RESUMO
Cytotoxicity and cellular uptake of spherical barium sulphate microparticles (diameter 1 µm) were studied with three different cell lines, i.e. THP-1 cells (monocytes; model for a phagocytosing cell line), HeLa cells (epithelial cells; model for a non-phagocytosing cell line), and human mesenchymal stem cells (hMSCs; model for non-phagocytosing primary cells). Barium sulphate is a chemically and biologically inert solid which allows to distinguish two different processes, e.g. the particle uptake and potential adverse biological reactions. Barium sulphate microparticles were surface-coated by carboxymethylcellulose (CMC) which gave the particles a negative charge. Fluorescence was added by conjugating 6-aminofluorescein to CMC. The cytotoxicity of these microparticles was studied by the MTT test and a live/dead assay. The uptake was visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The particle uptake mechanism was quantified by flow cytometry with different endocytosis inhibitors in THP-1 and HeLa cells. The microparticles were easily taken up by all cell types, mostly by phagocytosis and micropinocytosis, within a few hours. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is of primary importance in nanomedicine, drug delivery, and nanotoxicology. It is commonly assumed that cells take up only nanoparticles unless they are able to phagocytosis. Here, we demonstrate with chemically and biologically inert microparticles of barium sulphate that even non-phagocytosing cells like HeLa and hMSCs take up microparticles to a considerable degree. This has considerable implication in biomaterials science, e.g. in case of abrasive debris and particulate degradation products from implants like endoprostheses.
Assuntos
Sulfato de Bário , Fagocitose , Humanos , Células HeLa , Sulfato de Bário/farmacologia , Sulfato de Bário/metabolismo , Endocitose , Macrófagos/metabolismo , Tamanho da PartículaRESUMO
Most recently, we have described the G-protein coupled receptor 83 (GPR83), which is highly expressed by CD4(+)CD25(+) regulatory T cells (Tregs) to be involved in the induction of CD4(+)Foxp3(+) Tregs in the course of an ongoing immune response. Four GPR83 isoforms have been described. Here, we have shown that GPR83 isoform-4, which differs from GPR83 isoform-1 by 20 additional aminoacids in the second cytoplasmatic loop, is predominantly expressed by Tregs. Interestingly, GPR83 isoform-4 but not GPR83 isoform-1 retrovirally transduced T cells were able to interfere with inflammatory responses in vivo. Re-analysis of GPR83 transduced T cells revealed that this in vivo acquisition of suppressive activity was associated with the induction of Treg-associated molecules including Foxp3 in GPR83 isoform-4 but not GPR83 isoform-1 transduced CD4(+) T cells under inflammatory conditions. Our results suggest that the 20 additional aminoacids within GPR83 isoform-4 are involved in Treg induction during inflammatory immune responses.
Assuntos
Isoformas de Proteínas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Linfócitos T Reguladores/imunologia , Humanos , Fenótipo , Isoformas de Proteínas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
BACKGROUND: Regulatory T cells (T(regs)) have potential anti-inflammatory effects and are likely to be important in the pathogenesis of chronic inflammatory bowel disease (IBD). However, the induction and expansion of T(regs) at sites of mucosal inflammation are not yet fully understood and may involve antigen presentation by local dendritic cells (DCs) and/or intestinal epithelial cells (IECs). METHODS: To determine the unique ways in which the gut induces or expands T(regs), a transgenic mouse model that is based on the specific expression of a model autoantigen (influenza haemagglutinin (HA)) in the intestinal epithelium (VILLIN-HA) was used. Gut-associated DCs and IECs isolated from these mice were phenotypically and functionally characterised for the potential to interact with HA-specific T(regs) in vitro and in vivo. RESULTS: Intestinal self-antigen expression leads to peripheral expansion of antigen-specific CD4(+)Foxp3(+) T(regs). Although gut-associated DCs can induce antigen-specific CD4(+)Foxp3(+) T cell proliferation, in vivo depletion of DCs did not preclude proliferation of these cells. Interestingly, antigen presentation by primary IECs is sufficient to expand antigen-specific CD4(+)Foxp3(+) T(regs) efficiently. This is dependent on major histocompatibility complex class II, but, in contrast to DCs, is unlikely to require transforming growth factor beta and retinoic acid. CONCLUSION: This study provides experimental evidence for a new concept in mucosal immunity: in contrast to current thinking, expansion of T(regs) can be achieved independently of local DCs through antigen-specific IEC-T cell interactions.
Assuntos
Autoantígenos/imunologia , Células Epiteliais/imunologia , Fatores de Transcrição Forkhead/imunologia , Mucosa Intestinal/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva/métodos , Animais , Apresentação de Antígeno , Proliferação de Células , Células Cultivadas , Células Dendríticas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Modelos Animais , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.
Assuntos
Citomegalovirus/patogenicidade , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Muromonab-CD3/farmacologia , Mitógenos de Phytolacca americana/farmacologia , Sepse/tratamento farmacológico , Sepse/virologia , Adulto , Idoso , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40â¯nm, 420â¯nm, and 1⯵m) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release.
Assuntos
Sulfato de Bário/metabolismo , Células da Medula Óssea/citologia , Endocitose , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Fagocitose , Animais , Células da Medula Óssea/metabolismo , Fluorescência , Camundongos , Nanopartículas/ultraestrutura , Espectrometria por Raios XRESUMO
A universal influenza vaccine must provide protection against antigenically divergent influenza viruses either through broadly neutralizing antibodies or cross-reactive T cells. Here, intranasal immunizations with recombinant adenoviral vectors (rAd) encoding hemagglutinin (HA) and nucleoprotein (NP) in combination with rAd-Interleukin-(IL)-1ß or rAd-IL-18 were evaluated for their efficacy in BALB/c mice. Mucosal delivery of rAd-IL-1ß enhanced HA-specific antibody responses including strain-specific neutralizing antibodies. Nevertheless, the beneficial effects on the local T cell responses were much more impressive reflected by increased numbers of CD103+CD69+ tissue-resident memory T cells (TRM). This increased immunogenicity translated into superior protection against infections with homologous and heterologous strains including H1N1, pH1N1, H3N2, and H7N7. Inhibition of the egress of circulating T cells out of the lymph nodes during the heterologous infection had no impact on the degree of protection underscoring the unique potential of TRM for the local containment of mucosal infections. The local co-expression of IL-1ß and antigen lead to the activation of critical checkpoints in the formation of TRM including activation of epithelial cells, expression of chemokines and adhesion molecules, recruitment of lung-derived CD103+ DCs, and finally local TRM imprinting. Given the importance of TRM-mediated protection at mucosal barriers, this study has major implications for vaccine development.
Assuntos
Células Dendríticas/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Interleucina-1beta/metabolismo , Infecções por Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Adenoviridae/genética , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células Cultivadas , Feminino , Vetores Genéticos , Humanos , Imunidade Heteróloga , Memória Imunológica , Interleucina-18/genética , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de ÓrgãosRESUMO
Inflammatory bowel disease (IBD) is characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the etiology is poorly understood, it is widely accepted that loss of tolerance is involved in the development of IBD. Therefore, re-establishing tolerance or gut homeostasis is one of the key features in the development of new therapeutic strategies. Here we show that antigen targeting to DEC-205 on dendritic cells leads to an interleukin (IL)-10-dependent downregulation of C-X-C chemokine receptor 3 (CXCR3) expression on differentiated antigen-specific T helper type 1 (Th1) cells in vivo. This downregulation interferes with the migration of Th1 cells into the gut and protects mice against severe acute and relapsing intestinal inflammation. Moreover, CD4(+)CXCR3(+) T cells are highly enriched in the inflamed mucosa of IBD patients. Interference with this pathway may therefore be a promising approach for the treatment of IBD. In conclusion, we propose a hitherto undescribed mechanism by which IL-10 can act on effector T cells and orchestrate intestinal immune responses.
Assuntos
Antígenos CD/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Interleucina-10/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores CXCR3/imunologia , Receptores de Superfície Celular/imunologia , Células Th1/imunologia , Animais , Antígenos CD/genética , Antígenos CD4/genética , Antígenos CD4/imunologia , Movimento Celular , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Interleucina-10/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Receptores CXCR3/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Células Th1/patologiaRESUMO
Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic-polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro.
Assuntos
Adjuvantes Imunológicos/farmacologia , Fosfatos de Cálcio/farmacologia , Células Dendríticas/imunologia , Nanopartículas , Oligonucleotídeos/farmacologia , Receptores Toll-Like/agonistas , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Adjuvantes Imunológicos/química , Animais , Fosfatos de Cálcio/química , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Indutores de Interferon/química , Indutores de Interferon/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos/química , Poli I-C/química , Poli I-C/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
CD8(+) regulatory T cells appear impaired in number and/or function in some autoimmune diseases. However, the role of CD8(+) regulatory T cells in the pathogenesis of skin inflammation and psoriasis remains unknown. In this study, we set out to analyze the capability of CD8(+) regulatory T cells to inhibit skin inflammation in a murine model and to determine the frequency of CD8(+) regulatory T cells in patients with psoriasis. We demonstrate that murine fully competent CD8(+) regulatory T cells can be induced by stimulating naïve CD8(+) T cells in the presence of TGF-ß and retinoic acid (RA). Importantly, in vitro induced CD8(+) regulatory T cells significantly suppressed skin inflammation in vivo. Furthermore, we found that the frequency of regulatory CD8(+)CD25(+)Foxp3(+) T cells is decreased in peripheral blood but increased in lesional psoriatic skin of patients with psoriasis. Thus, our study suggests a previously unappreciated role of CD8(+)CD25(+)Foxp3(+) T cells in skin disorders, and induction of these cells in vitro may be an effective immunotherapy for skin inflammation.
RESUMO
BACKGROUND: Several studies have suggested that chronic inflammatory bowel disease may be a consequence of antigen specific recognition by appropriate T cells which expand and induce immunopathology. AIMS: We wished to investigate whether autoreactive CD4+ T cells can initiate the disease on recognition of enterocyte specific antigens directly and if induction of mucosal tolerance occurs. METHODS: Transgenic mice (VILLIN-HA) were generated that showed specific expression of haemagglutinin from influenza virus A exclusively in enterocytes of the intestinal epithelium. To investigate the impact of enterocyte specific haemagglutinin expression in an autoimmune environment, we mated VILLIN-HA mice with T cell receptor (TCR)-HA mice expressing an alpha/beta-TCR, which recognises an MHC class II restricted epitope of haemagglutinin, and analysed the HA specific T cells for induction of autoimmunity or tolerance. RESULTS: In VILLIN-HAxTCR-HA mice, incomplete central deletion of HA specific lymphocytes occurred. Peripheral HA specific lymphocytes showed an activated phenotype and increased infiltration into the intestinal mucosa, but not into other organs of double transgenic mice. Enterocyte specific lamina propria lymphocytes showed a dose dependent proliferative response on antigen stimulation whereas the proliferative capacity of intraepithelial lymphocytes was reduced. Mucosal lymphocytes from VILLIN-HAxTCR-HA mice secreted lower amounts of interferon gamma and interleukin (IL)-2 but higher levels of tumour necrosis factor alpha, monocyte chemoattractant protein 1, and IL-6. Mucosal immune reactions were accompanied by broad changes in the gene expression profile with expression of proinflammatory genes, but strikingly also a remarkable set of genes discussed in the context of peripheral induction of regulatory T cells, including IL-10, Nrp-1, and Foxp3. CONCLUSIONS: Enterocyte specific antigen expression is sufficient to trigger a specific CD4+ T cell response leading to mucosal infiltration. In our model, progression to overt clinical disease was counteracted most likely by induction of regulatory T cells.