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The study aimed to monitor the healing process in the canine skin following surgical incision and closure using staples or tissue glue and to compare them with the intradermal suture pattern. Surgically created skin incisions in 10 dogs were apposed with staples, tissue glue (n-butyl cyanoacrylate) and continuous intradermal pattern. The cosmetic appearance of the wounds was blindly evaluated on days 7, 14 and 28 and once a month until the end of the experiment, i.e., one year after the incision. Ultrasonographic and clinical evaluation was performed on days 0-10, 12, 14, 16, 18, 21, 24 and 28, once a week until the end of the 3rd month and once a month until the end of the experiment. Histopathological evaluation was performed on days 7, 14, 28, 180 and 365. The median time required for the performance of each technique differed significantly between techniques; stapling lasted 21 s, glue 2 min 16 s and intradermal 15 min 37 s. Cosmetic appearance with glue was statistically worse than staples and intradermal. The clinical appearance of intradermal was significantly better than glue and staples. No significant differences were found at histological evaluation; however, glue had the worst score throughout the experiment. The overall evaluation of the techniques showed that glue had the worst score compared to intradermal and staples, with the difference being statistically significant in the first postoperative week. Intradermal suture pattern is much better than glue application for skin closure in dogs, whilst is not significantly better than staples. Staples should be preferred when time is an important factor.
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The study aimed to compare incisional wound healing with intradermal suture patterns performed with (a) absorbable suture with burying of the knots and (b) nonabsorbable suture anchored with clips. Ten dogs were included in the study. Surgically created skin incisions were apposed with continuous intradermal suture pattern with 4/0 poliglecaprone 25 with burying of the knots and continuous intradermal pattern with 4/0 polypropylene with clips. Cosmetic, clinical, ultrasonographic and histological scores were evaluated. The intradermal pattern with clips was easier to perform and required significantly less time to complete than the intradermal suture with burying of the knots. Cosmetic, clinical, ultrasonographic and histological evaluation scores did not differ significantly between the techniques. Irrespective of the technique used, the cosmetic, ultrasonographic, clinical and histological appearances of the incisions improved over time. In conclusion, polypropylene was found to be a safe and effective suture material for use with intradermal suture pattern with clips in dogs and to have an easy and quick application. However, in our sample, its earlier removal from wounds than poliglecaprone 25 was not found to be associated with a supposedly beneficial effect on wound healing and scar appearance. Both suture materials can be useful in intradermal suture techniques in dogs.
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Mitochondrial activation and the production of mitochondrial reactive oxygen species (mROS) are crucial for CD4+ T cell responses and have a role in naïve cell signaling after TCR activation. However, little is known about mROS role in TCR-independent signaling and in recall responses. Here, we found that mROS are required for IL-12 plus IL-18-driven production of IFN-γ, an essential cytokine for inflammatory and autoimmune disease development. Compared to TCR stimulation, which induced similar levels of mROS in naïve and memory-like cells, IL-12/IL-18 showed faster and augmented mROS production in memory-like cells. mROS inhibition significantly downregulated IFN-γ and CD44 expression, suggesting a direct mROS effect on memory-like T cell function. The mechanism that promotes IFN-γ production after IL-12/IL-18 challenge depended on the effect of mROS on optimal activation of downstream signaling pathways, leading to STAT4 and NF-κB activation. To relate our findings to IFN-γ-driven lupus-like disease, we used Fas-deficient memory-like CD4+ T cells from lpr mice. Importantly, we found significantly increased IFN-γ and mROS production in lpr compared with parental cells. Treatment of WT cells with FasL significantly reduced mROS production and the activation of signaling events leading to IFN-γ. Moreover, Fas deficiency was associated with increased mitochondrial levels of cytochrome C and caspase-3 compared with WT memory-like cells. mROS inhibition significantly reduced the population of disease-associated lpr CD44hiCD62LloCD4+ T cells and their IFN-γ production. Overall, these findings uncovered a previously unidentified role of Fas/FasL interaction in regulating mROS production by memory-like T cells. This apoptosis-independent Fas activity might contribute to the accumulation of CD44hiCD62LloCD4+ T cells that produce increased IFN-γ levels in lpr mice. Overall, our findings pinpoint mROS as central regulators of TCR-independent signaling, and support mROS pharmacological targeting to control aberrant immune responses in autoimmune-like disease.
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Interleucina-18 , Linfócitos T , Animais , Linfócitos T CD4-Positivos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Camundongos , Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.
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Sepsis is a complex biphasic syndrome characterized by both pro- and anti-inflammatory immune states. Whereas early sepsis mortality is caused by an acute, deleterious pro-inflammatory response, the second sepsis phase is governed by acute immunosuppression, which predisposes patients to long-term risk for life-threatening secondary infections. Despite extensive basic research and clinical trials, there is to date no specific therapy for sepsis, and mortality rates are on the rise. Although IFN-ß is one of the most-studied cytokines, its diverse effects are not fully understood. Depending on the disease or type of infection, it can have beneficial or detrimental effects. As IFN-ß has been used successfully to treat diverse diseases, emphasis has been placed on understanding the role of IFN-ß in sepsis. Analyses of mouse models of septic shock attribute a pro-inflammatory role to IFN-ß in sepsis development. As anti-inflammatory treatments in humans with antibodies to TNF-α or IL1-ß resulted disappointing, cytokine modulation approaches were discouraged and neutralization of IFN-ß has not been pursued for sepsis treatment. In the case of patients with delayed sepsis and immunosuppression, there is a debate as to whether the use of specific cytokines would restore the deactivated immune response. Recent reports show an association of low IFN-ß levels with the hyporesponsive state of monocytes from sepsis patients and after endotoxin tolerance induction. These data, discussed here, project a role for IFN-ß in restoring monocyte function and reversing immunosuppression, and suggest IFN-ß-based additive immunomodulatory therapy. The dichotomy in putative therapeutic approaches, involving reduction or an increase in IFN-ß levels, mirrors the contrasting nature of the early hyperinflammatory state and the delayed immunosuppression phase.
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Fas induces massive apoptosis in T cells after repeated in vitro T cell receptor (TCR) stimulation and is critical for lymphocyte homeostasis in Fas-deficient (lpr) mice. Although the in vitro Fas apoptotic mechanism has been defined, there is a large conceptual gap between this in vitro phenomenon and the pathway that leads to in vivo development of lymphadenopathy and autoimmunity. A striking abnormality in lpr mice is the excessive proliferation of CD4+ and CD8+ T cells, and more so of the double-negative TCR+CD4-CD8-B220+ T cells. The basis of lpr T cell hyperproliferation remains elusive, as it cannot be explained by Fas-deficient apoptosis. T cell-directed p21 overexpression reduces hyperactivation/hyperproliferation of all lpr T cell subtypes and lymphadenopathy in lpr mice. p21 controls expansion of repeatedly stimulated T cells without affecting apoptosis. These results confirm a direct link between hyperactivation/hyperproliferation, autoreactivity, and lymphadenopathy in lpr mice and, with earlier studies, suggest that Fas apoptosis-independent pathways control lpr T cell hyperproliferation. lpr T cell hyperproliferation could be an indirect result of the defective apoptosis of repeatedly stimulated lpr T cells. Nonetheless, in this perspective, we argue for an alternative setting, in which lack of Fas would directly cause lpr T cell hyperactivation/hyperproliferation in vivo. We propose that Fas/Fas ligand (FasL) acts as an activation inhibitor of recurrently stimulated T cells, and that its disruption causes overexpansion of T cells in lpr mice. Research to define the underlying mechanism of this Fas/FasL effect could resolve the phenotype of lpr mice and lead to therapeutics for related human syndromes.
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M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-ß production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-ß expression, and p21 knockdown in human monocytes corroborated its role in IFN-ß regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.
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Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica , Interferon beta/metabolismo , Macrófagos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Sepse/imunologia , Idoso , Animais , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Lipopolissacarídeos , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/metabolismo , Fenótipo , Ligação Proteica , Multimerização Proteica , Sepse/terapia , Fator de Transcrição RelA/metabolismoRESUMO
Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cell responses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4(+) CD8(+) and CD4(-)CD8(-) lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity.
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Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Interferon gama/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Ovalbumina/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Vaccinia virus/imunologiaRESUMO
BACKGROUND: The ultrasonographic appearance of the normal canine haired skin examined using high-frequency ultrasonography has not been described. HYPOSTHESIS/OBJECTIVES: To describe the echogenicity of normal canine haired skin using high-frequency (50 MHz) ultrasonography and to compare ultrasonographic with histological measurements of skin thickness using snap-frozen tissue biopsy samples. ANIMALS: Ten normal healthy beagle dogs. METHODS: Ultrasonographic examination was performed on eight cutaneous sites by use of a 50 MHz polyvinylidene difluoride transducer. The skin echogenicity was evaluated, and the mean of 10 skin thickness measurements was calculated. Ultrasonography results were compared with histological findings of skin cryosections stained with haematoxylin and eosin, as well as with histometric measurements of skin thickness. Differences in the ultrasonographic and histological measurements among biopsy sites, age and sex of the animals were also examined. RESULTS: The skin layers and hair follicles could be identified with high-frequency ultrasound biomicroscopy in all eight examination sites of all 10 dogs. There was a highly significant, positive association between the ultrasonographic and histological measurements (P < 0.001) of skin thickness. For both ultrasonographic and histological skin thickness measurements, there were no statistically significant differences between sex, age or among the different examination sites. CONCLUSIONS AND CLINICAL IMPORTANCE: Cutaneous ultrasound biomicroscopy using a 50 MHz transducer is a useful tool for the following applications: (i) to identify the skin layers (including the epidermis, dermis and subcutaneous fat); (ii) to demonstrate the hair follicles in various areas of the haired skin; and (iii) to measure the thickness of normal canine skin accurately.
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Cabelo/anatomia & histologia , Microscopia Acústica/veterinária , Pele/anatomia & histologia , Animais , Cães , Feminino , MasculinoRESUMO
OBJECTIVE: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. METHODS: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. RESULTS: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. CONCLUSION: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.
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Artrite Experimental/imunologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Materiais Biomiméticos/administração & dosagem , Domínio Catalítico/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/química , Edema/induzido quimicamente , Edema/patologia , Feminino , Membro Posterior , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Peptídeos/administração & dosagem , Cicatrização/imunologiaRESUMO
There is growing evidence about the role of mesenchymal stem cells (MSCs) as cancer stem cells in many sarcomas. Nevertheless, little is still known about the cellular and molecular mechanisms underlying MSCs transformation. We aimed at investigating the role of p53 and p21, two important regulators of the cell cycle progression and apoptosis normally involved in protection against tumorigenesis. Mesenchymal stem cells from wild-type, p21(-/-)p53(+/+), and p21(-/-)p53(+/-) mice were cultured in vitro and analyzed for the appearance of tumoral transformation properties after low, medium, and high number of passages both in vitro and in vivo. Wild-type or p21(-/-)p53(+/+) MSCs did not show any sign of tumoral transformation. Indeed, after short-term in vitro culture, wild-type MSCs became senescent, and p21(-/-)p53(+/+) MSCs showed an elevated spontaneous apoptosis rate. Conversely, MSCs carrying a mutation in one allele of the p53 gene (p21(-/-)p53(+/-) MSCs) completely lost p53 expression after in vitro long-term culture. Loss of p53 was accompanied by a significant increase in the growth rate, gain of karyotypic instability, loss of p16 expression, and lack of senescence response. Finally, these cells were able to form fibrosarcomas partially differentiated into different mesenchymal lineages when injected in immunodeficient mice both after subcutaneous and intrafemoral injection. These findings show that MSCs are very sensitive to mutations in genes involved in cell cycle control and that these deficiencies can be at the origin of some mesodermic tumors.
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Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Células-Tronco Mesenquimais/patologia , Células-Tronco Neoplásicas/patologia , Proteína Supressora de Tumor p53/genética , Animais , Western Blotting , Transformação Celular Neoplásica/metabolismo , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Citometria de Fluxo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Mutantes , Mutação , Células-Tronco Neoplásicas/metabolismoRESUMO
p21 is a cell-cycle inhibitor that is also known to suppress autoimmunity. Here, we provide evidence of a novel role for p21 as an inhibitor of macrophage activation. LPS stimulation of p21-deficient peritoneal macrophages induced increased activation compared with controls, with elevated production of proinflammatory mediators such as TNF-alpha and IL-1beta. The enhanced activity of LPS-stimulated p21-deficient macrophages correlated with increased activity of the transcription factor NF-kappaB. LPS stimulation of p21-deficient macrophages led to increased IkappaBalpha kinase activity, and increased IkappaBalpha phosphorylation and degradation, resulting in elevated NF-kappaB activity. The effect of p21 in macrophage activation was independent of its cell-cycle inhibitory role. p21(-/-) mice showed greater sensitivity to LPS-induced septic shock than did WT mice, indicating that p21 contributes to maintenance of a balanced response to inflammatory stimuli and suggesting biological significance for the role of p21 in macrophage activation. Our findings project a role for p21 in the control of NF-kappaB-associated inflammation, and suggest that therapeutic modulation of p21 expression could be beneficial in inflammation-associated diseases.
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Inibidor de Quinase Dependente de Ciclina p21/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Choque Séptico/imunologia , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Feminino , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , NF-kappa B/metabolismo , Choque Séptico/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A new cytokinin receptor homologue, MsHK1, was isolated from Medicago sativa root nodules. MsHK1 expression was induced in alfalfa seedlings by exogenous application of the cytokinin trans-zeatin. Transcript accumulation was detected in different plant organs. MsHK1 expression was induced by salt stress in alfalfa roots, leaves and nodules, and transcript accumulation in the vascular bundles pointed to a putative role in osmosensing for MsHK1 and/or other close cytokinin receptor homologues. Expression in the meristem and the invasion zone of the nodule suggest a role for cytokinin receptors in cytokinin sensing during nodule cell division and differentiation.
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Medicago sativa/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Sequência de Aminoácidos , Southern Blotting , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hibridização In Situ , Medicago sativa/genética , Medicago sativa/metabolismo , Dados de Sequência Molecular , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Homologia de Sequência de Aminoácidos , Zeatina/farmacologiaRESUMO
Here we report the isolation of a new cytokinin receptor homologue, LaHK1, from lupin (Lupinus albus) root nodules. LaHK1 transcript accumulation was detected in different plant organs, and expression was analyzed throughout nodule development. We observed notably higher expression in nodule primordia and young nodules compared to the root or to mature nodules. We also detected elevated transcript accumulation in naturally senescent nodules and in senescent nodules subjected to foliar dark stress. The results could be an indication of a putative role of this cytokinin receptor homologue in nodule development, from morphogenesis through senescence.
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Citocininas/metabolismo , Lupinus/genética , Proteínas de Plantas/genética , Receptores de Superfície Celular/genética , Nódulos Radiculares de Plantas/genética , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Lupinus/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/fisiologia , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
Development of autoantibodies and lupus-like autoimmunity by 129/Sv x C57BL/6 p21(-/-) mice has established that cell cycle deregulation is one the defective pathways leading to break of tolerance. Memory T cell accumulation is thought to be related to tolerance loss in murine lupus models. We studied T cell memory responses in C57BL/6 p21(-/-) mice that develop lupus-like disease manifestations. p21 did not affect primary proliferation of naive T cells, and was required for cycling control, but not for apoptosis of activated/memory T cells. When we induced apoptosis by secondary TCR challenge, surviving memory T cells depended on p21 for proliferation control. Under conditions of secondary T cell stimulation that did not cause apoptosis, p21 was also needed for regulation of activated/memory T cell expansion. The requirement for p21 in the control of T cell proliferation of activated/memory T cells suggests that in addition to apoptosis, cycling regulation by p21 constitutes a new pathway for T cell homeostasis. Concurring with this view, we found accumulation in p21(-/-) mice of memory CD4(+) T cells that showed increased proliferative potential after TCR stimulation. Furthermore, OVA immunization of p21(-/-) mice generated hyperresponsive OVA-specific T cells. Overall, the data show that p21 controls the proliferation of only activated/memory T cells, and suggest that p21 forms part of the memory T cell homeostasis mechanism, contributing to maintenance of tolerance.
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Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/imunologia , Homeostase/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Autoimunidade/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos KnockoutRESUMO
Class I PI3K catalyzes formation of 3-poly-phosphoinositides. The family is divided into IA isoforms, activated by Tyr kinases and the IB isoform (PI3Kgamma), activated by G protein-coupled receptors. Mutations that affect PI3K are implicated in chronic inflammation, although the differential contribution of each isoform to pathology has not been elucidated. Enhanced activation of class IA-PI3K in T cells extends CD4+ memory cell survival, triggering an invasive lymphoproliferative disorder and systemic lupus. As both IA- and IB-PI3K isoforms regulate T cell activation, and activated pathogenic CD4+ memory cells are involved in triggering systemic lupus, we examined whether deletion of IB could reduce the pathological consequences of increased IA-PI3K activity. IB-PI3Kgamma deficiency did not abolish invasion or lymphoproliferation, but reduced CD4+ memory cell survival, autoantibody production, glomerulonephritis, and systemic lupus. Deletion of the IB-PI3Kgamma isoform thus decreased survival of pathogenic CD4+ memory cells, selectively inhibiting systemic lupus development. These results validate the PI3Kgamma isoform as a target for systemic lupus erythematosus treatment.
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Linfócitos T CD4-Positivos/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Isoenzimas/deficiência , Isoenzimas/imunologia , Nefropatias/etiologia , Nefropatias/imunologia , Nefropatias/patologia , Lúpus Eritematoso Sistêmico/complicações , Camundongos , Camundongos TransgênicosRESUMO
Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.
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Inibidores Enzimáticos/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Nefrite Lúpica/prevenção & controle , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/farmacologia , Tiazolidinedionas/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos MutantesRESUMO
Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes depends on chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non-obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti-CCR5 antibody. This did not influence peri-insulitis advancement, but inhibited beta-cell destruction and diabetes. These data demonstrate a role of CCR5-dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.
Assuntos
Linfócitos B/imunologia , Diabetes Mellitus/imunologia , Receptores CCR5/imunologia , Linfócitos T/imunologia , Animais , Movimento Celular/imunologia , Quimiocinas/genética , Quimiocinas/imunologia , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , RNA/genética , RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca(2+) flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Galpha(i) pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.
Assuntos
Quimiocinas CXC/fisiologia , Proteínas/fisiologia , Receptores CXCR4/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Animais , Cálcio/metabolismo , Quimiocina CXCL12 , Proteínas de Ligação a DNA/fisiologia , Hormônio do Crescimento/farmacologia , Humanos , Janus Quinase 1 , Proteínas Tirosina Quinases/fisiologia , Receptores CXCR4/antagonistas & inibidores , Fator de Transcrição STAT3 , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/fisiologia , Regulação para CimaRESUMO
Anti-DNA autoantibody production is a key factor in lupus erythematosus development; nonetheless, the link between glomerular anti-DNA autoantibody deposition and glomerulonephritis development is not understood. To study the inflammatory and destructive processes in kidney, we used IFN-gamma(+/-) MRL/lpr mice which produce high anti-DNA Ab levels but are protected from kidney disease. The results showed that defective macrophage recruitment to IFN-gamma(+/-) mouse kidney was not caused by decreased levels of monocyte chemoattractant protein-1, a chemokine that controls macrophage migration to MRL/lpr mouse kidney. To determine which IFN-gamma-producing cell type orchestrates the inflammation pathway in kidney, we transferred IFN-gamma(+/+) monocyte/macrophages or T cells to IFN-gamma(-/-) mice, which do not develop anti-DNA autoantibodies. The data demonstrate that IFN-gamma production by infiltrating macrophages, and not by T cells, is responsible for adhesion molecule up-regulation, macrophage accumulation, and inflammation in kidney, even in the absence of autoantibody deposits. Therefore, in addition to monocyte chemoattractant protein-1, macrophage-produced IFN-gamma controls macrophage migration to kidney; the degree of IFN-gamma production by macrophages also regulates glomerulonephritis development. Our findings establish the level of IFN-gamma secretion by macrophages as a link between anti-DNA autoantibody deposition and glomerulonephritis development, outline the pathway of the inflammatory process, and suggest potential treatment for disease even after autoantibody development.