RESUMO
It was reported that TNF receptor type II signaling, which has the capacity to stimulate CD4+ forkhead box P3+ (Foxp3+) regulatory T cells (Tregs), activated the noncanonical NF-κB pathway in an IKKα-dependent manner. Therefore, we studied the role of IKKα in the homeostasis of Treg population. To this end, we generated a mouse strain with conditional knockout of IKKα in CD4 cells (Ikkα(f/f):CD4.Cre) that showed a >60% reduction in the number of Tregs in the thymus and peripheral lymphoid tissues, whereas the number of Foxp3- effector T cells (Teffs) remained at a normal level. The function of Tregs deficient in IKKα was examined using Rag1(-/-) mice cotransferred with naive CD4 cells (nCD4s). Although wild-type (WT) Tregs inhibited colitis induced by transfer of WT nCD4s, IKKα-deficient Tregs failed to do so, which was associated with their inability to reconstitute Rag1(-/-) mice. Furthermore, nCD4s deficient in IKKα also failed to reconstitute Rag1(-/-) mice and were defective in proliferative responses in vitro and in vivo. Thus, our study reveals a novel role of IKKα in the maintenance of a normal Treg population and in the control of expansion of CD4 T cells. These properties of IKKα may be exploited as therapeutic strategies in the treatment of major human diseases.
Assuntos
Linfócitos T CD4-Positivos/citologia , Quinase I-kappa B/fisiologia , Linfócitos T Reguladores/citologia , Animais , Proliferação de Células , Colite/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/genética , Homeostase , Quinase I-kappa B/metabolismo , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timo/metabolismoRESUMO
Several lines of evidence indicate the instability of CD4(+)Foxp3(+) regulatory T cells (Tregs). We have therefore investigated means of promoting the stability of Tregs. In this study, we found that the proportion of Tregs in mouse strains deficient in TNFR2 or its ligands was reduced in the thymus and peripheral lymphoid tissues, suggesting a potential role of TNFR2 in promoting the sustained expression of Foxp3. We observed that upon in vitro activation with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab, Foxp3 expression by highly purified mouse Tregs was markedly downregulated. Importantly, TNF partially abrogated this effect of TCR stimulation and stabilized Foxp3 expression. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Furthermore, TNF was not able to maintain Foxp3 expression by TNFR2-deficient Tregs. In a mouse colitis model induced by transfer of naive CD4 cells into Rag1(-/-) mice, the disease could be inhibited by cotransfer of wild-type Tregs, but not by cotransfer of TNFR2-deficient Tregs. Furthermore, in the lamina propria of the colitis model, most wild-type Tregs maintained Foxp3 expression. In contrast, an increased number of TNFR2-deficient Tregs lost Foxp3 expression. Thus, our data clearly show that TNFR2 is critical for the phenotypic and functional stability of Tregs in the inflammatory environment. This effect of TNF should be taken into account when designing future therapy of autoimmunity and graft-versus-host disease by using TNF inhibitors.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Ativação Linfocitária/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Animais Congênicos , Linfócitos T CD4-Positivos/transplante , Colite/microbiologia , Colite/patologia , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/imunologia , Proteínas de Homeodomínio/genética , Mucosa Intestinal/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Linfopenia/etiologia , Linfopenia/imunologia , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/transplante , Timo/imunologia , Timo/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. In this article, we present a novel role for two potent alarmins, human ß-defensin 2 and 3 (HBD2 and 3), in promoting IFN-α production by human plasmacytoid dendritic cells. We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA-induced IFN-α production in a TLR9-dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal titration calorimetry studies to elucidate the nature of HBD3/CpG complexes demonstrate involvement of enthalpy-driven interactions, in addition to hydrophobic interactions, with the formation of complexes at a molar ratio of 2:1 defensin/CpG. The i.v. administration of HBD3/CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α, and IL-10 in serum, associated with an increased recruitment of APCs in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses to OVA, compared with OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus, the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells, resulting in an enhanced immune response.
Assuntos
Células Dendríticas/metabolismo , Inflamação/imunologia , Interferon-alfa/biossíntese , beta-Defensinas/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Transporte Biológico , Células Cultivadas , Ilhas de CpG , DNA/metabolismo , Células Dendríticas/imunologia , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Interferon-alfa/sangue , Interferon gama/sangue , Interleucina-12/sangue , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1ß and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1ß remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor l-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1ß in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.
Assuntos
Núcleo Celular/metabolismo , Canais de Cloreto/metabolismo , Macrófagos/metabolismo , Proteínas Mitocondriais/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Canais de Cloreto/genética , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Interferon gama/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Alarmins are endogenous, constitutively available, damage-associated molecular patterns that upon release can mobilize and activate various leukocytes for the induction of innate and adaptive immune responses. For our immune system to function appropriately, it relies on navigating various leukocytes to distinct places at the right time. The direction of cell migration is determined by chemotactic factors that include classical chemoattractants, chemokines, certain growth factors, and alarmins. This viewpoint provides an overview of alarmin-induced cell migration. Alarmins are capable of inducing the migration of diverse types of leukocytes and nonleukocytes either directly by triggering specific receptors or indirectly by inducing production of chemokines through the activation of various leukocytes via pattern recognition receptors. The receptors used by alarmins to directly induce cell migration can either be Gαi protein-coupled receptors or receptors such as the receptor for advanced glycation end products; however, the intracellular signaling events responsible for the direct chemotactic activities of alarmins are, to date, only partially elucidated. Given that alarmins act in concert with chemokines to regulate the recruitment and trafficking of leukocytes, these damage-associated molecular patterns are potentially involved in diverse biological processes as discussed in this viewpoint.
Assuntos
Fatores Quimiotáticos/imunologia , Leucócitos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Imunidade Adaptativa , Animais , Comunicação Celular/imunologia , Movimento Celular/imunologia , Humanos , Imunidade Inata , Imunomodulação , Transdução de Sinais/imunologiaRESUMO
CD4(+) T cells stimulate immune responses through distinct patterns of cytokine produced by Th1, Th2 or Th17 cells, or inhibit immune responses through Foxp3-expressing regulatory T cells (Tregs). Paradoxically, effector T cells were recently shown to activate Tregs, however, it remains unclear which Th subset is responsible for this effect. In this study, we found that Th17 cells expressed the highest levels of TNF among in vitro generated Th subsets, and most potently promoted expansion and stabilized Foxp3 expression by Tregs when co-transferred into Rag1(-/-) mice. Both TNF and IL-2 produced by Th17 cells contributed to this effect. The stimulatory effect of Th17 cells on Tregs was largely abolished when co-transferred with TNFR2-deficient Tregs. Furthermore, Tregs deficient in TNFR2 also supported a much lower production of IL-17A and TNF expression by co-transferred Th17 cells. Thus, our data indicate that the TNF-TNFR2 pathway plays a crucial role in the reciprocal stimulatory effect of Th17 cells and Tregs. This bidirectional interaction should be taken into account when designing therapy targeting Th17 cells, Tregs, TNF and TNFR2.
Assuntos
Ativação Linfocitária/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologia , Transferência Adotiva , Animais , Antígenos CD4/imunologia , Citocinas/imunologia , Fatores de Transcrição Forkhead/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Significant efforts have been devoted to the development of nanoparticular delivering systems targeting tumors. However, clinical application of nanoparticles is hampered by insufficient size homogeneity, difficulties in reproducible synthesis and manufacturing, frequent high uptake in the liver, systemic toxicity of the carriers (particularly for inorganic nanoparticles), and insufficient selectivity for tumor cells. We have found that properly modified synthetic analogs of transmembrane domains of membrane proteins can self-assemble into remarkably uniform spherical nanoparticles with innate biological activity. Self-assembly is driven by a structural transition of the peptide that adopts predominantly a beta-hairpin conformation in aqueous solutions, but folds into an alpha-helix upon spontaneous fusion of the nanoparticles with cell membrane. A 24-amino acid peptide corresponding to the second transmembrane helix of the CXCR4 forms self-assembled particles that inhibit CXCR4 function in vitro and hamper CXCR4-dependent tumor metastasis in vivo. Furthermore, such nanoparticles can encapsulate hydrophobic drugs, thus providing a delivery system with the potential for dual biological activity.
Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Relação Dose-Resposta a Droga , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/prevenção & controle , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Nanopartículas/ultraestrutura , Tamanho da Partícula , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/química , Receptores CXCR4/metabolismoRESUMO
We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1ß, and PGE(2), which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.
Assuntos
Neurônios/efeitos dos fármacos , Dor/fisiopatologia , Transdução de Sinais/fisiologia , Receptores Toll-Like/metabolismo , Aminoquinolinas/farmacologia , Anilidas/farmacologia , Animais , Ácidos Araquidônicos/farmacologia , Western Blotting , Cálcio/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Cinamatos/farmacologia , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Endocanabinoides , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Imidazóis/farmacologia , Camundongos , Microscopia Confocal , Neurônios/metabolismo , Poli I-C/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/agonistasRESUMO
Our previous study showed that TNFR2 is preferentially expressed by CD4(+)FoxP3(+) regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4(+)FoxP3(-) conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3(-) CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs, which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Fatores de Transcrição Forkhead , Imunidade Inata , Ativação Linfocitária/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/prevenção & controle , Linhagem Celular Tumoral , Células Cultivadas , Anergia Clonal/genética , Anergia Clonal/imunologia , Técnicas de Cocultura , Feminino , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/deficiência , Técnicas de Introdução de Genes , Imunidade Inata/genética , Ativação Linfocitária/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores Tipo II do Fator de Necrose Tumoral/genética , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3(+) cells present in CD25(high), CD25(low) and even CD25(-) subsets of CD4(+) cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4(+)CD25(+) Treg included all of the FOXP3(+) cells present in the CD4(+)CD25(high) subset as well as a substantial proportion of the FOXP3(+) cells present in the CD4(+)CD25(low) subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4(+)CD25(+)TNFR2(+) subset than in the CD4(+)CD25(high) subset. In addition, similar levels of FOXP3(+) cells were identified in both the CD4(+)CD25(+)TNFR2(+) and CD4(+)CD25(+)CD127(low/-) subsets. Furthermore, the CD4(+)CD25(+)TNFR2(+) subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4(+)CD25(+)TNFR2(+) cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4(+)CD25(+)TNFR2(-) and CD4(+)CD25(-)TNFR2(+) T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.
Assuntos
Fatores de Transcrição Forkhead/análise , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/classificação , Adulto , Apresentação de Antígeno , Antígenos CD/análise , Antígeno CTLA-4 , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-7/análise , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/análise , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
IL-24 (melanoma differentiation associated gene 7 product) is a member of the IL-10 cytokine family that has been reported to possess anti-tumor activity. IL-24 is produced by immune tissues and its expression can be induced in human peripheral blood mononuclear cells by pathogen-associated molecules. While immune cells are known to produce IL-24, the response of immune cells to IL-24 is unclear. Using recombinant human IL-24, we demonstrated that IL-24 induces human monocyte and neutrophil migration, in vitro. An in vivo chemotaxis model showed that IL-24 attracted CD11b positive myeloid cells. To further characterize the chemotactic IL-24 response and type(s) of receptor(s) utilized by IL-24, we treated monocytes with signaling pathway inhibitors. IL-24-induced migration was reduced by pertussis toxin treatment, thus implicating G-protein coupled receptors in this process. Additionally, MEK and JAK inhibitors markedly decreased monocyte migration toward IL-24. These results suggest that IL-24 activates several signaling cascades in immune cells eliciting migration of myeloid cells, which may contribute to the known anti-cancer effects of IL-24.
Assuntos
Movimento Celular/efeitos dos fármacos , Interleucinas/farmacologia , Células Mieloides/efeitos dos fármacos , Animais , Antígeno CD11b/biossíntese , Células Cultivadas , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Toxina Pertussis/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Benzamidas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Tiazóis/uso terapêutico , Dedos de Zinco , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Primers do DNA , DNA de Neoplasias/metabolismo , Humanos , Ativação TranscricionalRESUMO
Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression-for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and Trem2 expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that TREM1 expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.
RESUMO
Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5-1 microg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G-induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein-coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 microg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.
Assuntos
Proteínas Sanguíneas/farmacologia , Proteínas de Transporte , Catepsinas/farmacologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito , Monócitos/fisiologia , Neutrófilos/fisiologia , Animais , Anticorpos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/isolamento & purificação , Cálcio/metabolismo , Catepsina G , Catepsinas/imunologia , Catepsinas/isolamento & purificação , Fatores Quimiotáticos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Grânulos Citoplasmáticos/química , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Humanos , Isoflurofato/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/química , Toxina Pertussis , Serina Endopeptidases , Linfócitos T/fisiologia , Trombina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.
Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Aspartato-tRNA Ligase , Autoantígenos/fisiologia , Células Dendríticas/fisiologia , Histidina-tRNA Ligase/fisiologia , Miosite/imunologia , Aminoacil-RNA de Transferência , Receptores de Quimiocinas/fisiologia , Linfócitos T/fisiologia , Movimento Celular , Humanos , Interleucina-2/farmacologia , Receptores CCR2 , Receptores CCR3 , Receptores CCR5/fisiologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
An intact chemotactic response is vital for leukocyte trafficking and host defense. Opiates are known to exert a number of immunomodulating effects in vitro and in vivo, and we sought to determine whether they were capable of inhibiting chemokine-induced directional migration of human leukocytes, and if so, to ascertain the mechanism involved. The endogenous opioid met-enkephalin induced monocyte chemotaxis in a pertussis toxin-sensitive manner. Met-enkephalin, as well as morphine, inhibited IL-8-induced chemotaxis of human neutrophils and macrophage inflammatory protein (MIP)-1alpha, regulated upon activation, normal T expressed and secreted (RANTES), and monocyte chemoattractant protein 1, but not MIP-1beta-induced chemotaxis of human monocytes. This inhibition of chemotaxis was mediated by delta and micro but not kappa G protein-coupled opiate receptors. Calcium flux induced by chemokines was unaffected by met-enkephalin pretreatment. Unlike other opiate-induced changes in leukocyte function, the inhibition of chemotaxis was not mediated by nitric oxide. Opiates induced phosphorylation of the chemokine receptors CXCR1 and CXCR2, but neither induced internalization of chemokine receptors nor perturbed chemokine binding. Thus, inhibition of chemokine-induced chemotaxis by opiates is due to heterologous desensitization through phosphorylation of chemokine receptors. This may contribute to the defects in host defense seen with opiate abuse and has important implications for immunomodulation induced by several endogenous neuropeptides which act through G protein-coupled receptors.
Assuntos
Quimiotaxia/fisiologia , Monócitos/citologia , Monócitos/fisiologia , Neutrófilos/citologia , Neutrófilos/fisiologia , Receptores de Quimiocinas/fisiologia , Receptores Opioides delta/fisiologia , Receptores Opioides mu/fisiologia , Transdução de Sinais/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Humanos , Entorpecentes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.
Assuntos
Fatores Quimiotáticos/farmacologia , Fatores Quimiotáticos/fisiologia , Infecções/fisiopatologia , Inflamação/fisiopatologia , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologia , Animais , Linhagem Celular , Quimiotaxia de Leucócito/fisiologia , Infecções por HTLV-I/fisiopatologia , Humanos , Técnicas In Vitro , Camundongos , Monócitos/fisiologia , Neutrófilos/fisiologia , Oxirredução , Linfócitos T/fisiologiaRESUMO
BACKGROUND: The G-protein-coupled formylpeptide receptor (FPR) that mediates chemotaxis of phagocytic leucocytes induced by bacterial and host-derived chemotactic peptides is selectively expressed by highly malignant human gliomas and contributes to tumour growth and angiogenesis. As invasion of surrounding normal tissues is one of the important features of tumour malignancy, we investigated the function of FPR in the invasive behaviour of human glioblastoma cells. METHODS: Cells (FPR(+) and FPR(-)) were isolated by single-cell cloning from a human glioblastoma cell line U-87MG. The FPR expression was assayed by flow cytometry and reverse transcription PCR. The function of FPR was investigated by chemotaxis and calcium flux induced by FPR agonist fMLF. Tumour cell motility was assayed by a wound-healing model in vitro. The growth and invasive phenotype were observed with subcutaneous implantation of tumour cells in nude mice. Over-expression of FPR in FPR(-) cells was performed by transfection of a plasmid vector-containing human FPR gene. RESULTS: One of the glioma clones F9 that expressed high level of FPR showed a more 'motile' phenotype in vitro as compared with a clone G3 without FPR expression. Although F9 and G3 clones both formed subcutaneous tumours in nude mice, only F9 tumours invaded surrounding mouse connective tissues. Over-expression of FPR in G3 clone (G3F) increased the cell motility in vitro and the capacity of the cells to form more rapidly growing and invasive tumours in nude mice. We further found that, in addition to supernatant of necrotic tumour cells, foetal calf serum and human serum used in culture media contained FPR agonist activity and increased the motility of FPR-expressing glioblastoma cells. CONCLUSION: The expression of FPR is responsible for increased motility of human glioblastoma cells and their formation of highly invasive tumours.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Receptores de Formil Peptídeo/fisiologia , Animais , Neoplasias Encefálicas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Glioblastoma/genética , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Fenótipo , Receptores de Formil Peptídeo/agonistas , Receptores de Formil Peptídeo/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Transplante HeterólogoRESUMO
Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Quimiotaxia de Leucócito , Inflamação/imunologia , Queratinócitos/imunologia , Proteínas S100/metabolismo , Animais , Proteínas de Ligação ao Cálcio/imunologia , Linhagem Celular , Humanos , Inflamação/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Subpopulações de Linfócitos , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/imunologiaRESUMO
The G protein-coupled formylpeptide receptor (FPR), known to mediate phagocytic leucocyte chemotaxis in response to bacterial- and host-derived agonists, was expressed by tumor cells in specimens of surgically removed more highly malignant human gliomas. In human glioblastoma cell lines, FPR activation increased cell motility, tumorigenicity and production of angiogenic factors. In studies of the mechanistic basis for the selective expression of FPR in more highly malignant gliomas, we found that the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), while promoting the differentiation of human glioblastoma cells, downregulated FPR expression. Aza also reduced the global methylation levels in glioblastoma cells and activated the pathway of p53 tumor suppressor. Methylation-specific polymerase chain reaction revealed that Aza treatment of tumor cells reduced the methylation of p53 promoter, which was accompanied by increased expression of p53 gene and protein. In addition, overexpression of p53 in glioblastoma cells mimicked the effect of Aza treatment as shown by increased cell differentiation but reduction in FPR expression, the capacity of tumor sphere formation in soft agar and tumorigenesis in nude mice. Furthermore, Aza treatment or overexpression of the wild-type p53 in glioblastoma cells increased the binding of p53 to FPR promoter region shown by chromatin immunoprecipitation. These results indicate that increased methylation of p53 gene retains human glioblastoma cells at a more poorly differentiated phase associated with the aberrant expression of FPR as a tumor-promoting cell surface receptor.