RESUMO
Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.
Assuntos
Leucócitos Mononucleares , Linfócitos T , Humanos , Camundongos , Animais , Estudo de Associação Genômica Ampla , ImunoglobulinasRESUMO
BACKGROUND: CD40 is a compelling target for cancer immunotherapy, however, attempts to successfully target this pathway have consistently been hampered by dose-limiting toxicity issues in the clinic that prevents the administration of efficacious doses. METHODS: Here, using cytokine and cytokine receptor depletion strategies in conjunction with a potent CD40 agonist, we investigated mechanisms underlying the two primary sources of CD40 agonist-associated toxicity, hepatotoxicity and cytokine release syndrome (CRS). RESULTS: We demonstrate that CD40 agonist -induced hepatotoxicity and CRS are mechanistically independent. Historical data have supported a role for interleukin-6 (IL-6) in CRS-associated wasting, however, our findings instead show that an inflammatory cytokine network involving TNF, IL-12p40, and IFNγ underlie this process. Deficiency of TNF or IFNγ did not influence CD40-induced hepatitis however loss of IL-12p40 significantly decreased circulating concentrations of liver enzymes and reduced the frequency of activated CD14+MHCII+ myeloid cells in the liver, indicating a role for IL-12p40 in liver pathology. CONCLUSIONS: As clinical research programs aim to circumnavigate toxicity concerns while maintaining antitumor efficacy it will be essential to understand which features of CD40 biology mediate antitumor function to develop both safe and efficacious agonists.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Imunoterapia/métodos , Subunidade p40 da Interleucina-12/efeitos adversos , Animais , Feminino , Humanos , CamundongosRESUMO
Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Ensaios Clínicos como Assunto , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Intervalo Livre de Doença , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Cultura Primária de Células , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genéticaRESUMO
The extracellular matrix (ECM) is a highly dynamic structure that is present in all tissues and continuously undergoes controlled remodelling. This process involves quantitative and qualitative changes in the ECM, mediated by specific enzymes that are responsible for ECM degradation, such as metalloproteinases. The ECM interacts with cells to regulate diverse functions, including proliferation, migration and differentiation. ECM remodelling is crucial for regulating the morphogenesis of the intestine and lungs, as well as of the mammary and submandibular glands. Dysregulation of ECM composition, structure, stiffness and abundance contributes to several pathological conditions, such as fibrosis and invasive cancer. A better understanding of how the ECM regulates organ structure and function and of how ECM remodelling affects disease progression will contribute to the development of new therapeutics.
Assuntos
Matriz Extracelular/química , Matriz Extracelular/metabolismo , Morfogênese , Neoplasias/patologia , Animais , Endopeptidases/metabolismo , Células Epiteliais/química , Células Epiteliais/metabolismo , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Fibrose/patologia , HumanosRESUMO
Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.
Assuntos
Adenoma/patologia , Sistemas Computacionais , Neoplasias Intestinais/patologia , Microscopia Confocal/métodos , Células Mieloides/patologia , Animais , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Structural changes to the airways are features of severe asthma. The bronchial epithelium facilitates this remodeling process. Learning about the changes that develop in the airway epithelium could improve our understanding of asthma pathogenesis and lead to new therapeutic approaches. OBJECTIVE: We sought to determine the feasibility and relevance of air-liquid interface cultures of bronchial epithelium derived from endobronchial biopsy specimens of patients with different severities of asthma for studying the airway epithelium. METHODS: Human bronchial epithelial cells derived from endobronchial biopsy specimens of patients with mild and severe asthma were maintained in culture for 21 days in an air-liquid interface to reproduce a fully differentiated airway epithelium. Initially, features of remodeling that included epithelial and subepithelial layers, as well as mucus production, were assessed in paraffin-embedded endobronchial biopsy specimens to evaluate morphologic characteristics of asthmatic patients' epithelia. Ex vivo differentiated epithelia were then analyzed for morphology and function based on ultrastructural analysis, IL-8 release, lipoxin A(4) generation, mucin production, and lipoxygenase gene expression. RESULTS: Morphologic and inflammatory imbalances initially observed in endobronchial biopsy specimens obtained from patients with severe or mild asthma persisted in the air-liquid interface reconstituted epithelium throughout the differentiation process to 21 days. Epithelium from patients with severe asthma produced greater levels of mucin, released more IL-8, and produced lower levels of lipoxin A(4) than that from patients with mild asthma. Expression of 15-lipoxygenase 2 was increased in epithelium from patients with severe asthma, whereas expression levels of MUC5AC, MUC5B, 5-lipoxygenase, and 15-lipoxygeanse 1 were similar to those of patients with mild asthma. CONCLUSION: Ex vivo cultures of fully differentiated bronchial epithelium from endobronchial biopsy specimens maintain inherent phenotypic differences specifically related to the severity of asthma.
Assuntos
Remodelação das Vias Aéreas , Asma/patologia , Brônquios/patologia , Mucosa Respiratória/patologia , Adulto , Idoso , Asma/imunologia , Asma/fisiopatologia , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Progressão da Doença , Estudos de Viabilidade , Feminino , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Lipoxinas/genética , Lipoxinas/metabolismo , Lipoxigenase/genética , Lipoxigenase/metabolismo , Masculino , Pessoa de Meia-Idade , Mucinas/metabolismo , Mucosa Respiratória/imunologia , Adulto JovemRESUMO
ß-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/ß-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active ß-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.
Assuntos
Arrestinas/metabolismo , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Via de Sinalização Wnt , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células , Separação Celular , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Intestinais/genética , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição 4 , Ensaio Tumoral de Célula-Tronco , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
BACKGROUND: Clinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane x Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan. RESULTS: PXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies. CONCLUSION: Our results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions.
Assuntos
Camptotecina/análogos & derivados , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores de Esteroides/genética , Camptotecina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Irinotecano , Receptor de Pregnano X , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , TransfecçãoRESUMO
Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Animais , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proliferação de Células , Claudinas , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclina D1/genética , Ciclina D1/metabolismo , Imunofluorescência , Células HT29 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Carga Tumoral , Regulação para CimaRESUMO
The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant progastrin sequentially activated the transcription of Wnt and Notch target genes in progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of progastrin targeting for the clinical management of CRC.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Colorretais/metabolismo , Gastrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Gastrinas/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/biossíntese , Precursores de Proteínas/deficiência , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Notch/biossíntese , Receptores Notch/genética , Proteínas Serrate-Jagged , Transdução de Sinais , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima , beta Catenina/biossíntese , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor-gamma (PPARgamma) ligands that are widely used in type II diabetes treatment. In addition to their ability to improve glucose homeostasis, TZDs possess anti-inflammatory properties and inhibit growth of many cells, particularly cancerous airway epithelial cells. However, the functional effects of PPARgamma ligands on nonmalignant human bronchial epithelial cells have never been investigated. In the present study, we questioned whether PPARgamma ligands may regulate proliferation of human bronchial epithelial cells, and we studied their potential molecular mechanisms. We found that synthetic PPARgamma agonists, rosiglitazone (RGZ) and troglitazone (TGZ), induced proliferation of human bronchial epithelial cells, whereas the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), inhibited cell growth. RGZ and TGZ (10 microM) induced a rapid and transient intracellular Ca(2+) mobilization from thapsigargin-sensitive intracellular stores, whereas 15d-PGJ(2) (5 microM) did not induce any Ca(2+) signal. The PPARgamma antagonist GW-9662 did not inhibit any biological responses, but it reversed the effect of 15d-PGJ(2) on cell growth. Using RT-PCR, we detected mRNA expression of the GPR40 receptor, a G protein-coupled receptor recently identified as a receptor for free fatty acids and TZDs, in human bronchial epithelial cells. Downregulation of GPR40 by small-interfering RNA led to a significant inhibition of TZD-induced Ca(2+) mobilization and proliferation. This study provides evidence for the proliferative effect of anti-diabetic drug TZDs in nonmalignant human bronchial epithelial cells through GPR40 receptor activation, involving an intracellular Ca(2+) signaling pathway.
Assuntos
Cromanos/farmacologia , Hipoglicemiantes/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Adenocarcinoma , Anilidas/farmacologia , Brônquios/citologia , Sinalização do Cálcio/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/genética , Mucosa Respiratória/metabolismo , Rosiglitazona , TroglitazonaRESUMO
In this study, we investigated the synthesis of lipoxins (LXs) and their anti-inflammatory effects in different human airway epithelial cell culture models. After cell incubation with exogenous 5(S),6(R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, LXA(4) was detected in supernatants of differentiated human bronchial epithelial cells by contrast to non-differentiated cells. Exogenous LXA(4) significantly inhibited tumor necrosis factor-alpha (TNF-alpha)-induced interleukin-8 (IL-8) release in the different epithelial cell types and the potency of inhibition was dependent of the accessibility of the specific LXA(4) receptor, formyl-peptide receptor like-1 (FPRL-1) expressed by all these cells. Immunohistochemistry analysis on human bronchial biopsies showed a high expression of FPRL-1 in the epithelium. Finally, an FPRL-1 receptor antagonist, boc-2 peptide reversed LXA(4) effect on IL-8 generation. Together, these findings indicate that differentiated human bronchial epithelium synthesizes LX in vivo which could have autocrine actions through its specific receptor FPRL-1 to promote resolution of airway inflammation.
Assuntos
Células Epiteliais/metabolismo , Lipoxinas/biossíntese , Anti-Inflamatórios não Esteroides/farmacologia , Western Blotting , Brônquios/citologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Células Epiteliais/efeitos dos fármacos , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Imunoquímica , Interleucina-8/biossíntese , Lipoxinas/farmacologia , Receptores de Formil Peptídeo/biossíntese , Receptores de Lipoxinas/biossíntese , Mucosa Respiratória/citologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cysteinyl leukotrienes and the T helper (Th)-2 cytokines IL-5 and IL-13 directly modulate human airway smooth muscle functions such as contraction and proliferation. We studied the effects of other lipid mediators involved in asthma pathophysiology such as prostaglandin D(2) (PGD(2)), lipoxin, and isoprostanes, and the cytokines, IL-5, IL-4, and IL-13 on human airway smooth muscle cell migration. Chemotaxis and chemokinesis of cultured airway smooth muscle cells from humans without asthma (second to fifth passages, n = 6) were studied using collagen-I-coated polycarbonate membranes in Transwell culture plates. Receptor expression and kinase activation were studied by flow cytometry, polymerase chain reaction, and Western blotting techniques. In contrast to LTE(4)- stimulated (10(-6) M) chemokinesis and LTE(4)-primed migration toward platelet-derived growth factor (PDGF), isoprostane 15-F(2t)-IsoP, and IL-5 were neither chemotactic nor chemokinetic. PGD(2) (10(-10)-10(-6) M) was a chemoattractant and primed migration toward PDGF through the DP(2)/CRTh(2) receptor. Although airway smooth muscle cells did not express the lipoxin A(4) cognate receptor, LTE(4)-primed migration toward PDGF was blocked by lipoxin A(4) (10(-6) M), suggesting that this is mediated through CysLT(1)R antagonism. IL-13 (10 ng/ml), but not IL-4 (0.1-100 ng/ml), augmented migration toward PDGF. This was associated with increased Src-kinase phosphorylation and up-regulation of PDGF-alpha and -beta receptors, and was attenuated by IL-13Ralpha- and IL-4Ralpha-neutralizing antibodies, an Src-kinase antagonist (PP1, 3 muM), a CysLT(1)R antagonist, montelukast (10(-6) M), and by lipoxin A(4) (10(-6) M). PGD(2) and IL-13 promote human airway smooth muscle migration. IL-13 can promote airway smooth muscle migration through Src-kinase and leukotriene-dependent pathways. This may contribute to the accumulation of smooth muscle cells in remodeled airway submucosa.
Assuntos
Movimento Celular/fisiologia , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Pulmão/anatomia & histologia , Músculo Liso/fisiologia , Prostaglandina D2/metabolismo , Células Th2/metabolismo , Asma/fisiopatologia , Células Cultivadas , Ativação Enzimática , Humanos , Interleucina-4/metabolismo , Isoprostanos/metabolismo , Lipoxinas/metabolismo , Músculo Liso/citologia , Transdução de Sinais/fisiologia , Células Th2/citologiaRESUMO
Aspiration of gastric acid commonly injures airway epithelium and, if severe, can lead to respiratory failure from acute respiratory distress syndrome. Recently, we identified cyclooxygenase-2 (COX-2)-derived prostaglandin E(2) (PGE(2)) and lipoxin A(4) (LXA(4)) as pivotal mediators in vivo for resolution of acid-initiated acute lung injury. To examine protective mechanisms for these mediators in the airway, we developed an in vitro model of acid injury by transiently exposing well-differentiated normal human bronchial epithelial cells to hydrochloric acid. Transmission electron microscopy revealed selective injury to superficial epithelial cells with disruption of cell attachments and cell shedding. The morphological features of injury were substantially resolved within 6 hours. Acid triggered and early marked increases in COX-2 expression and PGE(2) production, and acid-induced PGE(2) significantly increased epithelial LXA(4) receptor (ALX) expression. LXA(4) is generated in vivo during acute lung injury, and we observed that nanomolar quantities increased basal epithelial cell proliferation and potently blocked acid-triggered interleukin-6 release and neutrophil transmigration across well-differentiated normal human bronchial epithelial cells. Expression of recombinant human ALX in A549 airway epithelial cells uncovered ALX-dependent inhibition of cytokine release by LXA(4). Together, these findings indicate that injured bronchial epithelial cells up-regulate ALX in a COX-2-dependent manner to promote LXA(4)-mediated resolution of airway inflammation.
Assuntos
Brônquios/metabolismo , Células Epiteliais/fisiologia , Ácido Gástrico/fisiologia , Lipoxinas/fisiologia , Mucosa Respiratória/metabolismo , Brônquios/ultraestrutura , Adesão Celular , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Humanos , Ácido Clorídrico/toxicidade , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Microscopia Eletrônica de Transmissão , Nitrobenzenos/farmacologia , Receptores de Formil Peptídeo/biossíntese , Receptores de Lipoxinas/biossíntese , Mucosa Respiratória/ultraestrutura , Sulfonamidas/farmacologiaRESUMO
RATIONALE AND OBJECTIVES: Severe asthma is characterized by increased airway inflammation that persists despite therapy with corticosteroids. It is not, however, merely an exaggeration of the eosinophilic inflammation that characterizes mild to moderate asthma; rather, severe asthma presents unique features. Although arachidonic acid metabolism is well appreciated to regulate airway inflammation and reactivity, alterations in the biosynthetic capacity for both pro- and antiinflammatory eicosanoids in severe asthma have not been determined. METHODS: Patients with severe asthma were identified according to National Heart, Lung, and Blood Institute Severe Asthma Research Program criteria. Samples of whole blood from individuals with severe or moderate asthma were assayed for biosynthesis of lipoxygenase-derived eicosanoids. MEASUREMENTS AND MAIN RESULTS: The counterregulatory mediator lipoxin A4 was detectable in low picogram amounts, using a novel fluorescence-based detection system. In activated whole blood, mean lipoxin A4 levels were decreased in severe compared with moderate asthma (0.4 [SD 0.4] ng/ml vs. 1.8 [SD 0.8] ng/ml, p=0.001). In sharp contrast, mean levels of prophlogistic cysteinyl leukotrienes were increased in samples from severe compared with moderate asthma (112.5 [SD 53.7] pg/ml vs. 64.4 [SD 24.8] pg/ml, p=0.03). Basal circulating levels of lipoxin A4 were also decreased in severe relative to moderate asthma. The marked imbalance in lipoxygenase-derived eicosanoid biosynthesis correlated with the degree of airflow obstruction. CONCLUSIONS: Mechanisms underlying airway responses in severe asthma include underproduction of lipoxins. This is the first report of a defect in lipoxin biosynthesis in severe asthma, and suggests an alternative therapeutic strategy that emphasizes natural counterregulatory pathways in the airways.
Assuntos
Asma/metabolismo , Lipoxinas/biossíntese , Adulto , Asma/fisiopatologia , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Proteínas de Membrana/metabolismo , Receptores de Leucotrienos/metabolismo , Escarro/químicaRESUMO
Acute lung injury (ALI) is a severe illness with excess mortality and no specific therapy. In its early exudative phase, neutrophil activation and accumulation in the lung lead to hypoxemia, widespread tissue damage, and respiratory failure. In clinical trials, inhibition of proinflammatory mediators has not proven effective. In this study, we pursued a new investigative strategy that emphasizes mediators promoting resolution from lung injury. A new spontaneously resolving experimental murine model of ALI from acid aspiration was developed to identify endogenous proresolving mechanisms. ALI increased cyclooxygenase 2 (COX-2) expression in murine lung. Selective pharmacologic inhibition or gene disruption of COX-2 blocked resolution of ALI. COX-2-derived products increased levels of the proresolving lipid mediators lipoxin A4 (LXA4) and, in the presence of aspirin, 15-epi-LXA4. Both LXA4 and 15-epi-LXA4 interact with the LXA4 receptor (ALX) to mediate anti-inflammatory actions. ALX expression was markedly induced by acid injury and transgenic mice with increased ALX expression displayed dramatic protection from ALI. Together, these findings indicate a protective role in ALI for COX-2-derived mediators, in part via enhanced lipoxin signaling, and carry potential therapeutic implications for this devastating clinical disorder.
Assuntos
Lesão Pulmonar , Pulmão/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , DNA Complementar/genética , Modelos Animais de Doenças , Expressão Gênica , Humanos , Ácido Clorídrico/toxicidade , Mediadores da Inflamação/metabolismo , Lipoxinas/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Prostaglandina-Endoperóxido Sintases/genética , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/genética , Receptores de Lipoxinas/metabolismo , Transdução de SinaisRESUMO
Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.