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Antibody-forming response of cultured spleen cells to a protein antigen: function of antigen presenting cells in SAMP1 mice
International Congress Series 1260 (2004) 209 – 213
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Antibody-forming response of cultured spleen cells to a protein antigen: function of antigen presenting cells in SAMP1 mice Tomomi Kimura, Arata Kohdan, Tomohide Hosokawa * Department of Life Science, Kyoto University of Education, 1 Fukakusa-Fujinomori-cho, Fushimi, Kyoto 612-8522, Japan Received 15 June 2003; received in revised form 19 June 2003; accepted 4 September 2003
Abstract. SAMP1 and C3H/He mice were immunized with one or two intraperitoneal injections of ovalbumin (OVA) with alum adjuvant. Spleen cells from immunized as well as unprimed naı¨ve mice were cultured with OVA for 7 or 14 days. We observed little anti-OVA antibody response when spleen cells from unprimed naive mice were cultured with OVA. A small but substantial amount of OVA-specific IgG antibody was produced regardless of the presence of OVA in both C3H/He and SAMP1 spleen cell cultures when the donor mice had received a single OVA injection. OVA-specific IgG and IgA antibody responses induced by OVA added to cultures were clearly observed when the donor mice were immunized with two OVA injections. However, the antibody response of SAMP1 cells was much lower than that of C3H/He cells. It is possible that immune memory generation in SAMP1 mice is impaired. However, the concentration of IgG antibody against OVA in the peripheral blood of OVA-primed SAMP1 mice was as high as that of C3H/He mice. The depletion of adherent cells from the OVA-primed C3H/He spleen cells markedly reduced the OVA-specific IgG and IgA antibody responses, which were almost completely restored by adding peritoneal adherent cells from either C3H/He or SAMP1 mice to the culture. These results suggest that a defect in Th2 cells from SAMP1 mice is generally observed in the antibody response to T-dependent antigens. Furthermore, the antigen-presenting function of SAMP1 adherent cells is not impaired and is not involved as a major cause in the defective antibody-producing response to T-dependent antigens. D 2003 Elsevier B.V. All rights reserved. Keywords: T-dependent antigen; Th2 cell; Antigen presenting cell; Adherent cell; SAMP1
1. Introduction SAMP1 mice developed by Takeda et al. [3] suffer from accelerated senescence and a markedly short life span. They also show an impaired antibody-forming response to
* Corresponding author. Tel.: +81-75-644-8278; fax: +81-75-645-1734. E-mail address: [email protected] (T. Hosokawa). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01569-3
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T-dependent antigens from early life. In particular, the impaired antibody response was evident when spleen cells were stimulated in vitro with xenogenic red blood cells [1]. Some dysfunction of antigen-specific Th2 cells appeared to be involved in this impaired antibody response [2]. Although xenogenic red blood cells induce a primary antibody-forming response in cultured spleen cells, the response does not seem to be a typical T-dependent antibody response. Thus, we used the protein OVA as a typical Tdependent antigen in order to reinvestigate the impaired antibody response of SAMP1 cells. We report here that the spleen cells of young SAMP1 mice primed to OVA showed an impaired antibody-forming response against OVA in culture. This impaired antibody response of SAMP1 cells could be attributed to some dysfunction of the Th2 cells themselves or to a possible dysfunction of the adherent antigen presenting cells. Therefore, the anti-OVA antibody response of SAMP1 spleen cells was compared to that of C3H/He cells. Thus, non-adherent spleen cells from either C3H/He or SAMP1 mice primed to OVA were cultured with or without peritoneal adherent cells from either C3H/He or SAMP1 naı¨ve mice in the presence and absence of OVA. 2. Materials and methods 2.1. Mice SAMP1/Kue (SAMP1) and C3H/HeSlc (C3H/He) mice were maintained under conventional breeding conditions. Three- to five-month-old mice of both gender were used. 2.2. Immunization with OVA Mice were immunized with a single (50 Ag bolus) or two (10 Ag each injection, 3 weeks apart) intraperitoneal injections of OVA (Sigma, St. Louis, MO, USA) with alum adjuvant. They were then killed to obtain OVA-primed spleen cells 3 weeks after the single or 10 days after the second injection. 2.3. Depletion of adherent cells Adherent cells from the spleen were depleted by the adherence of spleen cells to Sephadex G10 beads (Pharmacia Biotec, Tokyo, Japan) packed in a column. 2.4. Cell culture Spleen cells were suspended in RPMI 1640 medium supplemented with 2 mM Lglutamine, 5% fetal bovine serum and 5 10 5 M 2-mercaptoethanol. The cells were then cultured for 1 – 2 weeks at 5 106 cells/well in 24-well culture plates in a humidified atmosphere of 5% CO2 in air at 37j. Culture supernatants were collected at the end of the incubation, and were assayed for IgG, IgM and IgA antibodies to OVA by ELISA. AntiOVA IgM was not detected in any of the cultures.
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Fig. 1. Time course of the IgG antibody production in SAMP1 and C3H/He mice induced by the first and second injections of OVA.
2.5. Statistical analysis Non-parametric U-tests and Student’s t-test were used. Differences between two groups were considered statistically significant if the P value was less than 0.05. 3. Results 3.1. Antigen specific IgG antibody response of SAMP1 mice SAMP1 and C3H/He mice received two injections of 10 Ag OVA. Peripheral blood was then collected from each mouse at days 7 and 21 after the first injection and at day 10 after the second OVA injection. The blood was assayed for IgG antibodies to OVA. Although they produced much less anti-OVA antibody than the C3H/He mice at 7 days after the first OVA injection, the SAMP1 mice produced as much anti-OVA IgG antibody as the C3H/ He mice at 21 days after the first and at 10 days after the second OVA injection (Fig. 1).
Fig. 2. Immune memory expression of anti-OVA IgG and IgA antibodies in OVA-primed spleen cells in vitro.
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Fig. 3. Antigen-presenting activity of peritoneal adherent cells from SAMP1 and C3H/He mice.
Therefore, we obtained spleens from donor mice at 21 days after the first or 10 days after the second immunization. 3.2. Poor expression of immune memory to T-dependent antigen in cultured SAMP1 spleen cells OVA-primed spleen cells were cultured with varying concentrations of OVA to induce the antibody response of the memory cells. Since the optimal concentration of OVA was 0.1 Ag/ml (data not shown), we used it to induce the memory expression in all subsequent experiments. OVA-specific IgG and IgA antibody responses induced by OVA in culture were clearly observed when the donor mice were immunized with two OVA injections (Fig. 2). However, the antibody response of the SAMP1 spleen cells was much lower than that of the C3H/He cells. OVA-specific IgG and IgA antibody responses induced by OVA in culture increased from 7 to 14 days when the donor C3H/He mice were immunized with two OVA injections. 3.3. Antigen presenting activity of SAMP1 adherent cells is comparable to that of C3H/He cells Non-adherent spleen cells from either C3H/He or SAMP1 mice immunized with a single or two OVA injections were cultured with peritoneal adherent cells from either C3H/He or SAMP1 mice in the presence of OVA. The depletion of adherent cells from the primed C3H/He spleen cells markedly reduced the OVA-specific IgG and IgA antibody responses, which were almost completely restored by adding peritoneal adherent cells from either C3H/He or SAMP1 mice to the culture (Fig. 3). 4. Discussion Cultured spleen cells from antigen primed SAMP1 mice showed a very weak antibodyforming response to the priming protein antigen. These results are consistent with our earlier findings that SAMP1 mice showed a defective and low antibody-forming response to T-dependent antigen [1]. Since SAMP1 adherent cells showed as high antigen presenting activity as C3H/He adherent cells, the low antibody response to the T-dependent antigen
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OVA may be attributed to some dysfunction in the Th2 cells themselves. The concentration of anti-OVA IgG antibody in serum of donor SAMP1 mice was as high as that of the C3H/ He mice, regardless of the number of immunization. Therefore, the in vivo priming with OVA seemed to have been enough to generate immune memory cells specific to the antigen. In SAMP1 mice, the expression of immune memory may be impaired. References [1] T. Hosokawa, M. Hosono, K. Higuchi, et al., Immune responses in newly developed short-lived SAM mice. I: Age-associated early decline in immune activities of cultured spleen cells, Immunology 62 (1987) 419 – 423. [2] T. Hosokawa, M. Hosono, K. Hanada, et al., Immune responses in newly developed short-lived SAM mice. II: Selectively impaired T-helper cell activity in in vitro antibody response, Immunology 62 (1987) 425 – 429. [3] T. Takeda, M. Hosokawa, S. Takeshita, et al., A new murine model of accelerated senescence, Mech. Ageing Dev. 17 (1981) 183 – 194.
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Antibody-forming response of cultured spleen cells to a protein antigen: function of antigen presenting cells in SAMP1 mice Tomomi Kimura, Arata Kohdan, Tomohide Hosokawa * Department of Life Science, Kyoto University of Education, 1 Fukakusa-Fujinomori-cho, Fushimi, Kyoto 612-8522, Japan Received 15 June 2003; received in revised form 19 June 2003; accepted 4 September 2003
Abstract. SAMP1 and C3H/He mice were immunized with one or two intraperitoneal injections of ovalbumin (OVA) with alum adjuvant. Spleen cells from immunized as well as unprimed naı¨ve mice were cultured with OVA for 7 or 14 days. We observed little anti-OVA antibody response when spleen cells from unprimed naive mice were cultured with OVA. A small but substantial amount of OVA-specific IgG antibody was produced regardless of the presence of OVA in both C3H/He and SAMP1 spleen cell cultures when the donor mice had received a single OVA injection. OVA-specific IgG and IgA antibody responses induced by OVA added to cultures were clearly observed when the donor mice were immunized with two OVA injections. However, the antibody response of SAMP1 cells was much lower than that of C3H/He cells. It is possible that immune memory generation in SAMP1 mice is impaired. However, the concentration of IgG antibody against OVA in the peripheral blood of OVA-primed SAMP1 mice was as high as that of C3H/He mice. The depletion of adherent cells from the OVA-primed C3H/He spleen cells markedly reduced the OVA-specific IgG and IgA antibody responses, which were almost completely restored by adding peritoneal adherent cells from either C3H/He or SAMP1 mice to the culture. These results suggest that a defect in Th2 cells from SAMP1 mice is generally observed in the antibody response to T-dependent antigens. Furthermore, the antigen-presenting function of SAMP1 adherent cells is not impaired and is not involved as a major cause in the defective antibody-producing response to T-dependent antigens. D 2003 Elsevier B.V. All rights reserved. Keywords: T-dependent antigen; Th2 cell; Antigen presenting cell; Adherent cell; SAMP1
1. Introduction SAMP1 mice developed by Takeda et al. [3] suffer from accelerated senescence and a markedly short life span. They also show an impaired antibody-forming response to
* Corresponding author. Tel.: +81-75-644-8278; fax: +81-75-645-1734. E-mail address: [email protected] (T. Hosokawa). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01569-3
210
T. Kimura et al. / International Congress Series 1260 (2004) 209–213
T-dependent antigens from early life. In particular, the impaired antibody response was evident when spleen cells were stimulated in vitro with xenogenic red blood cells [1]. Some dysfunction of antigen-specific Th2 cells appeared to be involved in this impaired antibody response [2]. Although xenogenic red blood cells induce a primary antibody-forming response in cultured spleen cells, the response does not seem to be a typical T-dependent antibody response. Thus, we used the protein OVA as a typical Tdependent antigen in order to reinvestigate the impaired antibody response of SAMP1 cells. We report here that the spleen cells of young SAMP1 mice primed to OVA showed an impaired antibody-forming response against OVA in culture. This impaired antibody response of SAMP1 cells could be attributed to some dysfunction of the Th2 cells themselves or to a possible dysfunction of the adherent antigen presenting cells. Therefore, the anti-OVA antibody response of SAMP1 spleen cells was compared to that of C3H/He cells. Thus, non-adherent spleen cells from either C3H/He or SAMP1 mice primed to OVA were cultured with or without peritoneal adherent cells from either C3H/He or SAMP1 naı¨ve mice in the presence and absence of OVA. 2. Materials and methods 2.1. Mice SAMP1/Kue (SAMP1) and C3H/HeSlc (C3H/He) mice were maintained under conventional breeding conditions. Three- to five-month-old mice of both gender were used. 2.2. Immunization with OVA Mice were immunized with a single (50 Ag bolus) or two (10 Ag each injection, 3 weeks apart) intraperitoneal injections of OVA (Sigma, St. Louis, MO, USA) with alum adjuvant. They were then killed to obtain OVA-primed spleen cells 3 weeks after the single or 10 days after the second injection. 2.3. Depletion of adherent cells Adherent cells from the spleen were depleted by the adherence of spleen cells to Sephadex G10 beads (Pharmacia Biotec, Tokyo, Japan) packed in a column. 2.4. Cell culture Spleen cells were suspended in RPMI 1640 medium supplemented with 2 mM Lglutamine, 5% fetal bovine serum and 5 10 5 M 2-mercaptoethanol. The cells were then cultured for 1 – 2 weeks at 5 106 cells/well in 24-well culture plates in a humidified atmosphere of 5% CO2 in air at 37j. Culture supernatants were collected at the end of the incubation, and were assayed for IgG, IgM and IgA antibodies to OVA by ELISA. AntiOVA IgM was not detected in any of the cultures.
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Fig. 1. Time course of the IgG antibody production in SAMP1 and C3H/He mice induced by the first and second injections of OVA.
2.5. Statistical analysis Non-parametric U-tests and Student’s t-test were used. Differences between two groups were considered statistically significant if the P value was less than 0.05. 3. Results 3.1. Antigen specific IgG antibody response of SAMP1 mice SAMP1 and C3H/He mice received two injections of 10 Ag OVA. Peripheral blood was then collected from each mouse at days 7 and 21 after the first injection and at day 10 after the second OVA injection. The blood was assayed for IgG antibodies to OVA. Although they produced much less anti-OVA antibody than the C3H/He mice at 7 days after the first OVA injection, the SAMP1 mice produced as much anti-OVA IgG antibody as the C3H/ He mice at 21 days after the first and at 10 days after the second OVA injection (Fig. 1).
Fig. 2. Immune memory expression of anti-OVA IgG and IgA antibodies in OVA-primed spleen cells in vitro.
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Fig. 3. Antigen-presenting activity of peritoneal adherent cells from SAMP1 and C3H/He mice.
Therefore, we obtained spleens from donor mice at 21 days after the first or 10 days after the second immunization. 3.2. Poor expression of immune memory to T-dependent antigen in cultured SAMP1 spleen cells OVA-primed spleen cells were cultured with varying concentrations of OVA to induce the antibody response of the memory cells. Since the optimal concentration of OVA was 0.1 Ag/ml (data not shown), we used it to induce the memory expression in all subsequent experiments. OVA-specific IgG and IgA antibody responses induced by OVA in culture were clearly observed when the donor mice were immunized with two OVA injections (Fig. 2). However, the antibody response of the SAMP1 spleen cells was much lower than that of the C3H/He cells. OVA-specific IgG and IgA antibody responses induced by OVA in culture increased from 7 to 14 days when the donor C3H/He mice were immunized with two OVA injections. 3.3. Antigen presenting activity of SAMP1 adherent cells is comparable to that of C3H/He cells Non-adherent spleen cells from either C3H/He or SAMP1 mice immunized with a single or two OVA injections were cultured with peritoneal adherent cells from either C3H/He or SAMP1 mice in the presence of OVA. The depletion of adherent cells from the primed C3H/He spleen cells markedly reduced the OVA-specific IgG and IgA antibody responses, which were almost completely restored by adding peritoneal adherent cells from either C3H/He or SAMP1 mice to the culture (Fig. 3). 4. Discussion Cultured spleen cells from antigen primed SAMP1 mice showed a very weak antibodyforming response to the priming protein antigen. These results are consistent with our earlier findings that SAMP1 mice showed a defective and low antibody-forming response to T-dependent antigen [1]. Since SAMP1 adherent cells showed as high antigen presenting activity as C3H/He adherent cells, the low antibody response to the T-dependent antigen
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OVA may be attributed to some dysfunction in the Th2 cells themselves. The concentration of anti-OVA IgG antibody in serum of donor SAMP1 mice was as high as that of the C3H/ He mice, regardless of the number of immunization. Therefore, the in vivo priming with OVA seemed to have been enough to generate immune memory cells specific to the antigen. In SAMP1 mice, the expression of immune memory may be impaired. References [1] T. Hosokawa, M. Hosono, K. Higuchi, et al., Immune responses in newly developed short-lived SAM mice. I: Age-associated early decline in immune activities of cultured spleen cells, Immunology 62 (1987) 419 – 423. [2] T. Hosokawa, M. Hosono, K. Hanada, et al., Immune responses in newly developed short-lived SAM mice. II: Selectively impaired T-helper cell activity in in vitro antibody response, Immunology 62 (1987) 425 – 429. [3] T. Takeda, M. Hosokawa, S. Takeshita, et al., A new murine model of accelerated senescence, Mech. Ageing Dev. 17 (1981) 183 – 194.