RESUMO
The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif-/- and K14-Cre+/tg; Miffl/fl) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.
Assuntos
Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Pele/citologia , Pele/imunologia , Animais , Antracenos/toxicidade , Antígenos CD/genética , Antígenos CD/metabolismo , Carcinogênese , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Queratinócitos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Piperidinas/toxicidade , Piridinas/toxicidade , Receptores CXCR/genética , Receptores CXCR/metabolismoRESUMO
BACKGROUND: One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. OBJECTIVES: The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. METHODS: Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. RESULTS: Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. CONCLUSION: We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines.
Assuntos
Tecido Adiposo/metabolismo , Tecido Adiposo/cirurgia , Queratinócitos/metabolismo , Lipectomia , Adipócitos/metabolismo , Adolescente , Adulto , Idoso , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemAssuntos
Procedimentos Cirúrgicos Cardíacos , Movimento Celular/fisiologia , Células Progenitoras Endoteliais/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Monitorização Intraoperatória/métodos , Procedimentos Cirúrgicos Cardíacos/tendências , Humanos , Monitorização Intraoperatória/tendênciasRESUMO
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a structurally unique inflammatory cytokine that controls cellular signaling in human physiology and disease through extra- and intracellular processes. Macrophage migration inhibitory factor has been shown to mediate both disease-exacerbating and beneficial effects, but the underlying mechanism(s) controlling these diverse functions are poorly understood. METHODS AND RESULTS: Here, we have identified an S-nitros(yl)ation modification of MIF that regulates the protective functional phenotype of MIF in myocardial reperfusion injury. Macrophage migration inhibitory factor contains 3 cysteine (Cys) residues; using recombinant wtMIF and site-specific MIF mutants, we have identified that Cys-81 is modified by S-nitros(yl)ation whereas the CXXC-derived Cys residues of MIF remained unaffected. The selective S-nitrosothiol formation at Cys-81 led to a doubling of the oxidoreductase activity of MIF. Importantly, S-nitrosothiol-MIF formation was measured both in vitro and in vivo and led to a decrease in cardiomyocyte apoptosis in the reperfused heart. This decrease was paralleled by a S-nitrosothiol-MIF- but not Cys81 serine (Ser)-MIF mutant-dependent reduction of infarct size in an in vivo model of myocardial ischemia/reperfusion injury. CONCLUSIONS: S-nitros(yl)ation of MIF is a pivotal novel regulatory mechanism, providing enhanced activity resulting in increased cytoprotection in myocardial reperfusion injury.
Assuntos
Fatores Inibidores da Migração de Macrófagos/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Cisteína/metabolismo , Citoproteção , Humanos , Camundongos , Traumatismo por Reperfusão Miocárdica/etiologia , Óxido Nítrico/metabolismoRESUMO
BACKGROUND: Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study. METHODS: This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers. RESULTS: In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies. CONCLUSION: We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/citologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Células-Tronco Mesenquimais/citologia , Retalhos Cirúrgicos/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/sangue , Quimiocina CXCL12/farmacologia , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Feminino , Humanos , Técnicas In Vitro , Fatores Inibidores da Migração de Macrófagos/farmacologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Estudos Retrospectivos , Cicatrização/fisiologia , Adulto JovemRESUMO
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.
Assuntos
Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Quimiotaxia/efeitos dos fármacos , Glucocorticoides , Humanos , Concentração Inibidora 50 , Fatores Inibidores da Migração de Macrófagos/genética , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.