RESUMO
Human monocytes and macrophages are two major myeloid cell subsets with similar and distinct functions in tissue homeostasis and immune responses. GM-CSF plays a fundamental role in myeloid cell differentiation and activation. Hence, we compared the effects of GM-CSF on the expression of several immune mediators by human monocytes and monocyte-derived macrophages obtained from healthy donors. We report that GM-CSF similarly elevated the expression of CD80 and ICAM-1 and reduced HLA-DR levels on both myeloid cell subsets. However, GM-CSF increased the percentage of macrophages expressing surface IL-15 but reduced the proportion of monocytes carrying surface IL-15. Moreover, GM-CSF significantly increased the secretion of IL-4, IL-6, TNF, CXCL10, and IL-27 by macrophages while reducing the secretion of IL-4 and CXCL10 by monocytes. We show that GM-CSF triggered ERK1/2, STAT3, STAT5, and SAPK/JNK pathways in both myeloid subsets. Using a pharmacological inhibitor (U0126) preventing ERK phosphorylation, we demonstrated that this pathway was involved in both the GM-CSF-induced increase and decrease of the percentage of IL-15+ macrophages and monocytes, respectively. Moreover, ERK1/2 contributed to GM-CSF-triggered secretion of IL-4, IL-6, TNF, IL-27 and CXCL10 by macrophages. However, the ERK1/2 pathway exhibited different roles in monocytes and macrophages for the GM-CSF-mediated impact on surface makers (CD80, HLA-DR, and ICAM-1). Our data demonstrate that GM-CSF stimulation induces differential responses by human monocytes and monocyte-derived macrophages and that some but not all of these effects are ERK-dependent.
Assuntos
Interleucina-27 , Monócitos , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Sistema de Sinalização das MAP Quinases , Interleucina-15 , Molécula 1 de Adesão Intercelular , Interleucina-27/farmacologia , Interleucina-4 , Interleucina-6 , Macrófagos , Antígenos HLA-DRRESUMO
BACKGROUND: T lymphocytes exhibit numerous alterations in relapsing-remitting (RRMS), secondary progressive (SPMS), and primary progressive multiple sclerosis (PPMS). The NKG2D pathway has been involved in MS pathology. NKG2D is a co-activating receptor on subsets of CD4+ and most CD8+ T lymphocytes. The ligands of NKG2D are expressed at low levels in normal tissues but are elevated in MS postmortem brain tissues compared with controls. Whether the NKG2D pathway shows specific changes in different forms of MS remains unclear. METHODS: We performed unsupervised and supervised flow cytometry analysis to characterize peripheral blood T lymphocytes from RRMS, SPMS, and PPMS patients and healthy controls (HC). We used an in vitro microscopy approach to assess the role of NKG2D in the interactions between human CD8+T lymphocytes and human astrocytes. RESULTS: Specific CD8+, CD4+, and CD4-CD8- T cell populations exhibited altered frequency in MS patients' subgroups. The proportion of NKG2D+ T lymphocytes declined with age in PPMS patients but not in RRMS and HC. This reduced percentage of NKG2D+ cells was due to lower abundance of γδ and αß CD4-CD8- T lymphocytes in PPMS patients. NKG2D+ T lymphocytes were significantly less abundant in RRMS than in HC; this was caused by a decreased frequency of CD4-CD8- and CD8+ T lymphocytes and was not linked to age. Blocking NKG2D increased the motility of CD8+ T lymphocytes co-cultured with astrocytes expressing NKG2D ligand. Moreover, preventing NKG2D from interacting with its ligands increased the proportion of CD8+ T lymphocytes exhibiting a kinapse-like behavior characterized by short-term interaction while reducing those displaying a long-lasting synapse-like behavior. These results support that NKG2D participates in the establishment of long-term interactions between activated CD8+ T lymphocytes and astrocytes. CONCLUSION: Our data demonstrate specific alterations in NKG2D+ T lymphocytes in MS patients' subgroups and suggest that NKG2D contributes to the interactions between human CD8+ T lymphocytes and human astrocytes.
Assuntos
Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Linfócitos T CD8-PositivosRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in numerous chronic inflammatory diseases, including multiple sclerosis (MS). GM-CSF impacts multiple properties and functions of myeloid cells via species-specific mechanisms. Therefore, we assessed the effect of GM-CSF on different human myeloid cell populations found in MS lesions: monocyte-derived macrophages (MDMs) and microglia. We previously reported a greater number of interleukin (IL)-15+ myeloid cells in the brain of patients with MS than in controls. Therefore, we investigated whether GM-CSF exerts its deleterious effects in MS by increasing IL-15 expression on myeloid cells. We found that GM-CSF increased the proportion of IL-15+ cells and/or IL-15 levels on nonpolarized, M1-polarized and M2-polarized MDMs from healthy donors and patients with MS. GM-CSF also increased IL-15 levels on human adult microglia. When cocultured with GM-CSF-stimulated MDMs, activated autologous CD8+ T lymphocytes secreted and expressed significantly higher levels of effector molecules (e.g. interferon-γ and GM-CSF) compared with cocultures with unstimulated MDMs. However, neutralizing IL-15 did not attenuate enhanced effector molecule expression on CD8+ T lymphocytes triggered by GM-CSF-stimulated MDMs. We showed that GM-CSF stimulation of MDMs increased their expression of CD80 and ICAM-1 and their secretion of IL-6, IL-27 and tumor necrosis factor. These molecules could participate in boosting the effector properties of CD8+ T lymphocytes independently of IL-15. By contrast, GM-CSF did not alter CD80, IL-27, tumor necrosis factor and chemokine (C-X-C motif) ligand 10 expression/secretion by human microglia. Therefore, our results underline the distinct impact of GM-CSF on human myeloid cells abundantly present in MS lesions.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Interleucina-27 , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-15 , Macrófagos/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Disease-causing AQP2 mutations induce nephrogenic diabetes insipidus (NDI), a condition that challenges the bodily water balance by producing large urinary volumes. In this study, we characterize three new AQP2 mutations identified in our lab from NDI patients (A120D, A130V, T179N) along the previously reported A47V variant. Using Xenopus oocytes, we compared the key functional and biochemical features of these mutations against classical recessive (R187C) and dominant (R254Q) forms, and once again found clear functional recovery features (increased protein stability and function) for all mutations under study. This behaviour, attributed to heteromerization to wt-AQP2, challenge the classical model to NDI which often depicts recessive mutations as ill-structured proteins unable to oligomerize. Consequently, we propose a revised model to the cell pathophysiology of AQP2-related NDI which accounts for the functional recovery of recessive AQP2 mutations.
Assuntos
Aquaporina 2/genética , Diabetes Insípido Nefrogênico/genética , Adulto , Animais , Aquaporina 2/metabolismo , Western Blotting , Humanos , Lactente , Masculino , Mutação/genética , Oócitos , Linhagem , Xenopus laevisRESUMO
Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Mutations in the protein induce nephrogenic diabetes insipidus (NDI), which challenges the water balance by producing large urinary volumes. Although recessive AQP2 mutations are believed to generate non-functional and monomeric proteins, the literature identifies several mild mutations which suggest the existence of mixed wt/mut tetramers likely to carry function in heterozygotes. Using Xenopus oocytes, we tested this hypothesis and found that mild mutants (V24A, D150E) can associate with wt-AQP2 in mixed heteromers, providing clear functional gain in the process (62 ± 17% and 63 ± 17% increases, respectively), conversely to the strong monomeric R187C mutant which fails to associate with wt-AQP2. In kidney cells, both V24A and D150E display restored targeting while R187C remains in intracellular stores. Using a collection of mutations to expand recovery analyses, we demonstrate that inter-unit contacts are central to this recovery process. These results not only present the ground data for the functional recovery of recessive AQP2 mutants through heteromerization, which prompt to revisit the accepted NDI model, but more importantly describe a general recovery process that could impact on all multimeric systems where recessive mutations are found.
Assuntos
Aquaporina 2/química , Aquaporina 2/genética , Mutação , Multimerização Proteica , Animais , Aquaporina 2/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Diabetes Insípido Nefrogênico/genética , Feminino , Genes Recessivos , Heterozigoto , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Camundongos , Oócitos/metabolismo , Água/metabolismo , Xenopus laevisRESUMO
This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.