RESUMO
Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.
Assuntos
Anticorpos Antivirais/análise , Infecções por Caliciviridae/prevenção & controle , Proteínas de Escherichia coli , Gastroenterite/prevenção & controle , Vírus Norwalk/imunologia , Vacinação , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Administração Oral , Animais , Toxinas Bacterianas/administração & dosagem , Relação Dose-Resposta Imunológica , Enterotoxinas/administração & dosagem , Escherichia coli , Fezes/química , Fezes/virologia , Feminino , Hemocianinas/imunologia , Imunoglobulina A/análise , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vírus Norwalk/química , Ovalbumina/imunologia , Vagina/imunologia , Vagina/virologia , Vírion/imunologiaRESUMO
In order to evaluate the functions of lymphocyte function antigen-1 (LFA-1) (CD11a/CD18) and Mac-1 (CD11b/CD18) on neonatal neutrophils, we examined neutrophil adhesion to and migration through human umbilical vein endothelial cell (HUVEC) monolayers in vitro. Transendothelial migration of adult neutrophils was greatly enhanced by preincubation of HUVEC with interleukin-1 (IL-1). This migration was significantly inhibited by monoclonal antibodies (MoAbs) against LFA-1 (CD11a) and Mac-1 (CD11b) subunits. Migration of neonatal neutrophils was markedly diminished compared to adult neutrophils, and MoAbs against LFA-1 further reduced migration. In contrast, anti-Mac-1 MoAb was not inhibitory. Adhesion of adult neutrophils was significantly enhanced by prestimulation of HUVEC with IL-1, and was significantly inhibited by MoAbs against LFA-1. Adhesion of neonatal neutrophils was near adult levels and comparably inhibited by anti-LFA-1 MoAb. In addition, adhesion of neonatal and adult neutrophils to purified ICAM-1 in artificial planar membranes was comparable and almost completely inhibited by anti-LFA-1 MoAb. Chemotactic stimulation induced Mac-1-dependent adhesion of adult neutrophils to endothelial cells, purified intercellular adherence molecule-1 (ICAM-1) and protein-coated glass. In marked contrast, adhesion of neonatal neutrophils to these substrates was not significantly increased by chemotactic stimulation. These findings indicate that diminished transendothelial migration by neonatal neutrophils is related to abnormal interactions of Mac-1 with ICAM-1 and possibly other endothelial ligands. These functional deficits may contribute to impaired inflammation and infectious susceptibility in human neonates.
Assuntos
Endotélio Vascular/citologia , Neutrófilos/citologia , Envelhecimento/fisiologia , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/fisiologia , Antígenos CD11 , Antígenos CD18 , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/farmacologia , Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Fatores Quimiotáticos/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiologia , Hemocianinas/farmacologia , Heterozigoto , Humanos , Recém-Nascido , Molécula 1 de Adesão Intercelular , Interleucina-1/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/imunologia , Neutrófilos/fisiologia , Receptores de Adesão de Leucócito/imunologia , Receptores de Adesão de Leucócito/fisiologiaRESUMO
The CD11b/CD18 (Mac-1) heterodimeric surface glycoprotein contributes to a broad range of adherence-dependent neutrophil inflammatory functions. Previous investigations have indicated that diminished expression or regulation of Mac-1 may underlie abnormalities of stimulated adhesion and chemotaxis of neonatal neutrophils in vitro and inflammatory deficits in human neonates. To define the pathogenic mechanisms contributing to these findings, we compared the distribution and translocation of Mac-1 in subcellular fractions of neonatal and adult neutrophils before and after chemotactic stimulation. The total cell content of Mac-1 and the proportions of Mac-1 in beta fractions (vitamin B12 binding protein-rich granules), pre-gamma fractions (gelatinase-rich granules), or gamma fractions (plasma membrane) of neonatal neutrophils were comparable with those of adult neutrophils. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 nmol/L, 37 degrees C, 15 minutes), neonatal neutrophils demonstrated (1) diminished translocation of Mac-1 from pre-gamma fractions (P less than .05), and (2) diminished surface expression of Mac-1 (P less than .05), as compared with healthy adult neutrophils. As shown in enzymatic and immunochemical assays, neonatal cells contained significantly (P less than .01) diminished levels of neutrophil gelatinase. In response to FMLP (0.1 to 10 nmol/L, 37 degrees C, 15 minutes), neonatal suspensions also released significantly (P less than .001) less gelatinase, as compared with adult neutrophil suspensions. These observations demonstrate that diminished mobilization of Mac-1 from gelatinase-rich granular pools in neonatal neutrophils is associated with abnormal surface expression of this glycoprotein after chemotactic stimulation. This abnormality may contribute, in part, to abnormal migratory properties of neonatal neutrophils in response to inflammatory stimuli.
Assuntos
Antígenos de Diferenciação/metabolismo , Recém-Nascido/sangue , Neutrófilos/metabolismo , Receptores de Adesão de Leucócito/metabolismo , Transporte Biológico , Compartimento Celular , Quimiotaxia de Leucócito , Grânulos Citoplasmáticos/metabolismo , Exocitose/efeitos dos fármacos , Gelatinases , Humanos , Lactoferrina/metabolismo , Antígeno de Macrófago 1 , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Pepsina A/metabolismo , Frações Subcelulares/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcobalaminas/metabolismoRESUMO
The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.