffan Ahlstedt, Research and Development Laboratories, Astra Lakemedel AB, S-151 85 Sodertaije, Sweden.
629
630
S. Ahlstedt & L. Lindholm
shown in various species of bacteria belonging to Enterobacteriaceae (Wober & Alaupovic, 1971). In the present study it was hoped to show whether the protein moiety of the endotoxin had any effect on the anti-O antibody response after repeated treatment of mice with different E. coli 0 antigens. Experiments were also performed comparing the anti-06 antibody responses obtained after a primary injection with E. coli 04 bacteria and a second injection with 06 bacteria with that using E. coli 06 bacteria for both the primary and secondary injections. In addition, the influence of thymusderived cells on the secondary immune response was studied. MATERIALS AND METHODS
Animals. 6-week-old male and female CBA mice, five animals per group, bred in our laboratory were used throughout the study. In some experiments irradiation (0-800 R) of animals or cell suspensions was performed utilizing a 60Co source with a dose rate of 200 per min.
Antigens. Formalin-killed or boiled E. coil bacteria of the 04 and 06 (WHO strains designated U 4/41 and Su 4344/41 respectively) were used. The protein moiety of the 04 endotoxin was extracted and oxidized as described by Wober & Alaupovic (1971). Purified lipopolysaccharide (LPS) of E. coli 04 serotype was prepared according to 0rskov, 0rskov, Jann, Jann, MUller-Seitz & Westphal (1967). The preparation was found free of protein according to Lowry analysis, i.e. less than 0-25 g/l which corresponded to less than 0-5 %4 protein in the LPS. Sheep or human red blood cells (RBC) were also used. Injections. Mice were injected intravenously with the antigens suspended in phosphate buffer saline, pH = 7-2 (PBS). Two subsequent injections were given at 5 or 30 days after the last injection. The formalin-killed or boiled E. coli-bacteria were administered intravenously in a dose of 107 organisms per injection. The protein moiety of the 04 endotoxin was given in the optimal dose, 10 pg per mouse. It was injected alone or together with boiled 04 bacteria, with 10 pg of purified 04-LPS, or with 107 human or 108 sheep RBC.
Cell transfer. In some experiments cell suspensions of thymus or spleen from immunized mice (107 formalin-killed E. coli 06 bacteria) or PBS treated animals were prepared. The cells, 2 x 10, were injected into E. coli 06 immunized animals together with bacteria. The bacterial injections were given 5-28 days apart.
Antibody assay. The local haemolysis technique as described by Cunningham & Szenberg (1968) was used. The antibody response estimated using sheep RBC coated with heated extract of E. coli 06 antigen (Ahlstedt, Holmgren & Hanson, 1973) was expressed as plaque-forming cells (PFC) per spleen after subtracting the background PFC. Direct (IgM) as well as indirect (IgG) PFC were registered. The indirect plaques were developed by addition of an optimal dilution (1: 40) of goat anti-mouse y-globulin serum (Miles Research Lab., Kankakee, Ill.). This concentration of the antiserum suppressed more than 90%4 of the direct plaques. Statistics. The statistical analysis of mean values was performed using the Wilcoxon rank sum test.
RESULTS Influence of pretreatment with E. coli 04 antigens on the PFC formation to formalin-killed E. coli 06 bacteria The influence of formalin-killed bacteria alone or combinations of protein extracted from the 04 endotoxin and boiled 04 bacteria or purified 04 LPS or human red blood cells or saline on the antibody response to formalin-killed E. coil 06 bacteria given 28 days later was studied. Direct PFC responses were induced with all the antigen combinations used. The relative capacities of the different antigens to induce direct PFC were (protein + boiled bacteria) > (formalin-killed bacteria) > (protein + human RBC) = (protein + LPS) > (protein + saline). Indirect PFC responses were also induced. In this instance the relative efficiencies of the different combination were (protein + boiled bacteria) = (protein + human RBC) > (formalin killed bacteria) > (protein + saline) = (protein + LPS). The results suggest the importance of the protein for the enhancement of the indirect PFC response but not for the direct PFC response (Table 1).
Antibody response
to
Escherichia coli
Table 1. The antibody response (PFC per spleen) to E. colU 06 after a first injection of various E. coli 04 antigens and a second injection of formalin-killed E. coli 06 bacteria
O
R
0
antigens
631 z-
I
IgM-PFC
IgG-PFC
Formalin-killed 04 bacteria Boiled 04 bacteria + protein 04 LPS + protein Human RBC +protein Protein None
24,000 ± 2000* 30,200 ± 4500 13,700 ± 1800 14,400 ± 3000 6800 ±1300 7500 ±1000
32,000 ± 5000 40,300 ± 5100 20,200 ±2200 40,300±4200 22,400 ± 3800 0
50 R
200 R 400 R
(a) L. O
50 R
Effect of the protein moiety of the E. coli 04 endotoxin on PFC response to sheep red blood cells The effect of the protein moiety of the E. coli 04 endotoxin on the secondary PFC response to sheep RBC antigen was analysed. A prior injection of formalin-killed E. coil 04 bacteria on day 0 increased the antibody response about two-fold to sheep administered on day 5 (P< 0.01). The simultaneous injection of the protein moiety isolated from the 04 endotoxin together with sheep RBC on day 0 increased the indirect PFC response to sheep RBC given at day 5. However, this increase was less than the one described above, although significant (P< 0 05) (Table 2).
100 R
Irradiation sensitivity of spleen cells responding to E. coli 06 bacteria after the first and second injections with formalin-killed bacteria To determine the irradiation sensitivity of the response to formalin-killed 06 bacteria spleen cell suspensions were prepared from mice injected with formalin-killed E. coil 04 bacteria 5 days after antigen administration. These cells were irradiated with 0400 R and injected into irradiated (800 R)
mmo.p-
R
* Mean+ s.e.
L=
200 R 400 R :
(b)
100
1000
Figure 1. The PFC response on day 4 in irradiated (800 R) recipient mice, injected with 108 formalin-killed E. coli bacteria and 4 x 107 irradiated (0-400 R) spleen cells from donor mice given 108 formalin-killed E. coli bacteria 5 days previously Open bars, direct IgM-PFC; solid bars, indirect IgG-PFC; brackets indicate the standard error. (a) E. coli 04 bacteria were given to the donor mice and E. coli 06 bacteria to the recipient mice. (b) E. coli 06 bacteria were given both to the donor and the recipient mice.
recipient mice together with formalin-killed E. coli 06 bacteria. This resulted in PFC responses related to the irradiation dose on the cells and to the number of indirect PFC induced. The numbers of direct PFC induced were considerably lower than those of indirect PFC (P< 0 01). Similar direct PFC responses were registered for the animals given cells irradiated 0-100 R (Fig. la).
PFC response
Day 9
Secondary injection day 5
Direct PFC
Indirect PFC
108 sheep RBC
11,825 ± 2700*
20,900 ±4000
108 sheep RBC
12,375 ± 2500
33,275 ± 6100
108 sheep RBC
21,450 ± 2900
54,800 ± 5800
*
10000
PFC
Table 2. Adjuvant effect of the protein isolated from endotoxin on the PFC response against sheep red blood cells measured as PFC/spleen 4 days after the last injection
108 sheep RBC 108 sheep RBC + 10ug protein 107 formalin-killed E. coli 04 bacteria
momml
100 R
Primary antigen
Primary injection day 0
-
Mean+ s.e.
632
S. Ahlstedt & L. Lindholm 50000 r-
days compared with the primary antibody response. When the injections were spaced 28 days apart an accentuated response was seen (P
Antibody response to Escherichia coil 0 antigens and influence of the protein moiety of the endotoxin S. AHLSTEDT * & L. LINDHOL M Department of Immunology, Institute of Medical Microbiology, University of Gdteborg, Goteborg, Sweden
Received 23 December 1976; acceptedfor publication 9 March 1977
Summary. Exposure of mice to different serotypes of
A secondary response to E. coli 06 bacteria was also noted in agreement with previous results. It was found that this immune response could be reduced drastically by injecting primed thymocytes, simultaneously with the second injection.
E. coli bacteria, either 04 or 06, resulted in an enhanced indirect IgG-PFC response to the alternate bacteria. This effect seemed to be mediated by a protein connected with the endotoxin structure. This protein moiety had some weak adjuvant activities and increased the antibody response against sheep red blood cell about two-fold. This effect was not likely to be due to any contamination with the B-cell mitogen lipid A, a constituent of the endotoxin from which the protein was isolated. In addition, experiments were performed in which irradiated spleen cells (0-400 R) from mice injected with E. coli 04 bacteria were transferred to irradiated (800 R) recipients together with E. coil 06 bacteria. Decreasing numbers of antibody forming cells with increasing irradiation dose were found. The parallel experiment employing E. coli 06 bacteria for both primary and secondary antigen injections revealed an increased immune response for an irradiation dose of 50 R, showing that suppressor cells are more irradiation sensitive than the other cells involved in this immune response, but that the effect of such cells is possibly overcome by the influence of the protein residue isolated from endotoxin.
INTRODUCTION The adjuvant effect of the endotoxins on the antibody response to protein antigens is well established (Johnson, Gainer & Landy, 1956). In contrast, endotoxin has been claimed not to enhance antibody formation against other endotoxins (Neter, 1969). Previously it was shown, however, that patients with recurrent urinary tract infections formed more precipitating IgG antibodies than patients with their first infection in spite of varying infecting strains of Escherichia coil bacteria (Hanson, Holmgren, Jodal & Winberg, 1969). This observation of enhanced IgG antibody production has been confirmed in experimental systems using mice and rabbits (e.g. Ahlstedt & Holmgren, 1975; Ahlstedt & Kaijser, 1975). The mechanism for the altered antibody response described has not been determined. The effects are not due to serologically cross-reactive 0 antigens nor to shared core structures nor to the so-called common antigen in the cell wall (Ahlstedt & Holmgren, 1975). However, an immunologically identical protein structure linked to the endotoxin has been
* Present address: Astra Lakemedel AB, Research and Development Laboratories, SodertUlje, Sweden. Correspondence: Dr Staffan Ahlstedt, Research and Development Laboratories, Astra Lakemedel AB, S-151 85 Sodertaije, Sweden.
629
630
S. Ahlstedt & L. Lindholm
shown in various species of bacteria belonging to Enterobacteriaceae (Wober & Alaupovic, 1971). In the present study it was hoped to show whether the protein moiety of the endotoxin had any effect on the anti-O antibody response after repeated treatment of mice with different E. coli 0 antigens. Experiments were also performed comparing the anti-06 antibody responses obtained after a primary injection with E. coli 04 bacteria and a second injection with 06 bacteria with that using E. coli 06 bacteria for both the primary and secondary injections. In addition, the influence of thymusderived cells on the secondary immune response was studied. MATERIALS AND METHODS
Animals. 6-week-old male and female CBA mice, five animals per group, bred in our laboratory were used throughout the study. In some experiments irradiation (0-800 R) of animals or cell suspensions was performed utilizing a 60Co source with a dose rate of 200 per min.
Antigens. Formalin-killed or boiled E. coil bacteria of the 04 and 06 (WHO strains designated U 4/41 and Su 4344/41 respectively) were used. The protein moiety of the 04 endotoxin was extracted and oxidized as described by Wober & Alaupovic (1971). Purified lipopolysaccharide (LPS) of E. coli 04 serotype was prepared according to 0rskov, 0rskov, Jann, Jann, MUller-Seitz & Westphal (1967). The preparation was found free of protein according to Lowry analysis, i.e. less than 0-25 g/l which corresponded to less than 0-5 %4 protein in the LPS. Sheep or human red blood cells (RBC) were also used. Injections. Mice were injected intravenously with the antigens suspended in phosphate buffer saline, pH = 7-2 (PBS). Two subsequent injections were given at 5 or 30 days after the last injection. The formalin-killed or boiled E. coli-bacteria were administered intravenously in a dose of 107 organisms per injection. The protein moiety of the 04 endotoxin was given in the optimal dose, 10 pg per mouse. It was injected alone or together with boiled 04 bacteria, with 10 pg of purified 04-LPS, or with 107 human or 108 sheep RBC.
Cell transfer. In some experiments cell suspensions of thymus or spleen from immunized mice (107 formalin-killed E. coli 06 bacteria) or PBS treated animals were prepared. The cells, 2 x 10, were injected into E. coli 06 immunized animals together with bacteria. The bacterial injections were given 5-28 days apart.
Antibody assay. The local haemolysis technique as described by Cunningham & Szenberg (1968) was used. The antibody response estimated using sheep RBC coated with heated extract of E. coli 06 antigen (Ahlstedt, Holmgren & Hanson, 1973) was expressed as plaque-forming cells (PFC) per spleen after subtracting the background PFC. Direct (IgM) as well as indirect (IgG) PFC were registered. The indirect plaques were developed by addition of an optimal dilution (1: 40) of goat anti-mouse y-globulin serum (Miles Research Lab., Kankakee, Ill.). This concentration of the antiserum suppressed more than 90%4 of the direct plaques. Statistics. The statistical analysis of mean values was performed using the Wilcoxon rank sum test.
RESULTS Influence of pretreatment with E. coli 04 antigens on the PFC formation to formalin-killed E. coli 06 bacteria The influence of formalin-killed bacteria alone or combinations of protein extracted from the 04 endotoxin and boiled 04 bacteria or purified 04 LPS or human red blood cells or saline on the antibody response to formalin-killed E. coil 06 bacteria given 28 days later was studied. Direct PFC responses were induced with all the antigen combinations used. The relative capacities of the different antigens to induce direct PFC were (protein + boiled bacteria) > (formalin-killed bacteria) > (protein + human RBC) = (protein + LPS) > (protein + saline). Indirect PFC responses were also induced. In this instance the relative efficiencies of the different combination were (protein + boiled bacteria) = (protein + human RBC) > (formalin killed bacteria) > (protein + saline) = (protein + LPS). The results suggest the importance of the protein for the enhancement of the indirect PFC response but not for the direct PFC response (Table 1).
Antibody response
to
Escherichia coli
Table 1. The antibody response (PFC per spleen) to E. colU 06 after a first injection of various E. coli 04 antigens and a second injection of formalin-killed E. coli 06 bacteria
O
R
0
antigens
631 z-
I
IgM-PFC
IgG-PFC
Formalin-killed 04 bacteria Boiled 04 bacteria + protein 04 LPS + protein Human RBC +protein Protein None
24,000 ± 2000* 30,200 ± 4500 13,700 ± 1800 14,400 ± 3000 6800 ±1300 7500 ±1000
32,000 ± 5000 40,300 ± 5100 20,200 ±2200 40,300±4200 22,400 ± 3800 0
50 R
200 R 400 R
(a) L. O
50 R
Effect of the protein moiety of the E. coli 04 endotoxin on PFC response to sheep red blood cells The effect of the protein moiety of the E. coli 04 endotoxin on the secondary PFC response to sheep RBC antigen was analysed. A prior injection of formalin-killed E. coil 04 bacteria on day 0 increased the antibody response about two-fold to sheep administered on day 5 (P< 0.01). The simultaneous injection of the protein moiety isolated from the 04 endotoxin together with sheep RBC on day 0 increased the indirect PFC response to sheep RBC given at day 5. However, this increase was less than the one described above, although significant (P< 0 05) (Table 2).
100 R
Irradiation sensitivity of spleen cells responding to E. coli 06 bacteria after the first and second injections with formalin-killed bacteria To determine the irradiation sensitivity of the response to formalin-killed 06 bacteria spleen cell suspensions were prepared from mice injected with formalin-killed E. coil 04 bacteria 5 days after antigen administration. These cells were irradiated with 0400 R and injected into irradiated (800 R)
mmo.p-
R
* Mean+ s.e.
L=
200 R 400 R :
(b)
100
1000
Figure 1. The PFC response on day 4 in irradiated (800 R) recipient mice, injected with 108 formalin-killed E. coli bacteria and 4 x 107 irradiated (0-400 R) spleen cells from donor mice given 108 formalin-killed E. coli bacteria 5 days previously Open bars, direct IgM-PFC; solid bars, indirect IgG-PFC; brackets indicate the standard error. (a) E. coli 04 bacteria were given to the donor mice and E. coli 06 bacteria to the recipient mice. (b) E. coli 06 bacteria were given both to the donor and the recipient mice.
recipient mice together with formalin-killed E. coli 06 bacteria. This resulted in PFC responses related to the irradiation dose on the cells and to the number of indirect PFC induced. The numbers of direct PFC induced were considerably lower than those of indirect PFC (P< 0 01). Similar direct PFC responses were registered for the animals given cells irradiated 0-100 R (Fig. la).
PFC response
Day 9
Secondary injection day 5
Direct PFC
Indirect PFC
108 sheep RBC
11,825 ± 2700*
20,900 ±4000
108 sheep RBC
12,375 ± 2500
33,275 ± 6100
108 sheep RBC
21,450 ± 2900
54,800 ± 5800
*
10000
PFC
Table 2. Adjuvant effect of the protein isolated from endotoxin on the PFC response against sheep red blood cells measured as PFC/spleen 4 days after the last injection
108 sheep RBC 108 sheep RBC + 10ug protein 107 formalin-killed E. coli 04 bacteria
momml
100 R
Primary antigen
Primary injection day 0
-
Mean+ s.e.
632
S. Ahlstedt & L. Lindholm 50000 r-
days compared with the primary antibody response. When the injections were spaced 28 days apart an accentuated response was seen (P
