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Immunoregulation and Apoptosis Induction of Nitric Oxide in the Human Mixed Lymphocytes Culture
Immunoregulation and Apoptosis Induction of Nitric Oxide in the Human Mixed Lymphocytes Culture T. Fujiwara, H. Fujita, Y. Okimura, and K. Utsumi ABSTRACT Nitric oxide (NO) is a multifunctional molecule in a variety of physiologic and pathologic processes. Its precise effect on human T lymphocyte responses against alloantigens are not yet fully known, although it has been reported that NO is antiproliferative and can cause apoptosis in several cell types. To address these issues, we analyzed the effects of an NO donor on mixed lymphocyte cultures (MLC) and on apoptosis induction in T lymphocytes activated with alloantigens. NOC 18 was used as an NO donor. The MLC was performed with human peripheral blood mononuclear cells isolated from healthy volunteers. Cell division and interleukin (IL)-2 production were measured with CFSE labeling and an EIA kit, respectively. After cells were incubated with NOC 18 for 24 hours, DNA fragmentation was assessed using the diphenylamine assay. Pre-culture of cells with NOC 18 for 24 hours resulted in significant inhibition of cell proliferation and IL-2 production in MLC. NOC 18 induced DNA fragmentation of cells harvested from an MLC following 7 days of the culture, in a dose-dependent manner, whereas it never exerted any influence on DNA fragmentation of freshly isolated cells. A chemical NO donor, NOC-18, may have immunosuppressive ability when treatment of responder cells occurs before the beginning of the MLC and may induce apoptosis of alloantigen-activated T lymphocytes.
N
ITRIC OXIDE (NO) is a multifunctional molecule that acts in a variety of physiologic and pathologic processes. The diverse effects of NO concerning alloimmune responses as an immunoregulatory or inflammatory mediator, has been suggested during the last one and a half decades,1 but is presently inconclusive. It has not been determined whether NO is beneficial or harmful to an allograft. One possible reason for these controversies may be the different expression of inducible NO synthase 2 (NOS2) between human and mouse cells. It has been reported that activated human macrophages express NOS2 and produce NO at lower levels than are observed with activated mouse macrophages.2 Given these considerations, we examined the direct actions of NO on human T lymphocytes as an immunosuppressant, with antiproliferative and apoptosis induction effects.
was purchased also from Dojindo laboratories. All other materials used were of the highest commercially available grades. Proliferative responses and interleukin (IL)-2 production were estimated in mixed lymphocyte cultures. The mixed lymphocyte culture (MLC) was composed of responder and mitomycin C-treated stimulator cells, both of which were obtained from healthy adult volunteers and isolated by the density gradient centrifugation. Cells were stained with carboxyfluorescein diacetate succinimydyl ester (CFSE; Molecular Probes Inc., Eugene, Ore) according to the manufacture’s instruction. Following the culture, fluorescence was analyzed using a FACS flow cytometer (BD). Cell proliferation was designated as the percentage of cells that lost any CFSE fluorescence. After 3 days of MLC, interleukin IL-2 activities in the supernates were assayed with an EIA kit (Cayman Chemical, Ann Arbor, Mich).
Analysis of DNA Fragmentation Following the incubation of cells in the presence of NOC 18 for 24 hours, the extent of DNA fragmentation was determined by a
MATERIALS AND METHODS Reagents NOC 18, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate, was used as a NO chemical donor (Dojindo Laboratories, Inc., Kumamoto, Japan). 2-(4-carboxyphenyl)-4,4,5,5,tetramethyl-imidazoline-1—imidazoline-1-oxyl 3-oxide (carboxy-PTIO)
From the Department of Surgery, Kurashiki Medical Center, Kurashiki, Okayama Prefecture, Japan. Address reprint requests to Dr T. Fujiwara, Department of Surgery, Kurashiki Medical Center, 250 Bakuro-cho, Kurashiki, Okayama Prefecture 710-8522, Japan. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.170
Transplantation Proceedings, 38, 3211–3213 (2006)
3211
3212
FUJIWARA, FUJITA, OKIMURA ET AL
A 㻋㻈㻌 㻜㻓
dependent manner (Fig 1B), although simultaneous addition resulted in no inhibition (data not shown).
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Fig 1. Immunosuppressive effects of NOC 18 pretreatment in MLC: Cells were incubated with NOC 18 for 24 hours, and then cocultured with stimulator cells. Cell proliferation on day 7 of the MLC was assayed using CFSE labeling (A). IL-2 production of the supernatant after 3-day culture was measured with EIA (B). auto; cocultured with MMC-treated autologous cells. Asterisks (*) mean statistically significant difference from the corresponding value of the control (without NOC 18) (P ⬍ .05). Data shown are mean ⫾ SEM of four independent experiments.
Apoptosis Induction of NOC 18
NOC 18 had no effect on DNA fragmentation of freshly isolated MoNC, (Fig 2A). MLC cells harvested on day 7 of culture were spontaneously dying, also in apoptotic fashion. NOC 18 enhanced DNA fragmentation in the MLC cells in a dose-dependent manner (Fig 2B). The percentage of DNA fragmentation of MLC cells treated with 200 mol of NOC 18 for 24 hours was 40.7 ⫾ 4.8%, and that without NOC 18 (control) was 30.0 ⫾ 4.8%. Both immunosuppression and DNA fragmentation induced with NOC 18 were inhibited by adding NO-trapping agents, such as carboxy-PTIO or oxyhemoglobin (data not shown), suggesting that NO released from NOC 18 might be responsible for these effects. DISCUSSION
One explanation for this immunosuppressive mode of NOC 18 may be due to its long half-life. NOC 18 has been reported to release NO slowly and for a long time is to the medium.4 The results indicated that NOC 18 could induce apoptosis only on alloantigen-activated T lymphocytes, that is, activation-induced cell death.5 In summary, pretreatment of cells with NOC-18 for 24 hours before an MLC inhibited the response of T
spectrophotometric assay using diphenylamine, as previously described in detail.3 The percentage of DNA fragmentation (% DNA fragmentation) was calculated as the ratio of DNA in the supernate to the total DNA.
Statistical Analysis Data are expressed as the mean values ⫾ SD or SEM. Differences in measured variables between experimental and control groups were assessed using the two-tailed Student t-test. A value of ⬍.05 was considered significant.
RESULTS Immunosuppressive Effects of NOC 18 in MLC
NOC 18 had no effect on cell viability or apoptosis induction of freshly isolated mononuclear cells (MoNC), even at concentrations up to 500 mol. At first, we added NOC 18 to the MLC at the beginning of the culture to test the inhibition of the proliferative capacity of T lymphocytes. Unexpectedly, the simultaneous administration of NOC-18 resulted in no effect (data not shown). Following incubation with NOC 18 for 24 hours, cells were harvested, labeled with CFSE and cocultured with stimulator cells. Pretreatment of cells with NOC 18 caused significant inhibition of cell division. Figure 1A shows the results on day 7 of the MLC. Similar results were obtained concerning IL-2 production in the MLC. NOC 18 pretreatment suppressed the IL-2 concentrations of the supernates of MLC in a dose-
Fig 2. Apoptosis induction by NOC 18; The MoNC (A) and 7-day MLC cells (B) were incubated for 24 hours in the presence of the indicated concentration of NOC 18. The DNA fragmentation of the cells was measured after that. Asterisks (*) mean statistically significant difference from the corresponding value of the control (P ⬍ .0001). Data are mean ⫾ SD of seven separate experiments.
IMMUNOREGULATION
lymphocytes against alloantigens. NO induced apoptosis of alloantigen-activated T lymphocytes without any effect on the viability of nonactivated cells. REFERENCES 1. Langreher JM, Hoffman RA, Lancaster JJR, et al: Nitric oxide—a new endogenous immunomodulator. Transplantation 55: 1205, 1993 2. Chu SC, Marks-Konczalik HP, Wu TC, et al: Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS)
3213 gene: characterization of differences between human and mouse iNOS promoters. Biochem Biophys Res Commun 248:871, 1998 3. Yabuki M, Kariya S, Inai Y, et al: Molecular mechanisms of apoptosis in HL-60 cells induced by a nitric oxide-releasing compound. Free Radic Res 27:325, 1997 4. Hrabie JA, Klose JR, Wink DA, et al: New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem 58:1472, 1993 5. Wesselborg S, Janssen O, Kabelits D: Induction of activationdriven death (apoptosis) in activated not resting peripheral blood T cell. J Immunol 150:4338, 1993
N
ITRIC OXIDE (NO) is a multifunctional molecule that acts in a variety of physiologic and pathologic processes. The diverse effects of NO concerning alloimmune responses as an immunoregulatory or inflammatory mediator, has been suggested during the last one and a half decades,1 but is presently inconclusive. It has not been determined whether NO is beneficial or harmful to an allograft. One possible reason for these controversies may be the different expression of inducible NO synthase 2 (NOS2) between human and mouse cells. It has been reported that activated human macrophages express NOS2 and produce NO at lower levels than are observed with activated mouse macrophages.2 Given these considerations, we examined the direct actions of NO on human T lymphocytes as an immunosuppressant, with antiproliferative and apoptosis induction effects.
was purchased also from Dojindo laboratories. All other materials used were of the highest commercially available grades. Proliferative responses and interleukin (IL)-2 production were estimated in mixed lymphocyte cultures. The mixed lymphocyte culture (MLC) was composed of responder and mitomycin C-treated stimulator cells, both of which were obtained from healthy adult volunteers and isolated by the density gradient centrifugation. Cells were stained with carboxyfluorescein diacetate succinimydyl ester (CFSE; Molecular Probes Inc., Eugene, Ore) according to the manufacture’s instruction. Following the culture, fluorescence was analyzed using a FACS flow cytometer (BD). Cell proliferation was designated as the percentage of cells that lost any CFSE fluorescence. After 3 days of MLC, interleukin IL-2 activities in the supernates were assayed with an EIA kit (Cayman Chemical, Ann Arbor, Mich).
Analysis of DNA Fragmentation Following the incubation of cells in the presence of NOC 18 for 24 hours, the extent of DNA fragmentation was determined by a
MATERIALS AND METHODS Reagents NOC 18, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate, was used as a NO chemical donor (Dojindo Laboratories, Inc., Kumamoto, Japan). 2-(4-carboxyphenyl)-4,4,5,5,tetramethyl-imidazoline-1—imidazoline-1-oxyl 3-oxide (carboxy-PTIO)
From the Department of Surgery, Kurashiki Medical Center, Kurashiki, Okayama Prefecture, Japan. Address reprint requests to Dr T. Fujiwara, Department of Surgery, Kurashiki Medical Center, 250 Bakuro-cho, Kurashiki, Okayama Prefecture 710-8522, Japan. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.170
Transplantation Proceedings, 38, 3211–3213 (2006)
3211
3212
FUJIWARA, FUJITA, OKIMURA ET AL
A 㻋㻈㻌 㻜㻓
dependent manner (Fig 1B), although simultaneous addition resulted in no inhibition (data not shown).
㻍
㻍
㻙㻓
㻖㻓
B
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㻘㻓 㻔㻓㻓 㻱㻲㻦㻃㻔㻛㻃㻋䃒㻰㻌
㻕㻓㻓
Fig 1. Immunosuppressive effects of NOC 18 pretreatment in MLC: Cells were incubated with NOC 18 for 24 hours, and then cocultured with stimulator cells. Cell proliferation on day 7 of the MLC was assayed using CFSE labeling (A). IL-2 production of the supernatant after 3-day culture was measured with EIA (B). auto; cocultured with MMC-treated autologous cells. Asterisks (*) mean statistically significant difference from the corresponding value of the control (without NOC 18) (P ⬍ .05). Data shown are mean ⫾ SEM of four independent experiments.
Apoptosis Induction of NOC 18
NOC 18 had no effect on DNA fragmentation of freshly isolated MoNC, (Fig 2A). MLC cells harvested on day 7 of culture were spontaneously dying, also in apoptotic fashion. NOC 18 enhanced DNA fragmentation in the MLC cells in a dose-dependent manner (Fig 2B). The percentage of DNA fragmentation of MLC cells treated with 200 mol of NOC 18 for 24 hours was 40.7 ⫾ 4.8%, and that without NOC 18 (control) was 30.0 ⫾ 4.8%. Both immunosuppression and DNA fragmentation induced with NOC 18 were inhibited by adding NO-trapping agents, such as carboxy-PTIO or oxyhemoglobin (data not shown), suggesting that NO released from NOC 18 might be responsible for these effects. DISCUSSION
One explanation for this immunosuppressive mode of NOC 18 may be due to its long half-life. NOC 18 has been reported to release NO slowly and for a long time is to the medium.4 The results indicated that NOC 18 could induce apoptosis only on alloantigen-activated T lymphocytes, that is, activation-induced cell death.5 In summary, pretreatment of cells with NOC-18 for 24 hours before an MLC inhibited the response of T
spectrophotometric assay using diphenylamine, as previously described in detail.3 The percentage of DNA fragmentation (% DNA fragmentation) was calculated as the ratio of DNA in the supernate to the total DNA.
Statistical Analysis Data are expressed as the mean values ⫾ SD or SEM. Differences in measured variables between experimental and control groups were assessed using the two-tailed Student t-test. A value of ⬍.05 was considered significant.
RESULTS Immunosuppressive Effects of NOC 18 in MLC
NOC 18 had no effect on cell viability or apoptosis induction of freshly isolated mononuclear cells (MoNC), even at concentrations up to 500 mol. At first, we added NOC 18 to the MLC at the beginning of the culture to test the inhibition of the proliferative capacity of T lymphocytes. Unexpectedly, the simultaneous administration of NOC-18 resulted in no effect (data not shown). Following incubation with NOC 18 for 24 hours, cells were harvested, labeled with CFSE and cocultured with stimulator cells. Pretreatment of cells with NOC 18 caused significant inhibition of cell division. Figure 1A shows the results on day 7 of the MLC. Similar results were obtained concerning IL-2 production in the MLC. NOC 18 pretreatment suppressed the IL-2 concentrations of the supernates of MLC in a dose-
Fig 2. Apoptosis induction by NOC 18; The MoNC (A) and 7-day MLC cells (B) were incubated for 24 hours in the presence of the indicated concentration of NOC 18. The DNA fragmentation of the cells was measured after that. Asterisks (*) mean statistically significant difference from the corresponding value of the control (P ⬍ .0001). Data are mean ⫾ SD of seven separate experiments.
IMMUNOREGULATION
lymphocytes against alloantigens. NO induced apoptosis of alloantigen-activated T lymphocytes without any effect on the viability of nonactivated cells. REFERENCES 1. Langreher JM, Hoffman RA, Lancaster JJR, et al: Nitric oxide—a new endogenous immunomodulator. Transplantation 55: 1205, 1993 2. Chu SC, Marks-Konczalik HP, Wu TC, et al: Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS)
3213 gene: characterization of differences between human and mouse iNOS promoters. Biochem Biophys Res Commun 248:871, 1998 3. Yabuki M, Kariya S, Inai Y, et al: Molecular mechanisms of apoptosis in HL-60 cells induced by a nitric oxide-releasing compound. Free Radic Res 27:325, 1997 4. Hrabie JA, Klose JR, Wink DA, et al: New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem 58:1472, 1993 5. Wesselborg S, Janssen O, Kabelits D: Induction of activationdriven death (apoptosis) in activated not resting peripheral blood T cell. J Immunol 150:4338, 1993