RESUMEN
The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Ciclofosfamida/administración & dosificación , Sistema Enzimático del Citocromo P-450/biosíntesis , Neoplasias Hematológicas/tratamiento farmacológico , Adolescente , Adulto , Biomarcadores de Tumor/genética , Niño , Ciclofosfamida/efectos adversos , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Corazón/efectos de los fármacos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Trasplante de Células Madre , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
Omega-3 is known to enhance the effects of several chemotherapeutic agents and to exert several immunoregulatory actions In the present study, we evaluated the effects of a 21-day feeding regimen with omega-3-rich fish oil (FO) and its corresponding control, omega-6 rich corn oil (CO), on the BU-CY conditioning and the development of GVHD after BMT in mice. Before conditioning, FO, but not CO, feeding caused a significant attenuation in the number and functionality of splenic FoxP3+ T regulatory cells (Treg). FO feeding also enhanced the effects of the conditioning through severe depletion of Treg cells in the spleen and CD11b+ myeloid cells in both the BM and spleen. Consequently, FO-fed animals conditioned with BU-CY showed exacerbated GVHD following transplantation with allogeneic BM and splenic cells. In contrast, identical transplantation in CO-fed mice resulted in poor engraftment and body weight loss. Moreover, in standard-fed recipients, BMT with cells from FO-fed donors resulted in moderate GVHD and improved the survival time, whereas BMT with cells from CO-fed donors shortened the survival time and caused anemia. We conclude that food supplements should be considered in patients undergoing BMT and/or chemotherapy treatment.
Asunto(s)
Trasplante de Médula Ósea , Ácidos Grasos Omega-3/farmacología , Aceites de Pescado/microbiología , Factores de Transcripción Forkhead , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Depleción Linfocítica , Linfocitos T Reguladores/inmunología , Acondicionamiento Pretrasplante , Aloinjertos , Animales , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/patologíaRESUMEN
GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK-signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice.
Asunto(s)
Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Riñón/inmunología , Animales , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Femenino , Quinasas Janus/inmunología , Riñón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Transcripción STAT/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Transcriptoma/efectos de los fármacos , Acondicionamiento Pretrasplante , Inmunología del TrasplanteRESUMEN
It is now well established that the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARα) is expressed in different types of immune cells and plays a pivotal role in the regulation of age-related production of inflammatory cytokines. However, the role(s) of this receptor in the regulation of immune cell homoeostasis in ageing non-lymphoid and lymphoid organs has not yet been resolved. We examine this issue here by evaluating the hepatic and splenic immune status and immunoglobulin (Ig) production in male PPARα-null mice and their wild-type littermates at one and 2 years of age. In comparison with the age-matched control animals, PPARα-null mice exhibited age-related elevations in the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC) and splenocytes. Moreover, at 2 years of age, these alterations in hepatic immune cells were accompanied by significant increases in hepatic levels of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interferon-gamma (IFN-γ), in combination with the development of hepatic inflammatory loci containing mixtures of leucocytes. Alterations in splenocytes of old PPARα-null mice were also accompanied by increases in cellularity of both white and red pulps of the spleen. Furthermore, these same animals exhibited pronounced increases in the numbers of splenic plasma cells and enhanced production of Ig of different isotypes, including IgG1, IgG2a and IgE. Thus, our findings indicate that upon ageing, PPARα plays a crucial role in regulating the total numbers, compositions and functions of immune cells in both lymphoid and non-lymphoid immune organs of mice.
Asunto(s)
Envejecimiento/inmunología , Inmunoglobulinas/biosíntesis , Hígado/inmunología , PPAR alfa/inmunología , Bazo/inmunología , Animales , Citocinas/biosíntesis , Citocinas/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunoglobulinas/inmunología , Masculino , Ratones , Ratones Noqueados , PPAR alfa/deficienciaRESUMEN
Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1.8%). The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i.e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and alpha-galactosylceramide respectively) and produced immunoglobulin M and interferon-gamma. Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Hígado/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Proliferación Celular , Separación Celular/métodos , Supervivencia Celular , Células Cultivadas , Inmunocompetencia , Inmunoglobulina M/biosíntesis , Inmunofenotipificación , Interferón gamma/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunologíaRESUMEN
Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury- and silver-induced B cell activation and anti-nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody-secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H-2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long-term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Linfocitos B/inmunología , Mercurio/administración & dosificación , Plata/administración & dosificación , Xenobióticos/administración & dosificación , Animales , Anticuerpos Antinucleares/sangre , Autoinmunidad , Nucléolo Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Técnica de Placa Hemolítica , Inmunoglobulina E/análisis , Inmunoglobulina E/sangre , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Activación de Linfocitos , Ratones , Modelos Animales , Especificidad de la Especie , Bazo/inmunologíaRESUMEN
GVHD is a major complication in allogeneic SCT. Available GVHD models are mainly based on radiotherapy-conditioning and/or immune deficient mice. GVHD models based on chemotherapy-based regimens remain poorly studied, despite 50% of all transplantations being chemotherapy based. Our aim was to develop a GVHD model using chemotherapy as conditioning. Female BALB/c (H-2Kd) were conditioned with BU-CY and transplanted with 2 x 10(7) BM and 3 x 10(7) spleen cells from either C57BL/6 (H-2 Kb) mice (allogeneic setting) or from male BALB/c to serve as a control group for regimen-related toxicity and engraftment. GVHD manifestations and histopathological changes were evaluated. Chimerism and donor T cells presence in skin, intestine and liver were studied using FACS-, FISH analysis and immunohistochemistry. Allogeneic transplanted mice developed lethal GVHD starting from day+7 with both histological and clinical signs. Donor T cells accumulated in recipient skin and intestine with GVHD progression. BM-failure, apoptosis and T-lymphocyte infiltration into target organs were significantly higher in allogeneic when compared with the syngeneic group. No toxicity or GVHD signs were observed in the syngeneic setting. We report a mouse model of GVHD using BU-CY conditioning that represents the most common myeloablative-conditioning regimen in clinical SCT. This model can be utilized to study the role of conditioning on mechanisms underlying GVHD.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Acondicionamiento Pretrasplante/efectos adversos , Acondicionamiento Pretrasplante/métodos , Animales , Antineoplásicos Alquilantes/administración & dosificación , Busulfano/administración & dosificación , Ciclofosfamida/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Enfermedad Injerto contra Huésped/fisiopatología , Masculino , Ratones , Trasplante Homólogo/efectos adversosRESUMEN
Conditioning regimens are an important issue determining the outcome of hematopoietic stem cell transplantation (HSCT). Less toxicity, early engraftment and no relapse are the aims of efficient conditioning. Our objective was to investigate the long-term effects of BU-CY and their administration order on the toxicity and chimerism in a mouse model of HSCT. Female BALB/c mice were treated with either BU (15 mg/kg/day x 4)-CY (100 mg/kg/day x 2) or CY-BU. Treated mice were transplanted with Sca-1+ cells from male BALB/c mice. Until 90 days after HSCT, the animals were monitored for body weight and analyzed for cellular phenotype of the thymus, spleen and BM, total chimerism, the spleen chimerism of DCs and T regulatory (Treg) cells, and hepatotoxicity. BU-CY and CY-BU treatments exerted comparable myeloablative and immunosuppressive effects. The long-term engraftment of donor cells in the BM and thymus regeneration showed the same features in both groups. However, the two regimens differed; in general, hepatotoxicity and chimerism of DC and Treg cells. In the long term, BU-CY, but not CY-BU caused a marked decrease in body weight and a significant increase in the activities of the liver enzymes, particularly aspartate amino transferase (AST). We conclude that the alteration of the administration order of BU-CY to CY-BU not only gives the same level of engraftment but also reduces the toxicity of the conditioning regimen that might be valuable specially in young patients who are undergoing HSCT.
Asunto(s)
Busulfano/administración & dosificación , Ciclofosfamida/administración & dosificación , Trasplante de Células Madre Hematopoyéticas , Acondicionamiento Pretrasplante/métodos , Animales , Peso Corporal , Médula Ósea/efectos de los fármacos , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Quimerismo/efectos de los fármacos , Esquema de Medicación , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Hígado/efectos de los fármacos , Hígado/fisiología , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/anatomía & histología , Bazo/citología , Bazo/efectos de los fármacos , Timo/citologíaRESUMEN
The initiation and severity of systemic autoimmune diseases are influenced by a variety of genetic and environmental factors, in particular bacterial infections and products. Here, we have employed bacterial lipopolysaccharide (LPS), which non-specifically activates the immune system, to explore the involvement of innate immunity in mercury-induced autoimmunity in mice. Following treatment of mouse strains resistant [DBA/2 (H-2(d))] or susceptible [SJL(H-2(s))] to such autoimmunity with mercuric chloride and/or LPS or with physiological saline alone (control), their immune/autoimmune responses were monitored. Resistant DBA/2 mice were rendered susceptible to mercury-induced autoimmunity by co-administration of LPS, exhibiting pronounced increases in the synthesis of IgG1 and IgE, high titres of IgG1 deposits in the kidneys and elevated circulating levels of IgG1 antibodies of different specificities. Furthermore, the percentages of the T cells isolated from the spleens of DBA/2 mice exposed to both mercury and LPS that produced pro-inflammatory cytokines were markedly increased by in vitro stimulation with phorbol myristate acetate (PMA) and ionomycin, which was not the case for splenic T cells isolated from mice receiving mercuric chloride, LPS or saline alone. In addition, exposure of susceptible SJL mice to mercury in combination with LPS aggravated the characteristic features of mercury-induced autoimmunity, including increased synthesis of IgG1 and IgE, the production of IgG1 anti-nucleolar antibodies (ANolA) and the formation of renal deposits of IgG1. In summary, our findings indicate that activation of the innate immune system plays a key role in both the induction and severity of chemically induced autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Lipopolisacáridos/inmunología , Cloruro de Mercurio/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoinmunidad/efectos de los fármacos , Susceptibilidad a Enfermedades , Femenino , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Riñón/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
In susceptible mice, mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of immunoglobulin (Ig) G1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. We have previously hypothesized that mercury confers more adverse immunological effects on those mouse strains, which are genetically prone to develop spontaneous autoimmune diseases than on normal strains. In this study, we tested our hypothesis in tight skin 1 (Tsk1/+) mice, a murine model for human scleroderma. As a support for our hypothesis, we observed that in Tsk1/+ mice, B cells were spontaneously hyperactive and that treatment with mercury induced a strong immune/autoimmune response in these mice, but not in their non-Tsk (+/+) littermates. This response was characterized by the formation of high numbers of splenic IgG1, IgG2b and IgG3 antibody-secreting cells, increased serum levels of IgE, production of IgG1 antibodies against single-stranded DNA (ssDNA), trinitrophenol (TNP) as well as thyroglobulin and the development of renal IgG1 deposits. Neither Tsk1/+ mice nor F1 hybrid crosses between this strain, and mercury susceptible B10.S (H-2(s)) were able to produce IgG1-ANolA in response to mercury. Moreover, mercury-induced immune activation in Tsk1/+ was not able to potentiate the progression of skin fibrosis in this strain. Thus, exposure to mercury accelerates the immune dysregulation, but not the development of skin fibrosis in Tsk1/+ mice.
Asunto(s)
Fibrosis/inmunología , Cloruro de Mercurio/inmunología , Cloruro de Mercurio/toxicidad , Esclerodermia Sistémica/inmunología , Piel/efectos de los fármacos , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/efectos de los fármacos , Linfocitos B/inmunología , ADN de Cadena Simple/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosis/patología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/efectos de los fármacos , Isotipos de Inmunoglobulinas , Riñón/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Mutantes , Picratos/inmunología , Piel/inmunología , Piel/patología , Tiroglobulina/inmunologíaRESUMEN
Xenobiotic-metals such as mercury (Hg) and silver (Ag) induce an H-2 linked antinucleolar autoantibody (ANolA) production in susceptible mice. The mechanism for induction of ANolA synthesis is not well understood. However, it has been suggested that both metals interact with nucleolar proteins and reveal cryptic self-peptides to nontolerant autoreactive T cells, which in turn stimulate specific autoreactive B cells. In this study, we considered this suggestion and asked if mercury and silver display, if not identical, similar cryptic self-peptides, they would induce comparable ANolA responses in H-2 susceptible mice. We analysed the development of ANolA production in mercury- and/or silver-treated mice of H-2s, H-2q and H-2f genotypes. We found that while mercury stimulated ANolA synthesis in all strains tested, silver induced ANolA responses of lower magnitudes in only H-2s and H-2q mice, but not in H-2f mice. Resistance to silver in H-2f mice was independent of the dosage/time-period of silver-treatment and non-H-2 genes. Further studies showed that F1 hybrid crosses between silver-susceptible A.SW (H-2s) and -resistant A.CA (H-2f) mice were resistant to silver, but not mercury with regard to ANolA production. Additionally, the magnitudes of mercury-induced ANolA responses in the F1 hybrids were lower than those of their parental strains. The above differential ANolA responses to mercury and silver can be explained by various factors, including the different display of nucleolar cryptic peptides by these xenobiotics, determinant capture and coexistence of different MHC molecules. Our findings also suggest that the ability of a xenobiotic metal merely to create cryptic self-peptides may not be sufficient for the induction of an ANolA response.
Asunto(s)
Anticuerpos Antinucleares/biosíntesis , Autoinmunidad/inmunología , Mercurio/inmunología , Proteínas Nucleares/inmunología , Plata/inmunología , Animales , Cruzamientos Genéticos , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta Inmunológica , Femenino , Genes MHC Clase I/inmunología , Genotipo , Inmunoglobulina G/biosíntesis , Ratones , Ratones EndogámicosRESUMEN
Administration of mercuric chloride into susceptible rats and mice induces a systemic autoimmune disease, which is characterized by a T-cell-dependent polyclonal B-cell activation, an increase in serum levels of immunoglobulin (Ig)G1 and IgE, production of antibodies of different specificities and development of renal IgG deposits. A peculiar feature of mercury-induced autoimmunity is that the polyclonal B-cell activation spontaneously disappears in spite of continuous injection of mercury. The exact mechanism(s) for autoregulation of mercury-induced autoimmunity is not well understood. In the present study, we analysed the regulation of mercury-induced immune/autoimmune responses in mice and tested whether spontaneous downregulation of these responses is owing to a general immunosuppression. Mercury-susceptible [SJL (H-2s)] and -resistant [DBA/2 (H-2d)] mice were injected with mercury for 4, 10, 15 and 17 weeks. Immune/autoimmune responses were monitored in these mice. Thereafter, mercury-injected mice for 17 weeks were further immunized with horse red blood cells (HRBC) to study whether the subsequent humoral immune response to a foreign antigen is suppressed. We found that except for IgG1 anti-nucleolar antibody production and renal IgG1 deposition, other characteristics of mercury-induced autoimmunity were downregulated in SJL (H-2s) mice after chronic treatment with mercury. However, these mice did not show any reduction in the number of splenic antibody-secreting cells and/or in serum titres of specific IgM, IgG1 and IgG2a anti-HRBC antibodies in response to HRBC as compared with naïve mice. Similarly, in mercury-resistant DBA/2 (H-2d) mice, chronic treatment with mercury did not either suppress specific antibody responses against HRBC. Our findings show that the autoregulation of mercury-induced immune/autoimmune responses observed after chronic treatment with mercury is not owing to a general immunosuppression.
Asunto(s)
Autoanticuerpos/biosíntesis , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Cloruro de Mercurio/farmacología , Animales , Autoanticuerpos/genética , Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Eritrocitos/inmunología , Femenino , Caballos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interferón gamma/química , Interferón gamma/genética , Interleucina-4/biosíntesis , Interleucina-4/química , Interleucina-4/genética , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Cloruro de Mercurio/administración & dosificación , Ratones , Ratones Endogámicos DBA , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Bazo/metabolismoRESUMEN
In susceptible mice, the heavy metal ion mercury is able to induce a strong immune activation, which resembles a T helper 2 (Th2) type of immune response and is characterized by a polyclonal B cell activation, formation of high levels of IgG1 and IgE antibodies, production of autoantibodies of different specificities and development of renal IgG deposits. In the present study, we analysed the in vivo effects of mercury in nonobese diabetic (NOD) mice, which is believed to develop a spontaneous Th1 cell-mediated autoimmune diabetes similar to type 1 diabetes in humans. Three weeks of treatment with mercury induced a strong Th2 like immune/autoimmune response in NOD mice. This response was characterized by an intensive increase in splenic IgG1 antibody secreting cells, a marked elevation in serum IgE levels, a substantial increase in splenic IL-4 mRNA, but a significant decrease in splenic IFN-gamma mRNA. Mercury-induced IgG1 antibodies were mainly against ssDNA, TNP and thyroglobulin, but not against nucleolar antigen. Moreover, mercury-injected NOD mice developed high titres of IgG1 deposits in the kidney glomeruli. We further tested if the generated Th2 response could interfere with the development of insulitis and diabetes in NOD mice. We found that three weeks of treatment with mercury was also able to significantly suppress the development of insulitis and postpone the onset of diabetes in these mice. Thus, mercury-induced immune activation can counter-regulate the Th1 cell-mediated autoimmune responses and confer a partial protection against autoimmune diabetes in NOD mice.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Activación de Linfocitos/efectos de los fármacos , Mercurio/farmacología , Animales , Especificidad de Anticuerpos , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Células Cultivadas , Femenino , Inmunoglobulina G/biosíntesis , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Riñón/inmunología , Cinética , Ratones , Ratones Endogámicos NOD , ARN Mensajero/biosíntesis , Bazo/inmunología , Células Th2/inmunologíaRESUMEN
Previous studies have shown that genetic factors control the susceptibility to mercury-induced immunoglobulin (Ig)G1 antibody formation, IgE synthesis, renal IgG deposits and antinucleolar autoantibodies (ANolA) production in the susceptible mice. In this study, we examined the genetic control of resistance to these characteristics after HgCl2 injection in F1 hybrid crosses between the highly mercury resistant DBA/2 and mercury susceptible NZB (H-2d), SJL (H-2 s), A.CA (H-2f) and DBA/1 (H-2q) mice and also in backcross hybrids between (DBA/2 x SJL)F1 and SJL mice. We observed that mercury-induced immune/autoimmune manifestations were profoundly downregulated in most (if not all) of the F1 hybrids, indicating that the resistance to mercury was a dominant trait. Analysis of mercury-induced immune/autoimmune responses in the (DBA/2 x SJL) x SJL backcross hybrids suggested that only one gene or a cluster of genes determined the resistance to the ANolA production, whereas the resistance to other characteristics was controlled by two and/or three gene loci. By H-2 genotyping the backcross mice, it was found that H-2d haplotype per se could confer resistance to ANolA production. However, we did not find any significant association between the H-2d haplotype and the resistance to increase of IgG1 and IgE synthesis and the development of renal IgG1 deposits. Thus, while in DBA/2 mice, gene(s) in the H-2 loci strictly contribute to the inheritance of resistance to ANolA production; non-H-2 genes mainly govern the inheritance of unresponsiveness regarding other characteristics.
Asunto(s)
Autoinmunidad/genética , Cloruro de Mercurio/inmunología , Animales , Autoinmunidad/inmunología , Cruzamientos Genéticos , Femenino , Genes Dominantes , Antígenos H-2/genética , Masculino , Cloruro de Mercurio/administración & dosificación , Ratones , Ratones Endogámicos DBARESUMEN
Immune complexes from the synovial fluid of rheumatoid arthritis (RA) patients, or artificial rheumatoid factors (RF)-like (antibody--antibody) immune complexes, induce a remarkably intense, sustained and selective immunoglobulin (Ig)G1 response under certain experimental conditions in mice. Because the IgG1 antibody response is extraordinarily strong, the role of interleukin (IL)-4, important for IgG1 synthesis, was investigated. Both C57BL/6 and NZB IL-4-deficient mice produced IgG1--RF antibodies after injection with RF-like immune complexes, although the antibody levels were slightly delayed compared to wild type mice. This shows that IL-4 is not obligatory in RF-like immune complex induced IgG1--RF production. A discrepancy in the decline of serum IgG1--RF was noted between NZB and C57Bl/6 mice. Serum IgG1-RF declined 43 days postinjection (p.i.), in C57BL/6 mice whereas high serum levels of IgG1--RF were maintained more than 100 days in the NZB mice, indicating different regulatory mechanisms in these mice. To study if the affinity for mouse IgG increased with time in NZB mice and thus become more directed against self, the cross-reactivity of the IgG1--RF antibodies with IgG from other species was investigated early and late after injection. It was, however, found that the cross-reactivity with IgG of human, goat and rabbit origin did not change between the two time points.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Inmunoglobulina G/biosíntesis , Factor Reumatoide/biosíntesis , Animales , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo/aislamiento & purificación , Artritis Reumatoide/sangre , Autoantígenos/inmunología , Enfermedades Autoinmunes/sangre , Linfocitos T CD4-Positivos/inmunología , Reacciones Cruzadas , Femenino , Cabras , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Interleucina-4/fisiología , Isoantígenos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Conejos , Factor Reumatoide/genética , Factor Reumatoide/inmunología , Solubilidad , Especificidad de la Especie , Líquido Sinovial/química , Líquido Sinovial/inmunología , Factores de TiempoRESUMEN
Rheumatoid factor (RF)-like (antibody-antibody) immune complexes induce a selective and intensive immunoglobulin (Ig)G1-RF response after a single injection in mice. However, the longevity of the response differs between mouse strains: serum IgG1-RF antibody titres decline 40 days after injection in C57Bl/6 mice whereas levels are maintained for more than 100 days in NZB mice. In order to elucidate whether this difference was owing to a lower ability of NZB mice to clear the injected immune complexes, sections of kidney, spleen, liver and mesenteric lymph nodes were harvested at different time points after injection with RF-like immune complexes. Immunohistochemical staining revealed that NZB mice have a delayed clearance of the injected immune complexes, because the immune complexes are retained for more than 40 days in their spleens and 100 days in their kidneys, compared to only 14 days in C57Bl/6 mice. Germinal centres were also present for a longer period in the spleens of the NZB mice, accompanying the presence of the immune complexes, and were abnormally large compared to C57Bl/6 mice. The clearance of immune complexes from the spleen coincided with the decline in serum levels of IgG1-RF, indicating that prolonged retention of immune complexes is responsible for the sustained IgG1-RF response.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Centro Germinal/patología , Factor Reumatoide/metabolismo , Animales , Riñón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NZB , Especificidad de la Especie , Bazo/inmunologíaRESUMEN
Mercury-induced autoimmunity is characterized by a T cell-dependent B cell activation (mainly of IgG1 and IgE isotypes), production of anti-nucleolar autoantibodies (ANolA) and the formation of renal IgG deposits. The autoimmunity is to a large extent controlled by genetic factors. We studied 15 different inbred mouse strains of seven H-2 (mouse MHC) genotypes to determine the importance of H-2 and non-H-2 background genes in mercury-induced autoimmunity. The tested strains exhibited a diverse autoimmune response to mercury. In each H-2 genotype, there was at least one strain which responded to mercury by the production of high levels of IgG1 and IgE Ig as well as by the development of high titers of renal IgG1 deposits. Only mouse strains with H-2(s) and H-2(q) genotypes, irrespective of their background genes, produced ANolA after mercury treatment. Only SJL (H-2(s)) and A.SW (H-2(s)) mice were highly susceptible to all characteristics of mercury-induced autoimmunity. NZB (H-2(d)) mice were also highly susceptible, but they did not develop ANolA. Only the DBA/2 (H-2(d)) strain was found to be resistant to all tested mercury-induced autoimmune manifestations, suggesting that non-responsiveness to mercury in DBA/2 mice was largely influenced by non H-2 genes. These findings imply that H-2 genes mainly determine the susceptibility to mercury-induced ANolA production, whereas non-H-2 genes control the susceptibility to and the severity of the B cell activation and renal IgG deposition.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Antígenos H-2/genética , Mercurio/toxicidad , Animales , Anticuerpos Antinucleares/biosíntesis , Nucléolo Celular/inmunología , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Masculino , Ratones , Ratones Endogámicos , Especificidad de la EspecieRESUMEN
Mercury can induce a systemic autoimmune disease in susceptible mouse strains. H-2s mice are particularly susceptible to mercury-induced autoimmunity and other mouse strains are more or less resistant. T helper 1/T helper 2 (Th1/Th2) dichotomy has been proposed for resistance or susceptibility, respectively. In the current study we show that mercury treatment induced a full autoimmune response in both C57BL/6 (H-2b) wild-type and interleukin-4 (IL-4)-deficient mice. Antibody production of all isotypes were induced, except that in IL-4-deficient mice there was no immunoglobulin E (IgE) and very low levels of immunoglobulin G1 (IgG1) antibody synthesis. Autoantibodies of different specificities were produced. The granular pattern of all IgG subclasses deposits were detected in the kidneys. In contrast to mercury-treated H-2s seconds mice, we did not detect any anti-nucleolar autoantibodies in the sera of mercury-treated wild-type or IL-4-deficient mice. To further explore the role of Th1/Th2 cytokines in the mercury model, we performed anti-interferon-gamma antibody treatment in IL-4-deficient mice together with mercury treatment and found that the production of IgG2a and IgG3, but not IgG2b, antibodies was downregulated. This indicated that besides Th2-type cytokines, Th1-type and other cytokines were involved as well in mercury-induced autoimmune response. Thus, C57BL/6 mice with H-2b genotype are highly susceptible to mercury-induced autoimmunity, and the genetic susceptibility to mercury involves more than a predisposition of a Th1-or Th2-type response.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Citocinas/inmunología , Mercurio/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Anticuerpos Antinucleares/biosíntesis , Autoanticuerpos/biosíntesis , Regulación hacia Abajo/inmunología , Técnica del Anticuerpo Fluorescente Directa , Inmunoglobulina G/metabolismo , Interferón gamma/inmunología , Interleucina-4/inmunología , Riñón/inmunología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunologíaRESUMEN
Treatment with HgCl2 induces a systemic autoimmune disease in certain mice and rats. The major characteristic of this disease in mice with H-2s genotype is the production of anti-nucleolar autoantibodies (ANoIA). The exact mechanism(s) for the production and the functional role of mercury-induced ANoIA are not known. We have studied the ability of mercury-induced ANoIA to enter the living cells in vivo and in vitro. We found that in highly susceptible mice, treatment with mercury induced ANoIA capable of localizing in the nucleoli of kidney and liver cells in vivo. No detectable nucleoli localization of ANoIA were found in the cells of the heart, stomach, intestine and spleen. Consistent with the in vivo studies, mercury-induced ANoIA were also able to enter and translocate in the nucleoli of certain cells in vitro. The highest degree of antibody penetration was found in A-498 cells (a human kidney cell line) followed by 3T3 cells (a mouse fibroblast cell line), whereas the cells of lymphoid origin exhibited a very low degree of antibody penetration. Penetrated ANoIA could be recovered from the nucleoli of live 3T3 cells previously treated with ANoIA. The in vitro nucleolar translocation by ANoIA did not affect the DNA synthesis, but was found to be an active process dependent on time and temperature. Furthermore, pre-treatment of living cells with trypsin markedly inhibited both cell entry and nucleolar accumulation of ANoIA. Thus, mercury-induced ANoIA have a unique ability to transgress the membrane of certain living cells in vivo and in vitro, and to localize in the nucleoli.
Asunto(s)
Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Cloruro de Mercurio/farmacología , Animales , Línea Celular , Nucléolo Celular/inmunología , Proteínas Cromosómicas no Histona/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Riñón/citología , Riñón/inmunología , Masculino , RatonesRESUMEN
Mercury can induce systemic autoimmunity in susceptible mouse strains characterized by a T-cell-dependent polyclonal B-cell activation, increased serum levels of IgG1 and IgE antibodies, production of autoantibodies, and the formation of immune complexes in the kidneys. However, certain resistant mouse strains do not show any of the autoimmune manifestations after mercury injection. Th1/Th2 dichotomy has been proposed to be responsible for resistance and susceptibility, respectively. Immunosuppression has also been suggested in resistant animals after mercury injection. To test whether immunosuppression or a biased Th1-type response was induced by mercury in resistant DBA/2 mice, we injected DBA/2 mice with mercury for 1 or 3 weeks and then immunized the mice with horse red blood cells (HRBCs) to study whether the subsequent humoral response to HRBCs was inhibited or skewed to the production of antibodies of IgG2a isotype switched by Th1-type cytokines. We found that there was no reduction of the number of splenic antibody-producing cells in the subsequent response to HRBCs compared with saline-treated mice. By haemagglutination tests, the titers of HRBC-specific antibodies were the same after HRBCs injection in both mercury- and saline-treated DBA/2 mice. There was no increase in total serum IgG2a antibody. Sera of both mercury- and saline-treated mice immunized with HRBCs showed high titres of specific IgM, IgG1 and IgG2a anti-HRBCs antibodies. Surprisingly, 3-week treatment with mercury induced a reduction in the titres of specific IgG2a anti-HRBCs antibodies in DBA/2 mice after immunization with HRBCs. Our results demonstrated that mercury did not induce a general immunosuppression or a biased Th 1-type immune response in resistant DBA/2 mice. The nonresponsiveness in mice resistant to mercury-induced autoimmunity must be due to some other unknown mechanism(s).