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1.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298979

RESUMO

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.


Assuntos
Plaquetas/fisiologia , Antígenos CD18/fisiologia , Degranulação Celular , Córnea/irrigação sanguínea , Eritrócitos/fisiologia , Hiperemia/fisiopatologia , Mastócitos/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Vasculite/imunologia , Vênulas/metabolismo , Animais , Antígenos CD18/deficiência , Movimento Celular , Quimiotaxia de Leucócito , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Epitélio Corneano/fisiologia , Feminino , Hiperemia/sangue , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Microscopia Eletrônica , Modelos Animais , Fagocitose , Regeneração/fisiologia , Vasculite/sangue , Vênulas/patologia , Cicatrização/fisiologia
2.
BMC Immunol ; 22(1): 2, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407124

RESUMO

BACKGROUND: Dendritic cells (DCs) play a key role in shaping T cell responses. To do this, DCs must be able to migrate to the site of the infection and the lymph nodes to prime T cells and initiate the appropriate immune response. Integrins such as ß2 integrin play a key role in leukocyte adhesion, migration, and cell activation. However, the role of ß2 integrin in DC migration and function in the context of infection-induced inflammation in the gut is not well understood. This study looked at the role of ß2 integrin in DC migration and function during infection with the nematode worm Trichuris muris. Itgb2tm1Bay mice lacking functional ß2 integrin and WT littermate controls were infected with T. muris and the response to infection and kinetics of the DC response was assessed. RESULTS: In infection, the lack of functional ß2 integrin significantly reduced DC migration to the site of infection but not the lymph nodes. The lack of functional ß2 integrin did not negatively impact T cell activation in response to T. muris infection. CONCLUSIONS: This data suggests that ß2 integrins are important in DC recruitment to the infection site potentially impacting the initiation of innate immunity but is dispensible for DC migration to lymph nodes and T cell priming in the context of T. muris infection.


Assuntos
Antígenos CD18/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Animais , Antígenos CD18/deficiência , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Tricuríase/imunologia , Trichuris
3.
PLoS One ; 14(12): e0226126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815949

RESUMO

Salmonella can appear in the bloodstream within CD18 expressing phagocytes following oral ingestion in as little as 15 minutes. Here, we provide evidence that the process underlying this phenomenon is reverse transmigration. Reverse transmigration is a normal host process in which dendritic cells can reenter the bloodstream by traversing endothelium in the basal to apical direction. We have developed an in vitro reverse transmigration assay in which dendritic cells are given the opportunity to cross endothelial monolayers in the basal to apical direction grown on membranes with small pores, modeling how such cells can penetrate the bloodstream. We demonstrate that exposing dendritic cells to microbial components negatively regulates reverse transmigration. We propose that microbial components normally cause the host to toggle between positively and negatively regulating reverse transmigration, balancing the need to resolve inflammation with inhibiting the spread of microbes. We show that Salmonella in part overcomes this negative regulation of reverse transmigration with the Salmonella pathogenicity island-2 encoded type III secretion system, which increases reverse transmigration by over an order of magnitude. The SPI-2 type III secretion system does this in part, but not entirely by injecting the type III effector SpvC into infected cells. We further demonstrate that SpvC greatly promotes early extra-intestinal dissemination in mice. This result combined with the previous observation that the spv operon is conserved amongst strains of non-typhoidal Salmonella capable of causing bacteremia in humans suggests that this pathway to the bloodstream could be important for understanding human infections.


Assuntos
Carbono-Oxigênio Liases/metabolismo , Salmonella/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antígenos CD18/deficiência , Antígenos CD18/genética , Carbono-Oxigênio Liases/genética , Células Dendríticas/microbiologia , Células Dendríticas/fisiologia , Endotélio/citologia , Endotélio/microbiologia , Feminino , Intestinos/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Fagócitos/metabolismo , Fagócitos/microbiologia , Salmonella/patogenicidade
4.
Cell Rep ; 26(6): 1614-1626.e5, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30726742

RESUMO

ß2-integrins promote neutrophil recruitment to infected tissues and are crucial for host defense. Neutrophil recruitment is defective in leukocyte adhesion deficiency type-1 (LAD1), a condition caused by mutations in the CD18 (ß2-integrin) gene. Using a model of Citrobacter rodentium (CR)-induced colitis, we show that CD18-/- mice display increased intestinal damage and systemic bacterial burden, compared to littermate controls, ultimately succumbing to infection. This phenotype is not attributed to defective neutrophil recruitment, as it is shared by CXCR2-/- mice that survive CR infection. CR-infected CD18-/- mice feature prominent upregulation of IL-17 and downregulation of IL-22. Exogenous IL-22 administration, but not endogenous IL-17 neutralization, protects CD18-/- mice from lethal colitis. ß2-integrin expression on macrophages is mechanistically linked to Rac1/ROS-mediated induction of noncanonical-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome-dependent IL-1ß production, which promotes ILC3-derived IL-22. Therefore, ß2-integrins are required for protective IL-1ß-dependent IL-22 responses in colitis, and the identified mechanism may underlie the association of human LAD1 with colitis.


Assuntos
Antígenos CD18/genética , Citrobacter rodentium/patogenicidade , Colite/genética , Infecções por Enterobacteriaceae/genética , Interleucinas/genética , Macrófagos/imunologia , Animais , Antígenos CD18/deficiência , Antígenos CD18/imunologia , Citrobacter rodentium/imunologia , Colite/imunologia , Colite/microbiologia , Colite/mortalidade , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Feminino , Regulação da Expressão Gênica/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucinas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia , Interleucina 22
5.
FASEB J ; 32(1): 143-154, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28904019

RESUMO

Cathepsin B (CtsB) contributes to atherosclerosis and cancer progression by processing the extracellular matrix and promoting angiogenesis. Although CtsB was reported to promote and reduce angiogenesis, there is no mechanistic explanation that reconciles this apparent discrepancy. CtsB cleaves CD18 from the surface of immune cells, but its contribution to angiogenesis has not been studied. We developed an in vivo technique for visualization of immune cell transmigration from corneal vessels toward implanted cytokines. Wild-type (WT) leukocytes extravasated from limbal vessels, angiogenic stalks, and growing tip vessels and migrated toward the cytokines, indicating immune competence of angiogenic vessels. Compared to WT leukocytes, CtsB-/- leukocytes accumulated in a higher number in angiogenic vessels, but extravasated less toward the implanted cytokine. The accumulated CtsB-/- leukocytes in angiogenic vessels expressed more CD18. CD18-/- leukocytes extravasated later than WT leukocytes. However, once extravasated, CD18-/- leukocytes transmigrated more rapidly than their WT counterparts. These results suggest that, although CD18 facilitates efficient extravasation, outside of the vessel CD18 interaction with the extracellular matrix, it reduced transmigration velocity. Our results reveal an unexpected role for CtsB in leukocyte extravasation and transmigration, which advances our understanding of the complex contribution of CtsB to angiogenesis.-Nakao, S., Zandi, S., Sun, D., Hafezi-Moghadam, A. Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.


Assuntos
Antígenos CD18/metabolismo , Catepsina B/metabolismo , Leucócitos/patologia , Leucócitos/fisiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Animais , Antígenos CD18/deficiência , Antígenos CD18/genética , Catepsina B/deficiência , Catepsina B/genética , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/etiologia
6.
Vet Clin Pathol ; 46(3): 391-400, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28750142

RESUMO

BACKGROUND: Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice. OBJECTIVES: The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections. METHODS: Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin ß2 subunit (ITGB2) gene. RESULTS: The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4. CONCLUSIONS: Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway.


Assuntos
Antígenos CD18/genética , Doenças do Gato/genética , Deleção de Genes , Síndrome da Aderência Leucocítica Deficitária/veterinária , Animais , Antígenos CD18/deficiência , Gatos , Adesão Celular , Citometria de Fluxo/veterinária , Síndrome da Aderência Leucocítica Deficitária/genética , Leucocitose/genética , Leucocitose/veterinária , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
7.
Thromb Haemost ; 117(2): 325-338, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-27853810

RESUMO

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, αMß2) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that a genetic deficiency and a therapeutic modulation of Mac-1 regulate adipose tissue inflammation in a mouse model of diet-induced obesity (DIO). C57Bl6/J mice genetically deficient (Mac-1-/-) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1-/- mice presented with increased diet-induced weight gain, decreased insulin sensitivity in skeletal muscle and in the liver in insulin-clamps, insulin secretion deficiency and elevated glucose levels in fasting animals, and dyslipidaemia. Unexpectedly, accumulation of adipose tissue macrophages (ATMs) was unaffected, while gene expression indicated less inflamed adipose tissue and macrophages in Mac-1-/- mice. In contrast, inflammatory gene expression at distant locations, such as in skeletal muscle, was not changed. Treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype with increased expression of IL-6 and MCP-1, whereas accumulation of ATMs did not change. Finally, inhibition of Mac-1's adhesive interaction to CD40L by the peptide inhibitor cM7 did not affect myeloid cell accumulation in adipose tissue. We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, Mac-1 modulates inflammatory gene expression in macrophages. These findings question the net effect of integrin blockade in cardio-metabolic disease.


Assuntos
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Quimiotaxia , Dieta/efeitos adversos , Inflamação/metabolismo , Gordura Intra-Abdominal/metabolismo , Leucócitos/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/deficiência , Antígeno CD11b/genética , Antígenos CD18/deficiência , Antígenos CD18/genética , Adesão Celular , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Genótipo , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Inflamação/genética , Inflamação/patologia , Resistência à Insulina , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Antígeno de Macrófago 1/genética , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Aumento de Peso
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