RESUMO
BACKGROUND: Previous studies suggest that CD8(+) T cells are immunosuppressive after burn injury, but recent reports indicate that CD8(+) T cells have several functions similar to CD4(+) T cells, including the secretion of cytokines. This study uses HY male antigen in transgenic HY female mice to determine the antigen-specific response of activated CD8(+) T cells after burn injury. METHODS: HY TCR transgenic female mice underwent burn or sham injury. Seventy-two hours after the burn, splenocytes were stimulated with 20 micromol/L HY peptide for 16, 48, and 64 hours; cellular proliferation, intracellular interferon-gamma and interleukin-2, and apoptosis were measured. RESULTS: Burn injury significantly impaired proliferation to HY antigen (P < or =.05). Activated CD8(+) T cells from burned mice showed increased intracellular interferon-gamma and interleukin-2 16 hours after stimulation compared with sham (P < or =.05) and at no time was less than control mice. The percent of CD8(+) T cells decreased with the time of stimulation but was not due to apoptosis by Annexin V staining. CONCLUSIONS: Activated CD8(+) T cells express a T(h1)-like phenotype after burn injury. This provides evidence that CD8(+) T cells are not simply suppressive and that is consistent with data that CD4(+) T cells are primed for a T(h1) response after burn injury.
Assuntos
Queimaduras/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Th1/imunologia , Animais , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Baço/citologia , Baço/imunologia , Células Th1/citologiaRESUMO
BACKGROUND: The liver plays a critical regulatory role in the acute inflammatory response to injury, although the mechanisms of this regulation are not well understood. transforming growth factor-beta1 (TGF-beta1) is induced after burn injury and may contribute to an inhibitory or fatal effect on hepatocytes. We investigated the association over time between plasma concentration of TGF-beta1, expression of TGF-beta1 m-RNA in liver tissue, and histologic analysis of liver apoptosis after burn injury. METHODS: Male BALB/c mice were anesthetized and randomized to receive 0% (sham), moderate (approximately 25%) (M), or large (approximately 50%) (L) body surface area full-thickness contact burn, followed by resuscitation and analgesia. Animals were killed over a time course from 15 minutes to 24 hours after burn injury, and liver tissue and peripheral blood were collected. Plasma levels of TGF-beta1 (nanograms per milliliter) were measured by enzyme-linked immunosorbent assay. TGF-beta1 m-RNA was extracted from liver and measured by reverse transcription-polymerase chain reaction. Histology of liver apoptosis was examined after fixation and staining with TdT-mediated dUTP nick-end labeling (TUNEL) method. RESULTS: The plasma concentration of TGF-beta in burn group L was significantly increased at 4 hours after burn when compared with sham and M burn groups. This rise in plasma TGF-beta1 was preceded by an increase in hepatic TGF-beta1 m-RNA expression at 30 minutes, 1, 2, and 4 hours after burn in the L group. Histologic analysis found greater hepatocyte death in the L group than in the M group at 8 hours after burn. CONCLUSION: The levels of induced TGF-beta1 and TGF-beta1 m-RNA after L burn injury are higher and peak earlier than after M burn injury. Elevated TGF-beta1 may be associated with cell death in hepatocytes. The TGF-beta1 rise may be associated with hepatocyte injury and systemic response to massive burn.
Assuntos
Queimaduras/imunologia , Queimaduras/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Fígado/química , Fígado/patologia , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/sangue , Doença Aguda , Animais , Apoptose , Superfície Corporal , Queimaduras/patologia , Ensaio de Imunoadsorção Enzimática , Marcação In Situ das Extremidades Cortadas , Inflamação , Escala de Gravidade do Ferimento , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Crescimento Transformador beta/genéticaRESUMO
Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that mediates apoptotic cell death in human lymphocytes in vitro. To better understand the mechanism through which TGF-beta1 exerts its apoptotic effect, we investigated the role of TGF-beta1 in the relationship between burn injury and cell death of splenocytes in a mouse model of either 0%, 25%, or 40% full-thickness burns. Mice were killed and spleens were harvested at 15 and 30 minutes and at 1, 2, 4, 8, 12, and 24 hours after the burn. The spleens were divided and used for both histologic analyses with H-E stain and TUNEL stain and for total messenger RNA isolation and reverse transcriptase-polymerase chain reaction amplification. Amplified polymerase chain reaction products were analyzed for signal strength by electrophoresis. TGF-beta1 RNA expression was highest at 2 hours after the burn injuries in the 40% full-thickness burns and at 4 hours after the burn injuries in the 25% full-thickness burns. The relative increase in TGF-beta1 RNA was 3 times greater with the larger burn than with the smaller burn. In histologic analysis, splenocyte apoptotic cell death was observed at 4 to 24 hours after the burn in the 40% full-thickness burns but at only 4 to 12 hours in the 25% full-thickness burns. TGF-beta1 RNA peak expression was observed at different times after the burn in 25% and 40% full-thickness burns. Histologic analysis showed apoptotic cell death in proportion with respective messenger RNA expressions. This suggests that TGF-beta1 may be associated with apoptosis of splenocytes in vivo and that the effect of TGF-beta1 after a burn injury may be important in the immune system.
Assuntos
Apoptose , Queimaduras/metabolismo , Queimaduras/patologia , Baço/metabolismo , Baço/patologia , Fator de Crescimento Transformador beta/biossíntese , Análise de Variância , Animais , Sequência de Bases , Queimaduras/imunologia , Células Cultivadas , Modelos Animais de Doenças , Escala de Gravidade do Ferimento , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Distribuição Aleatória , Valores de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/análiseRESUMO
The liver plays a critical role in the inflammatory response to injury; however, the mechanisms by which the liver is affected and how it influences the rest of the immune system are not well understood. Partial hepatectomy is a direct injury to the liver, whereas a burn is an indirect injury to liver, but both injuries appear to produce damage to the liver. In this study, we used a mouse model of 25% total body surface area and 40% total body surface area full-thickness burns to investigate the mechanism of liver damage and response to burn injury by measuring levels of c-Jun messenger (m)RNA, NFkappaB nuclear protein, interleukin-6, transaminases, and liver tissue histology over time. c-Jun and NFkappaB are 2 transcription factors that are induced by partial hepatectomy and related to hepatocyte injury and growth. In both groups of mice with burns, expression of c-Jun mRNA and NFkappaB nuclear protein was activated within 30 minutes after the burn injury, followed by increased levels of interleukin-6 and, finally, elevated enzyme levels. Liver injuries were similar in both groups despite the magnitude of the burns. We believe that these gene products are initiated in the hepatocyte injury after a burn and that they precede other inflammatory responses such as cytokine release, plasma transaminase levels, and histologic changes.
Assuntos
Queimaduras/genética , Queimaduras/metabolismo , Citocinas/metabolismo , Genes jun/genética , Fígado/enzimologia , NF-kappa B/metabolismo , Alanina Transaminase/metabolismo , Amônia/sangue , Análise de Variância , Animais , Aspartato Aminotransferases/metabolismo , Queimaduras/patologia , Técnicas de Cultura , Citocinas/análise , Modelos Animais de Doenças , Expressão Gênica , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Necrose , RNA Mensageiro/análise , Distribuição Aleatória , Valores de ReferênciaRESUMO
BACKGROUND: Transforming growth factor-beta (TGF-beta) has been shown to be an inhibitor of immunoglobulin (Ig) synthesis and may contribute to decreased Ig synthesis after burn injury. This study investigated the relationship between TGF-beta and Ig synthesis after burn injury. METHODS: Twenty-four BALB/c mice received either a 30% body surface area full-thickness contact burn or no burn. Splenocytes were isolated 8 days after burn and were cultured with 0, 0.05 or 0.5 ng/mL TGF-beta. After culture, total IgG and total IgM were measured by enzyme-linked immunosorbent assay. The number of IgM-secreting cells per 10(5) cells was measured by enzyme-linked immunoabsorbent spot forming assay. Total IgM per IgM-secreting cell (pg/cell) was calculated. RESULTS: Total IgG, IgM, IgM-secreting cells, and B-cell number after culture were decreased by burn injury, and the decrease was exacerbated by the presence of TGF-beta. The total IgM per IgM-secreting cells, however, was significantly increased by TGF-beta at 0.5 ng/mL. CONCLUSION: These data demonstrates that TGF-beta does not specifically impair IgM secretion by committed IgM B cells but appears to decrease B-cell proliferation or clonal expansion.
Assuntos
Linfócitos B/citologia , Queimaduras/imunologia , Imunoglobulinas/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Animais , Contagem de Células , Divisão Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologiaRESUMO
BACKGROUND: Lack of skin for autograft continues to be problematic in patients with large burns. Allograft and xenograft have been used, but are prone to rapid rejection. Use of cultured keratinocytes (CK) and major histocompatibility complex (MHC) II "knockout" grafts leads to prolonged graft survival compared to allograft. Whether this prolongation is secondary to decreased priming efficacy or target recognition is unknown. Whether a combination of these techniques would generate a less immunogenic allograft remains to be determined. METHODS: CBA mice (n = 100) were flank-grafted with full thickness C57BL/6 (B6 FT), B6 cultured keratinocytes (B6 CK), B6 major histocompatibility complex II "knockout" full thickness (KO II FT), B6 major histocompatibility complex II "knockout" cultured keratinocytes (KO II CK), or a full thickness autograft (Auto). Three weeks after priming flank grafting, B6, MHC I (KO I), and KO II full thickness tail grafts were placed on each mouse. Tail graft rejection was assessed daily by an observer blinded to flank and tail-graft type. A 4-point grading system for graft color, hair loss, and texture was used. RESULTS: Animals primed with KO II CK flank grafts had increased survival of tail grafts over B6 FT flank grafted controls (12.3 +/- 1.05 vs 10.1 +/- 1.00, P < 0.05). Within flank graft groups, however, B6, KO I, and KO II tail graft survival was similar. CONCLUSIONS: KO II CK allografts decrease host priming compared to normal B6 FT allograft. MHC deletion (KO I or KO II) does not protect a target graft from rejection in a primed host. CK and KO techniques may offer a less immunogenic allograft and a readily available source of wound coverage in patients with extensive burns.
Assuntos
Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Queratinócitos/química , Queratinócitos/transplante , Transplante de Pele/métodos , Animais , Apresentação de Antígeno/imunologia , Queimaduras/imunologia , Queimaduras/terapia , Células Cultivadas , Feminino , Expressão Gênica/fisiologia , Sobrevivência de Enxerto/imunologia , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Mutagênese/fisiologia , Transplante de Pele/imunologia , CaudaRESUMO
BACKGROUND: Full-thickness (FT) and cultured keratinocyte (CK) allografts have been used as temporary skin replacements in patients with massive burns, but these grafts are ultimately rejected after restoration of host immunocompetence. Genetic engineering has permitted the creation of knockout (KO) mice deficient in class I or class II major histocompatibility antigens. This study examines the immunogenicity of such grafts to determine if these genetically modified keratinocytes could be used for permanent wound coverage. METHODS: Host sensitization to alloantigen was assessed by second-set rejection. CBA mice (n = 111) were primed with flank grafts consisting of FT and CK allografts from normal C57BL/6 donors, FT and CK class I KO allografts, FT and CK class II KO allografts, and CK autografts. Three weeks later, hosts were challenged with normal tail allografts and observed for second-set rejection. Median graft survival was analyzed by chi2 and Wilcoxon rank tests. In the second experiment, cytotoxic T lymphocytes (CTLs) were harvested from CBA mice (n = 28) 3 weeks after flank grafting. CTL effectors were tested on radiolabeled targets at various ratios in a 51Cr release assay. Dilution curves of CTL activity were compared by analysis of variance. RESULTS: Hosts primed with CK or FT allografts demonstrated accelerated rejection of second-set tail grafts compared with hosts covered with CBA autografts. CK knockout grafts were less immunogenic than FT knockout skin; class II KO allografts were considerably less immunogenic than class I KO allografts. CTL activity against the knockout CK allografts was negligible compared with that of hosts primed with normal allografts or FT knockout allografts. CONCLUSION: Although full-thickness knockout skin retains substantial immunogenicity, cultured keratinocytes deficient in class II antigens fail to prime for accelerated second-set rejection and do not elicit a CTL response in the graft recipient. This lack of immunogenicity may permit the indefinite survival of allogeneic knockout keratinocytes in patients requiring massive wound excision and coverage.
Assuntos
Queimaduras/imunologia , Queimaduras/cirurgia , Queratinócitos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Transplante de Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Transplante HomólogoRESUMO
INTRODUCTION: Burn injury delays allograft rejection and impairs the host defense against infection. These functions are mediated via the cytotoxic T-lymphocyte (CTL) response. The CTL response is divided into antigen recognition/processing and effector phases. Presensitization allows selective analysis of changes, induced by burn injury, in the effector limb of the CTL response in relation to time and burn size. METHODS: Anesthetized CBA mice were primed with either a flank allograft from C57BL/6 (B6) mice or an autograft (negative control). Five weeks after grafting, animals were anesthetized and received either a 0, 20, or 40% burn. Spleens were harvested 3, 7, 10, and 14 days after burn injury (n = 96), cocultured with B6 stimulator splenocytes, and assessed for CTL response to radiolabeled allogeneic targets in a 51Cr release assay. In experiment 2, spleens were harvested from unburned and 40% burned animals on Postburn Days 3 and 14. After triple staining, cells were analyzed by flow cytometry for CD4, CD8, and CD25 antigens. In experiment 3, splenocytes from 0 and 40% burned animals on Postburn Days 3 and 14, were cocultured with B6 stimulators for 5 days. Supernatants were evaluated for interleukin (IL)-2, IL-5, and interferon-gamma (IFN-gamma) using ELISA: RESULTS: The CTL response for 20 and 40% burned animals decreased 3 days postburn (-11.9 and -30.1%, P < 0.05), returned to baseline in 7-10 days, and was increased by 14 days postburn (15.8 and 22.6%, P < 0.05). The T-helper lymphocyte population (CD4) from 40% burn animals was significantly decreased on Postburn Days 3 and 14 (10.12 +/- 0.45% vs 11.78 +/- 0.29% and 10.19 +/- 0.24% vs 14.21 +/- 0.97%, respectively, P < 0.05). The CTL effector (CD8) splenocyte population was significantly higher in the burned animals on Postburn Day 14 (4.55% vs 3.71%, P < 0.05). On Postburn Day 3, average IL-5 production was higher in the burned animals (1.80 pg/ml vs 0.59 pg/ml, respectively, P < 0.05). The burn group, on Postburn Days 3 and 14, showed a decrease in mean IL-2 production (212.81 pg/ml vs 263.6 pg/ml and 342.7 pg/ml vs 421.4 pg/ml, respectively, P < 0.05). Mean IFN-gamma production on Postburn Days 3 and 14 was decreased in burned mice (263.75 pg/ml vs 285.57 pg/ml and 218.16 pg/ml vs 263.42 pg/ml, P < 0.05). CONCLUSIONS: Burn injury impairs the effector limb of the CTL response as a function of burn size in the immediate postburn period. CTL activity returns to baseline within 7-10 days postburn and has a rebound increase by Day 14. Early CTL suppression, after burn injury, may be due to a decrease in the T-helper subpopulation. The late increase in cytotoxicity may be secondary to an increase in the effector CTL population in the late postburn period. Burn injury causes a T-helper-2 phenotype as demonstrated by depressed IL-2 and IFN-gamma production and increased IL-5 production.
Assuntos
Queimaduras/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação de Antígeno , Queimaduras/patologia , Queimaduras/cirurgia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Receptores de Interleucina-2/metabolismo , Transplante de Pele/imunologia , Transplante de Pele/patologia , Baço/imunologia , Baço/patologia , Linfócitos T Citotóxicos/patologia , Fatores de Tempo , Transplante Autólogo , Transplante HomólogoRESUMO
OBJECTIVE: Early burn wound excision restores immunocompetence and improves patient survival, but the exact mechanisms have not yet been defined. Burn injury impairs cytotoxic T lymphocyte (CTL) activity as a function of burn size, increasing the risk of infection. The purpose of this study was to determine if early wound excision improved viral-specific CTL function. METHODS: Anesthetized C57BL/6 mice (n = 20) received 0%, 20%, or 40% total body surface area full-thickness contact burns and were inoculated 3 days later with intraperitoneal lymphocytic choriomeningitis virus. Eight days after infection, or 11 days after burn, CTL effectors (E) were harvested and tested against infected, radiolabeled L-Dh targets (T) in a 51Cr-release assay, at varied E:T ratios. Dilution curves of CTL activity were compared by analysis of variance. In the second experiment, mice (n = 18) underwent a 30% burn that was totally excised and grafted on postburn days (PBDs) 0, 3, and 7. Control groups included sham burn and no excision of a 30% burn. In the third experiment, mice (n = 22) received a 30% burn that was partially, completely, or not excised on PBD 3. Control groups included sham burn with and without excision. All groups were infected with intraperitoneal lymphocytic choriomeningitis virus on PBD 3. Viral-specific CTL activity was determined on PBD 11. RESULTS: Both 20% and 40% burn injury impaired viral-specific CTL function. Wound excision on PBDs 0 and 3, but not on PBD 7, partially restored CTL function. Total excision of the 30% burn improved CTL activity to a greater extent than did partial excision. CONCLUSION: Burn injury inhibits viral-specific CTL activity. Early, complete wound excision augments CTL function. Improved CTL activity after burn may reduce the risk of infection, providing an immunologic rationale for expeditious wound excision.
Assuntos
Queimaduras/imunologia , Queimaduras/cirurgia , Linfócitos T Citotóxicos/imunologia , Animais , Queimaduras/virologia , Feminino , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
The role of PGE2 in suppression of B-cell function after burn injury was investigated. Splenocytes from burned or sham-burned mice were isolated 8 days after burn injury and cultured with lipopolysaccharide with or without the addition of prostaglandin E2 (PGE2) or indomethacin (Indo). Anti-peptidoglycan polysaccharide immunoglobulin (Ig)M (specific antibody to a bacterial antigen), total IgM, and total IgG levels in culture supernatant and lymphocyte proliferation were measured. All B-cell functions were significantly suppressed by burn injury. PGE2 suppressed all B-cell functions except for IgG synthesis. Indo restored anti-peptidoglycan polysaccharide IgM to normal levels, but did not have a significant effect on suppressed proliferation and total IgM synthesis. IgG synthesis was increased by PGE2 and inhibited by Indo. Although not all B-cell suppression was accounted for by PGE2, this prostaglandin appeared to be a mechanism responsible for impaired antigen specific antibody response and isotype switching. Successful restoration of specific antibody synthesis to bacterial antigen suggests a potential therapeutic role for a cyclo-oxygenase blocking agent after burn injury.
Assuntos
Linfócitos B/fisiologia , Queimaduras/imunologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/fisiologia , Indometacina/farmacologia , Animais , Especificidade de Anticorpos , Antígenos de Bactérias , Linfócitos B/imunologia , Divisão Celular , Modelos Animais de Doenças , Imunoglobulina G/imunologia , Imunoglobulina G/fisiologia , Imunoglobulina M/imunologia , Imunoglobulina M/fisiologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
INTRODUCTION: Cultured epidermal autografts (CEAs) have been used for wound coverage in patients with massive burns and other skin defects. However, CEAs often display late breakdown, which may be immunologically mediated and initiated by persistent foreign fibroblasts used as a feeder layer to optimize keratinocyte growth. This study investigates whether these fibroblasts, previously shown to persist in vitro, survive after grafting and induce host sensitization to alloantigen. METHODS: CEAs from CBA donors (H-2k) were grown on allogeneic NIH 3T3 (H-2q) or syngeneic LTK (H-2k) fibroblasts, which were removed by trypsinization 7 days later. CBA mice (n = 85) were flank-grafted with NIH allografts (positive control), CEA/3T3s, CEA/LTKs, or CBA autografts (negative control). Hosts were challenged with second set NIH tail allografts 3 weeks later. Median graft survival was compared between groups by Wilcoxon rank and chi 2 analysis. Additional CBA mice (n = 15) received CEAs that were biopsied 0, 4, and 8 days after grafting. The presence of allogeneic fibroblasts was determined by Western immunoblotting, using KL295, a monoclonal antibody that recognizes H-2q (but not H-2k) class II histocompatibility antigens. RESULTS: Allogeneic fibroblasts persisted after grafting but decreased over time, as determined by alloantigen expression on Western immunoblots. Accelerated tail graft rejection occurred in hosts primed by NIH allografts (9 days, p < 0.05), as well as by CEAs growth with an allogeneic (10 days, p < 0.05) but not a syngeneic feeder layer (12 days, NS). Mice receiving flank autografts rejected second set tail allografts at 12 days. CONCLUSIONS: Immunogenic fibroblasts used to grow CEAs survive in vivo and sensitize the graft recipient for accelerated second-set rejection. These persistent cells may initiate an inflammatory response that may result in late graft breakdown and limit the utility of CEAs grown with a foreign fibroblast feeder layer.
Assuntos
Técnicas de Cultura , Epiderme/transplante , Fibroblastos/imunologia , Rejeição de Enxerto/imunologia , Imunologia de Transplantes , Animais , Western Blotting , Sobrevivência Celular , Células Epidérmicas , Epiderme/imunologia , Feminino , Fibroblastos/citologia , Isoantígenos , Queratinócitos/citologia , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Transplante de Pele/imunologia , Transplante Autólogo , Transplante HomólogoRESUMO
Although burn wound excision and grafting have been shown to improve patient survival, the effects on immune function, especially humoral immunity, are not completely understood. The purpose of this study was to investigate the effect of immediate and early wound excision on antibody synthesis and B-cell proliferation, specifically, antibody response to PGPS, a ubiquitous bacterial cell wall antigen. Thirty-six male BALB/c mice were divided into four groups. Sham mice received no burn, and remaining mice received a 30% body surface area full-thickness burn. Under general anesthesia, excision and grafting was performed either 6 or 72 hr after injury (BE&G6 and BE&G72 groups). A fourth control group received burn but did not undergo excision and grafting (Burn group). Splenocytes were isolated 8 days postburn and stimulated with 2.5 microgram/ml lipopolysaccharide. Anti-PGPS IgM, total IgM, and total IgG levels were determined by ELISA. B-cell proliferation, measured by [3H]-thymidine uptake, was expressed as stimulation index. All B-cell functions were significantly suppressed by burn injury. Immediate excision and grafting (BE&G6) restored anti-PGPS IgM synthesis to normal, while nonspecific B-cell functions did not change significantly. Early excision and grafting (BE&G72), however, failed to significantly improve any B-cell functions. Immediate but not early BE&G restored antibody synthesis to the bacterial cell wall antigen (PGPS). Immediate BE&G may therefore lead to a decrease in bacterial infection after burn injury.
Assuntos
Anticorpos Antibacterianos/biossíntese , Linfócitos B/imunologia , Queimaduras/imunologia , Queimaduras/cirurgia , Peptidoglicano/imunologia , Polissacarídeos Bacterianos/imunologia , Transplante de Pele/imunologia , Animais , Formação de Anticorpos , Antígenos de Bactérias/imunologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Fatores de Tempo , Transplante IsogênicoRESUMO
Both burn injury and intestinal ischemia have been proven to induce bacterial translocation from the gut. It is still unknown, however, whether the bacteria induces immune response in these different models. To assess this, we measured in vitro IgM synthesis to peptidoglycan polysaccharide (PGPS), a ubiquitous gut bacterial antigen, after burn injury or gut ischemia-reperfusion in a mouse model. Eighty-five BALB/c mice were divided into four groups. Gut ischemia was produced by placing a vessel loop around the superior mesenteric artery at celiotomy (group Isc; n = 31). After 45 minutes, the abdomen was reopened, and the vessel loop removed. All animals had visible gut ischemia. Control mice (group Isc-C; n = 15) underwent two sham operations. Burn injury was 25% body surface area full-thickness to the dorsum (group B; n = 27). Another control group (B-C; n = 12) was also used. Animals were euthanized 24 hours after recirculation or 5 days after the burn injury. All spleens were removed, and cell suspensions prepared. Cells were cultured in 2.5 micrograms/ml lipopolysaccharide for 5 days, and anti-PGPS IgM level in the supernatant was measured by an enzyme-linked immunosorbent assay. Intestinal ischemia produced a significant rise in in vitro anti-PGPS IgM synthesis per 10(5) lymphocytes, which is the principal immunoglobulin response to infection. However, anti-PGPS IgM in mice after burn injury was significantly decreased. This decreased IgM synthesis after burn injury compared to gut ischemia may represent continued immune impairment from the burn wound, and may account for organ dysfunction related to bacterial translocation after burn injury.
Assuntos
Translocação Bacteriana , Queimaduras/imunologia , Imunoglobulina M/biossíntese , Isquemia/imunologia , Estômago/irrigação sanguínea , Animais , Translocação Bacteriana/imunologia , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptidoglicano/imunologia , Peptidoglicano/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Traumatismo por Reperfusão/imunologia , Baço/citologia , Estômago/imunologiaRESUMO
Burn injury impairs cellular immunity, increases the risk of viral infection, and delays allograft rejection, but little is known about its effect on antigen processing and cytotoxic T-lymphocyte (CTL) function. This study examined the effect of burn injury on alloantigen sensitization with an in vivo model of second-set rejection and in vitro assays of CTL alloreactivity. Anesthetized CBA mice (n = 95) received a 0%, 20%, or 40% full-thickness contact burn that was partially excised 3 days later and covered with autograft or C57BL/6 allograft. Two weeks after the burn was inflicted, mice were challenged with second-set tail allografts, which were observed for rejection. Median graft survival times were compared by Wilcoxon rank and chi-squared analysis. Additional CBA mice (n = 24) underwent similar burn injury, excision, and grafting. Splenocytes were harvested 2 weeks later and were used as CTL effectors against radiolabeled targets. Dilution curves of target lysis were compared by analysis of variance. Forty percent burn injury prolonged unprimed allograft survival from 13 to 15 days (p < 0.01) but had a greater effect on primed allograft survival, which increased from 9 to 12.5 days (p < 0.01). Furthermore, a 40% burn eliminated the influence of priming, resulting in second-set graft survival similar to that of mice in an unburned, unprimed control group (12.5 vs. 13 days, NS). Whereas 20% burn injury did not inhibit CTL priming, a 40% burn profoundly impaired CTL function (p < 0.001), which recovered only after 6 days of in vitro allostimulation. Burn injury inhibits both alloantigen priming and the immunologic memory of CTLs as a function of burn size. This impairment in alloantigen processing helps to explain defects in cellular immunity and suggests a mechanism for prolonged allograft survival and decreased viral resistance after burn injury occurs.
Assuntos
Distinções e Prêmios , Queimaduras/imunologia , Isoantígenos/imunologia , Linfócitos T Citotóxicos/imunologia , Transplante de Tecidos , Imunologia de Transplantes/imunologia , Animais , Queimaduras/terapia , Distribuição de Qui-Quadrado , Modelos Animais de Doenças , Feminino , Sobrevivência de Enxerto/imunologia , Antígenos de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos CBA , Transplante de Tecidos/patologia , Transplante de Tecidos/fisiologiaRESUMO
BACKGROUND: Cultured keratinocyte (CK) and cadaveric skin allografts have prolonged survival in patients with massive thermal injury. It is unclear if this delayed rejection is due to impaired host responsiveness or decreased graft immunogenicity. Although burn injury has been shown to decrease parameters of allograft response, no studies have examined the effect of burn injury on alloantigen expression. This study investigated the effect of burn size on class II antigen expression in CK allografts as well as on tissue levels of interferon-gamma (IFN-gamma), the principle regulator of alloantigen expression. METHODS: Anesthetized CBA mice (n = 64) received a 0%, 20% partial-thickness (PT), 20% full-thickness (FT), or 40% FT contact burn. Forty-eight hours later, wounds were partially excised and covered with CK allografts from C57BL/6 donors. Five days after burn injury, grafts were analyzed for donor-specific class II antigen. Protein expression was determined by Western immunoblotting and quantified with video densitometry. Wound, serum, and unburned skin levels of IFN-gamma were determined by enzyme-linked immunosorbent assay. Groups were compared by Fisher's analysis of variance. RESULTS: As burn size increased, class II antigen expression decreased (p < 0.001). This corresponded with decreased wound and skin levels of IFN-gamma after 40% burn (p < 0.05); however, wound IFN-gamma was significantly elevated after 20% PT and FT burns (p < 0.01). Serum IFN-gamma increased as burn size increased (p < 0.01). CONCLUSIONS: Burn injury decreases the antigenicity of CK allografts, which partly explains delayed allograft rejection after burn injury. Although wound IFN-gamma increases after minor thermal injury, the profound decrease in wound and skin IFN-gamma after a major burn corresponds with diminished class II antigen expression. The decreased availability of IFN-gamma after major thermal injury provides a mechanism for limited allograft tolerance.
Assuntos
Queimaduras/imunologia , Genes MHC da Classe II/imunologia , Interferon gama/análise , Queratinócitos/imunologia , Animais , Feminino , Expressão Gênica , Humanos , Queratinócitos/transplante , Camundongos , Camundongos Endogâmicos CBARESUMO
Cultured keratinocyte (CK) allografts have limited antigenicity and have been used as a skin replacement in patients with massive thermal injury. Recent data indicate that CK grafts are more immunogenic than previously believed and could compromise wound healing in the immunocompetent host. The purpose of this study was to determine if the immunosuppression of burn injury might affect the alloantigen response and minimize sensitization to CK allografts. CBA mice received a 0%, 20%, or 40% burn that was partially excised three days later and grafted with a full-thickness (FT) skin allograft, CK allograft, or CK autograft. Two weeks postburn, mice received FT tail skin allografts, which were observed for rejection. We observed that FT and CK allografts primed the unburned host with equal efficacy. However, burn injury selectively minimized priming by CK allografts, resulting in delayed rejection of second-set allografts. With evidence that burn injury inhibits host sensitization to CK allografts, we then examined the effect of burn size on CTL alloreactivity. Additional CBA mice underwent burn injury, excision, and grafting as described above. Host splenocytes were harvested two weeks later and tested on radiolabeled targets for allospecific cytotoxicity. CTLs from unburned mice primed with FT allografts demonstrated the greatest CTL lysis, followed next by CTLs from unburned mice covered with CK allografts. Burn injury inhibited CTL activity as a function of wound size. Activity of CTLs from burned mice primed with CK allografts improved after in vitro allostimulation but remained below that of CTLs from unburned, unprimed mice. We conclude that burn injury selectively inhibits the allospecific response to CK allografts. The decreased immunogenicity of CK allografts, when used for burn wound coverage, may improve the long-term survival of allogeneic keratinocytes, enhancing their potential as a biologic skin replacement.
Assuntos
Queimaduras/imunologia , Rejeição de Enxerto , Memória Imunológica , Queratinócitos/imunologia , Transplante de Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Citotoxicidade Imunológica , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fatores de TempoRESUMO
We studied 75 BALB/c mice to examine the role of impaired immunoglobulin M (IgM) synthesis in the increased risk of bacterial infection after burn injury by investigating the kinetics of IgM synthesis to peptidoglycan polysaccharide (PGPS), a ubiquitous bacterial antigen. Splenocytes were isolated 1, 5, and 8 days postburn (PBD) and cultured with lipopolysaccharide for 5 days. Culture supernatant was collected and anti-PGPS IgM and total IgM levels were measured by ELISA. Total IgM-secreting cells were measured by ELISPOT assay. Total IgM and anti-PGPS IgM per IgM-secreting cell were calculated. On PBD 1, anti-PGPS IgM synthesis but not total IgM synthesis is increased in burned animals. By PBD 5, total IgM and anti-PGPS IgM synthesis in the burn group start to fall and by PBD 8, both are significantly decreased. The early increase in anti-PGPS IgM synthesis represents a positive response to bacterial challenge. However, the late nonspecific decrease in total IgM and anti-PGPS IgM synthesis suggests a potential mechanism for increased susceptibility to bacterial infection 5 to 10 days after burn injury.
Assuntos
Queimaduras/imunologia , Queimaduras/metabolismo , Imunoglobulina M/biossíntese , Peptidoglicano/imunologia , Animais , Infecções Bacterianas/imunologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Baço/metabolismoRESUMO
BACKGROUND: Cytotoxic lymphocytes (CTLs) are an important component of immune function, involved in antigen recognition and resistance to viral infection. Burn injury suppresses cell-mediated immunity, induces allograft tolerance, and increases the risk of viral infection, but the mechanisms are not well understood. This study analyzes the effect of burn size and burn wound excision on CTL activity. METHODS: Anesthetized CBA mice (n = 12) received a 0%, 20%, or 40% body surface area contact burn. Additional mice (n = 16) received a 40% burn that was totally, partially, or not excised 72 hours after burn. Excised areas were covered with normal, syngeneic skin. Two weeks later harvested splenocytes were cocultured with allogeneic stimulators. CTL activity was determined by a 51Cr release assay, in which CTL effectors were tested on allogeneic, radiolabeled targets. Dilution curves of CTL activity were compared by ANOVA: RESULTS: Both 20% and 40% burns significantly inhibited CTL activity (p < 0.05). Total but not partial excision of a 40% burn restored CTL activity (p < 0.01). Both total and partial wound excision also improved survival (p < 0.05). CONCLUSIONS: Burn injury inhibits CTL activity in a size-dependent manner, and total wound excision significantly improves both CTL function and survival after injury. This study suggests a mechanism for the immunosuppressive effects of burn injury and provides an immunologic rationale for early, complete burn wound excision.
Assuntos
Queimaduras/cirurgia , Linfócitos T Citotóxicos/fisiologia , Animais , Queimaduras/mortalidade , Queimaduras/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Período Pós-Operatório , Análise de Sobrevida , Fatores de TempoRESUMO
Recent reports suggest that long-term graft take of cultured epidermal autografts (CEAs) is less than 50% when late graft loss is considered. The characteristics of late CEA loss suggest that it may occur as a result of an immunologic reaction to persistent xenogeneic cells and/or proteins used to grow CEA. In this study we examined whether immunologically reactive, mouse 3T3 fibroblasts used as feeder layers can persist in primary, secondary, and tertiary human CEA. We cocultured keratinocytes from 11 separate burn patients with growth-arrested 3T3 fibroblasts. After removing visible 3T3 fibroblasts from CEA with trypsinization, we allowed CEA to reach confluence. We then harvested CEA either as primary, secondary, or tertiary cultures. We detected mouse fibroblasts using fluorescence activated cell sorting (FACS) with a monoclonal antibody specific for mouse major histocompatibility (MHC) antigens. We detected mouse MHC class II antigens by performing Western immunoblotting with another mouse MHC-specific monoclonal antibody. By FACS we identified mouse fibroblasts in 100, 75, and 62.5% of primary, secondary, and tertiary passage CEAs, respectively. Similarly by immunoblotting we found mouse MHC class II antigen in 100, 80, and 66.7% of primary, secondary, and tertiary CEAs. These results demonstrate that xenogeneic fibroblast feeder layers capable of generating immunogenic transplantation antigens persist in CEAs. The persistence of these cells and their antigen expression may contribute to CEA loss.
Assuntos
Epiderme/transplante , Fibroblastos/imunologia , Transplante de Pele/imunologia , Animais , Western Blotting , Separação Celular , Células Cultivadas , Células Epidérmicas , Epiderme/imunologia , Citometria de Fluxo , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Transplante Heterólogo/imunologiaRESUMO
It has been reported that cultured keratinocyte (CK) allografts are not rejected in mice, unlike in other species. Several reports have suggested that mouse CK allografts are incapable of stimulating a primary alloresponse, including sensitization of recipients to alloantigens. In this study, we investigated the immunogenicity of mouse CK allografts in vivo by determining whether CK allografts primed for a second set rejection response. First, we grafted mice with either CK allografts, CK autografts, full-thickness (FT) allografts, or no graft at all. We then regrafted mice 4 weeks later with a tail skin allograft. Mice grafted with CK allografts rejected second allografts as rapidly and as vigorously as mice grafted with FT flank allografts. Next, we tested whether CK allograft primed recipients for enhanced CTL responses. We found that mice grafted with CK allografts generated a significantly enhanced CTL alloreactive response after in vitro stimulation. The response was similar to that of mice grafted with FT skin allografts. With evidence that CK allografts primed, we biopsied wounds immediately after CK allografting and, using Western immunoblotting, found that CK allografts had substantial expression of MHC class II antigens in vivo. We conclude from the results of our studies that mouse CK allografts unequivocally prime recipients to alloantigens in vivo and suggest that a possible mechanism for alloantigen priming may be CK allograft expression of MHC class II antigens.