RESUMEN
Nematode infections induce the upregulation of mucin- and glycosylation-related genes in intestinal epithelial cells in vivo. However, the factor(s) that induce these changes in epithelial cells have not been fully elucidated. In the present study, we analysed the effects of the Th2 cytokines IL-4 and IL-13 and the excretory-secretory (ES) product of the nematode Nippostrongylus brasiliensis on the gene expression of the major mucin core peptide MUC2, the sialyltransferase ST3GalIV (Siat4c) and the sulphotransferase HS3ST1 in intestinal epithelium-derived IEC-6 cells by quantitative reverse transcription (RT)-PCR. The administration of IL-4 and IL-13 resulted in a significant upregulation of ST3GalIV and HS3ST1 gene transcription, but had no effect on MUC2, in IEC-6 cells. RT-PCR studies also demonstrated the constitutive expression of IL-13Ralpha1 and IL-4R in IEC-6 cells. On the other hand, the ES product induced upregulation of ST3GalIV, but not HS3ST1 or MUC2, while coadministration of IL-13 and the ES product induced a slight but significant upregulation of MUC2. Co-incubation of live N. brasiliensis adult worms with IEC-6 cells resulted in the upregulation of ST3GalIV and MUC2. These results suggested that HS3ST1 gene expression is strictly regulated by IL-4/IL-13, while ST3GalIV and MUC2 gene expressions are regulated by redundant mechanisms.
Asunto(s)
Íleon/parasitología , Interleucina-13/fisiología , Interleucina-4/fisiología , Mucina 2/biosíntesis , Nippostrongylus/patogenicidad , Sialiltransferasas/biosíntesis , Sulfotransferasas/biosíntesis , Animales , Antígenos Helmínticos/fisiología , Células Epiteliales/inmunología , Células Epiteliales/parasitología , Perfilación de la Expresión Génica , Íleon/inmunología , Masculino , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Infections with helminthic parasites occasionally induce pulmonary diseases with possible involvement of immunological mechanisms. In rats infected with the nematode Nippostrongylus brasiliensis, pulmonary granulomatous lesions develop and persist after the larvae have migrated through the lungs. To determine the pathogenesis of this lesion, we examined cytokine gene expression in the lungs using RT-PCR and in situ hybridization. Two weeks after infection, when fully developed lesions appeared, levels of IL-3 and of type2 cytokines IL-4, IL-5, IL-6 and IL-13 gene expression were markedly enhanced in whole lung homogenates. Those of IL-2 and IFN-gamma were also slightly increased 2 weeks postinfection. IL-12 mRNA level did not change after 2 weeks but was slightly increased after 4 weeks. Levels of IL-10 and proinflammatory cytokine TNF gene expression did not show significant changes, although a slight increase was observed in IL-1beta message after 2 weeks. In situ hybridization studies showed that lung granulomatous lesions were composed mainly of lymphoid cells expressing IL-3, IL-4 and IL-13 mRNA, but not IFN-gamma mRNA. IL-5 mRNA-expressing cells were fewer in number than these cells. RMCP II immunohistochemistry revealed that mast cells increased in number in the lung granulomas. From these results, it was concluded that the nematode infection-associated lung granuloma was a type 2 lesion.
Asunto(s)
Citocinas/genética , Enfermedades Pulmonares Parasitarias/inmunología , Pulmón/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Animales , Modelos Animales de Enfermedad , Expresión Génica , Pulmón/patología , Enfermedades Pulmonares Parasitarias/genética , Enfermedades Pulmonares Parasitarias/patología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Infecciones por Strongylida/genética , Infecciones por Strongylida/patologíaRESUMEN
Autoinfective strongyloidiasis is potentially fatal, yet the majority of infected individuals harbour asymptomatic and chronic infections. The role of humoral responses in modulating autoinfection was assessed by examining antibody isotype responses to filariform larval antigens amongst chronically infected ex-Far East Prisoners of War (exFEPOWs) with longstanding (> 30 years) infection. Serum immunoglobulin (Ig)G1, IgG4, IgE and IgA responses to whole Strongyloides stercoralis L3 extracts and their constituent antigenic components were characterized by ELISA and quantitative immunoblotting. Comparison of two groups of S. stercoralis infected exFEPOWs with and without detectable larvae in stool demonstrated novel trends. Significantly enhanced recognition of six immunodominant antigenic components by IgA was associated with undetectable larval output, as was enhanced IgE recognition of several components. Additionally, IgE and IgG4 exhibited parallel antigen recognition patterns. These findings are consistent with roles for IgA in modulating larval output, for IgE in regulating autoinfection, and for IgG4 in blocking IgE-mediated responses in human strongyloidiasis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/genética , Nippostrongylus , Infecciones por Strongylida/inmunología , Actinas/genética , Animales , Ganglios Linfáticos/metabolismo , Masculino , Mesenterio , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344RESUMEN
It has been reported that infection with Nippostrongylus brasiliensis induces villus atrophy with various histological alterations. In N. brasiliensis-infected rats, villus length in the jejunum was reduced significantly at day 10 p.i., when serum levels of rat mast cell protease (RMCP) II had increased significantly. To determine whether the villus atrophy is associated with enhancement of apoptosis, apoptotic nuclei were labelled using the nick end-labelling method. Numbers of labelled cells were markedly increased in the villus epithelium at 7-10 days p.i., while the numbers returned to normal 14 days p.i. when worms were rejected from the intestine and villus length became normal. Examination of the expression of the adhesion molecule E-cadherin showed granular immunoreactivity in the cytoplasm of atrophic villus epithelium with loss of normal localization to epithelial cell borders. In mast cell-deficient Ws/Ws rats, villus length was reduced as significantly as in +/+ counterparts at day 10 p.i. with marked increases in the numbers of apoptotic cells. These results suggested that villus atrophy was closely associated with enhanced apoptosis and loss of adhesion in epithelial cells. Mast cell activation appears not to be involved in these alterations.
Asunto(s)
Apoptosis , Mucosa Intestinal/patología , Intestino Delgado/patología , Nippostrongylus/parasitología , Infecciones por Strongylida/patología , Animales , Atrofia , Cadherinas/aislamiento & purificación , Adhesión Celular , Quimasas , Gránulos Citoplasmáticos , Mucosa Intestinal/parasitología , Intestino Delgado/parasitología , Masculino , Mastocitos/enzimología , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Proteínas Proto-Oncogénicas c-kit/genética , Ratas , Ratas Mutantes , Serina Endopeptidasas/sangre , Infecciones por Strongylida/parasitologíaRESUMEN
Ws/Ws rats are deficient in both mucosal- and connective tissue-type mast cells. To study the role of mast cells in active anaphylaxis, changes in vascular permeability in the trachea upon intravenous antigen challenge with Evans blue dye were examined in Ws/Ws, heterogenic Ws/+, and normal +/ + rats sensitized with the nematode Nippostrongylus brasiliensis. Antigen challenge resulted in fatal anaphylactic shock in some +/+ and Ws/+ rats, but not in Ws/Ws rats. Marked dye leakage developed within 30 min in the trachea of +/+ and Ws/+ rats, while Ws/Ws rats showed no substantial increases in the levels of vascular permeability. Ex vivo stimulation of sensitized lung fragments from +/+ animals with specific antigen induced significant releases of histamine and leukotriene (LT) C4, while sensitized Ws/Ws rat-lung fragments did not. In Ws/Ws rats, levels of nematode-specific IgE, IgG1 and IgG2a antibodies as well as levels of lung eosinophilia were not significantly different from those in +/+ rats. These results show that mast cell-deficient Ws/Ws rats fail to develop active anaphylaxis, and this is mediated probably by the lack of mast cell-derived mediators required for initiation of the reaction.
Asunto(s)
Anafilaxia/inmunología , Anafilaxia/parasitología , Pulmón/inmunología , Pulmón/parasitología , Mastocitos/inmunología , Mastocitos/parasitología , Nippostrongylus/inmunología , Animales , Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Permeabilidad Capilar , Recuento de Células , Liberación de Histamina , Inmunización , Leucotrieno C4/metabolismo , Pulmón/metabolismo , Pulmón/patología , Mastocitos/patología , Ratas , Ratas Mutantes , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/metabolismo , Infecciones por Strongylida/patología , Tráquea/irrigación sanguíneaRESUMEN
The effects of lactic dehydrogenase virus (LDV) infection on the protective immune responses to the nematode Nippostrongylus brasiliensis were studied. Mice with chronic LDV infection showed significantly higher levels of parasite egg production than non-LDV-infected (control) mice after N. brasiliensis infection. Concurrent LDV infection also suppressed peripheral blood eosinophilia and the lung mastocytosis induced by this nematode. LDV infection showed higher expression levels of the interferon-gamma (IFN-gamma) mRNA in lymph nodes compared with control mice before N. brasiliensis infection. In addition, the IgG2a production in LDV-infected mice was higher than that in control mice before and after N. brasiliensis infection. These results suggest that LDV infection modulates protective immune responses against N. brasiliensis infection by the activation of T-helper type 1 cells.
Asunto(s)
Infecciones por Arterivirus/inmunología , Virus Elevador de Lactato Deshidrogenasa , Nippostrongylus , Infecciones por Strongylida/inmunología , Animales , Infecciones por Arterivirus/parasitología , Enfermedad Crónica , Eosinófilos/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Yeyuno/inmunología , Recuento de Leucocitos , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Masculino , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Recuento de Huevos de Parásitos , ARN Mensajero/metabolismo , Infecciones por Strongylida/parasitologíaRESUMEN
Development of basophilic leukocytes was studied in the Mongolian gerbil, Meriones unguiculatus, after infection with the nematode Nippostrongylus brasiliensis. After infection, peripheral blood basophilia developed and peaked at 2 weeks. In bone marrow sections, numbers of alcian blue+/safranine- basophilic cells were increased. These cells did not bind berberine sulfate and were clearly distinguishable from the bone marrow-resident mast cells, safranine+ and berberine sulfate+. Alcian blue+/safranine- cells were identified by electron microscopy as basophilic myelocytes in various stages of maturation. In the early period of infection, these cells had round-to-oval granules with a homogenous electron-dense matrix, a well-developed Golgi apparatus and rough endoplasmic reticulum, and a nonsegmented nucleus. By enzyme cytochemical analysis, intense peroxidase activity was demonstrated in all of the specific granules as well as in the rough endoplasmic reticulum and Golgi apparatus. Two weeks after infection, the number of bone marrow basophilic cells further increased, forming distinct clusters or islands composed of up to 100 cells each. On electron micrographs, the basophilic cells in these clusters appeared to be late-stage basophilic myelocytes, ie, having an increased number of granules, a less-conspicuous Golgi apparatus and rough endoplasmic reticulum, a horseshoe-shaped-to-lobulated nucleus, and reduced peroxidase activity. Eosinophils and mast cells were rarely found in the basophilic cell clusters. Four weeks after infection, the clusters had disappeared. These results show that gerbil basophilic myelocytes tend to form cell clusters in the bone marrow during their active proliferation. The comparative paucity of other cell lineages in basophilic cell clusters suggests that basophilia is generated from differentiation/proliferation of precommitted basophil progenitors independently from cells of other lineages.
Asunto(s)
Basófilos/patología , Médula Ósea/patología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Nippostrongylus , Infecciones por Strongylida/patología , Infecciones por Strongylida/fisiopatología , Azul Alcián , Animales , Basófilos/fisiología , Basófilos/ultraestructura , Médula Ósea/fisiopatología , Colorantes , Gránulos Citoplasmáticos/patología , Gránulos Citoplasmáticos/ultraestructura , Gerbillinae , Recuento de Leucocitos , Microscopía Electrónica , Fenazinas , Infecciones por Strongylida/sangre , Factores de TiempoRESUMEN
Certain nematode infections induce eosinophil infiltration and granulomatous responses in the lungs. To examine the role of mast cells in the development of lung lesions, normal +/+ and genetically mast cell-deficient Ws/Ws rats were infected with the nematode Nippostrongylus brasiliensis. In +/+ rats, numbers of eosinophils in bronchoalveolar lavage fluid (BALF) increased significantly 3-7 days after infection, and granulomatous responses composed of histiocytes/ macrophages and multinucleate giant cells were triggered in the lungs 3-14 days after infection. Challenge infection, which was carried out on day 28 after primary infection, induced much higher levels of granulomatous response than after primary infection, suggesting that the response is mediated at least in part by an immunological mechanism. In Ws/Ws rats, both the eosinophil percentage in BALF and the size of the granulomas in the lungs were significantly smaller than in +/+ rats after primary as well as after challenge infection. The amount of rat mast cell protease (RMCP) II in +/+ rat BALF was increased 1 day after primary infection and more significantly after challenge infection, suggesting that lung mucosal mast cells were activated more markedly after the challenge infection. In Ws/Ws rats, RMCP II was undetectable throughout the observation period. The time course of nematode migration in the lungs did not differ in +/+ and Ws/Ws rats. These results suggest that mast cell activation might be relevant to eosinophil infiltration and granulomatous response in the lungs, although the responses do not affect lung migration of the nematode.
Asunto(s)
Granuloma/inmunología , Tolerancia Inmunológica/genética , Parasitosis Intestinales/inmunología , Pulmón/inmunología , Mastocitos/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Animales , Granuloma/genética , Granuloma/patología , Parasitosis Intestinales/genética , Parasitosis Intestinales/patología , Pulmón/patología , Ratas , Ratas Mutantes , Infecciones por Strongylida/genética , Infecciones por Strongylida/patologíaRESUMEN
Worm expulsion of, and IgE and interferon (IFN)-gamma responses to, Nippostrongylus brasiliensis were studied in 2 rat strains, Brown Norway (BN) and Fischer (F)-344. BN rats expelled the majority of worms by day 14 post-infection (p.i.) with approximately 6% of worms surviving for at least 3 weeks. In F-344 rats, worm expulsion was delayed by 2 days relative to that in BN, while the numbers of residual worms were significantly fewer than in BN, suggesting that different immune mechanisms are involved in early and late phases of immunity. Total serum IgE, as well as in vitro IgE production by mesenteric lymph node (MLN) cells, was increased 2 weeks p.i., the levels being markedly higher in BN than in F-344 rats. Serum rat mast cell protease II was also increased more significantly in BN than in F-344 rats. In contrast, production of IgG2a and IFN-gamma by MLN and spleen cells was found to be higher in F-344 than in BN rats. These results indicate that the early worm expulsion is correlated with the host IgE and mast cell responsiveness, whereas the persistence of infection in the late period may be controlled by different immune mechanisms.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Interferón gamma/biosíntesis , Nippostrongylus , Infecciones por Strongylida/inmunología , Animales , Anticuerpos Antihelmínticos/biosíntesis , Formación de Anticuerpos , Quimasas , Ensayo de Inmunoadsorción Enzimática , Heces/microbiología , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/sangre , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Intestino Delgado/parasitología , Masculino , Nippostrongylus/inmunología , Nippostrongylus/aislamiento & purificación , Recuento de Huevos de Parásitos , Anafilaxis Cutánea Pasiva , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Serina Endopeptidasas/análisis , Factores de TiempoRESUMEN
The number of goblet cells in the small intestines of C3H/HeN mice increased rapidly following their infection with about 500 third-stage larvae (L3) of the intestinal nematode Nippostrongylus brasiliensis. The number of goblet cells reached its peak on day 9 postinfection (p.i.). Worm burdens in the hosts' small intestines were determined following a challenge infection with encysted metacercariae of the intestinal trematodes Echinostoma trivolvis or E. caproni on days 8 and 16 after primary infections with N. brasiliensis. All metacercariae of E. trivolvis or E. caproni used to challenge the hosts on day 8 p.i. were expelled. Considerable numbers of E. trivolvis (48.6%) and E. caproni (67.1%) remained in the intestines of hosts challenged with these echinostomes on day 16 p.i. All the controls used for E. trivolvis and E. caproni infections without primary infections with N. brasiliensis showed recovery rates greater than 70%. An enzyme-linked immunosorbent assay (ELISA) showed that the IgM titer rose remarkably and plateaued on day 11 p.i. No marked rise in the IgG or IgA titer occurred during the experiment. These results indicate that mucins increased by hyperplastic goblet cells associated with primary infections with N. brasiliensis are responsible for a rapid expulsion of the worms of the challenge infection with E. trivolvis or E. caproni from the mouse host.
Asunto(s)
Equinostomiasis , Mucosa Intestinal/citología , Mucosa Intestinal/parasitología , Mucinas/biosíntesis , Infecciones por Strongylida , Animales , Interacciones Huésped-Parásitos , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
Although the immune responses to intestinal nematode infection have been well studied and have been shown to be strongly driven by Th2-associated cytokines in mice, such information has been limited with respect to rats. We investigated changes in levels of the mRNAs encoding interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-10, and gamma interferon in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis by reverse transcription-PCR in comparison with immunoglobulin E (IgE)/IgG2a antibody, eosinophil, basophil, and mucosal mast cell responses. In the two rat strains used, Brown Norway and Fischer-344, which show different responses to allergens, serum IgE increased to much higher levels in the former than in the latter 2 weeks after infection. Intestinal mastocytosis was observed much earlier and more intensely in Brown Norway rats than in Fischer-344 rats, but the degrees of peripheral eosinophilia and basophilia did not differ between the two strains. In both strains, IL-3, IL-4, and IL-5 mRNA expression increased and peaked around 7 to 14 days after infection, while expression of IL-2, IL-10, and gamma interferon mRNAs did not change notably throughout the experimental period. The highest IL-4 mRNA expression was observed slightly earlier in Brown Norway than in Fischer-344 rats, but levels of IL-3 and IL-5 mRNAs peaked synchronously in both strains. The amounts of mRNAs encoding these three cytokines were always higher in Brown Norway than in Fischer-344 rats. It is suggested that in rats, Th2 or Th2-like cells are also induced after nematode infection, and IgE elevation is mainly related to increased IL-4 gene expression.
Asunto(s)
Citocinas/biosíntesis , Nippostrongylus/inmunología , ARN Mensajero/biosíntesis , Infecciones por Strongylida/inmunología , Animales , Secuencia de Bases , Citocinas/genética , Interferón gamma/genética , Masculino , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Especificidad de la Especie , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pI 8.5, showed maximum enzyme activity at pH 5.5 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64, completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis, a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and IgG1, production of anti-cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response.
Asunto(s)
Anticuerpos Antihelmínticos/biosíntesis , Cisteína Endopeptidasas/inmunología , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Nippostrongylus/inmunología , Secuencia de Aminoácidos , Animales , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Masculino , Datos de Secuencia Molecular , Nippostrongylus/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74 kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.
Asunto(s)
Acetilcolinesterasa/análisis , Proteínas del Helminto/análisis , Nippostrongylus/metabolismo , Acetilcolinesterasa/inmunología , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Antihelmínticos/inmunología , Gránulos Citoplasmáticos/enzimología , Aparato de Golgi/enzimología , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Inmunoglobulina E/inmunología , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Masculino , Nippostrongylus/anatomía & histología , Nippostrongylus/inmunología , Ratas , Ratas Sprague-DawleyRESUMEN
Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Parasitosis Intestinales/patología , Intestino Delgado/patología , Mastocitos/fisiología , Nippostrongylus , Infecciones por Strongylida/patología , Animales , Recuento de Células Sanguíneas , Médula Ósea/patología , Recuento de Células , Movimiento Celular , Células Cultivadas , Concanavalina A/farmacología , Medios de Cultivo Condicionados/farmacología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Parasitosis Intestinales/sangre , Mastocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit , Ratas , Ratas Endogámicas BN , Ratas Mutantes , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptores del Factor Estimulante de Colonias/deficiencia , Receptores del Factor Estimulante de Colonias/genética , Eliminación de Secuencia , Factor de Células Madre , Infecciones por Strongylida/sangre , Linfocitos T/metabolismoRESUMEN
Infections of intestinal nematodes induce the T cell-dependent proliferation of intestinal mucosal mast cells (MMC). To examine whether nematode-induced MMC proliferation is affected by the site of infestation, adult-stage nematode Nippostrongylus brasiliensis (NB) was transplanted into the normal infection site, the duodenum, or into heterotopic sites, the peritoneal cavity (i.p.) or subcutaneous tissue (s.c.), of rats. Two weeks after duodenal inoculation, MMC numbers in the small intestine had increased 6.5-fold. In contrast, i.p. and s.c. inoculation induced only slight increases of intestinal MMC. After i.p. inoculation, worm granulomas developed in the connective tissues adhering to stomach and duodenum, and large numbers of mast cells appeared around the granulomas. The majority of the latter mast cells showed histochemical features similar to MMC: they were formalin sensitive, berberine sulfate-, alcian blue+/safranine-, and rat mast cell protease (RMCP) II+. After s.c. inoculation, worm granulomas developed at the inoculation site, but the number of mast cells around the granulomas was not significantly increased. These results suggest that intense proliferation of MMC or MMC-like cells is induced only by the infections on mucosa or in mucosa-associated tissues.
Asunto(s)
Mucosa Intestinal/parasitología , Mastocitos/patología , Nippostrongylus/patogenicidad , Infecciones por Strongylida/patología , Animales , Basófilos , División Celular , Cromatografía Líquida de Alta Presión , Duodeno/parasitología , Duodeno/patología , Eosinófilos , Granuloma/patología , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Mastocitos/inmunología , Cavidad Peritoneal/parasitología , Cavidad Peritoneal/patología , Ratas , Ratas Sprague-Dawley , Piel/parasitología , Piel/patología , Infecciones por Strongylida/sangre , VirulenciaRESUMEN
IgE and IgG2a antibody production and interferon (IFN)-gamma secretion were studied in rats infected with the gut nematode Nippostrongylus brasiliensis by in vitro cultivation of mononuclear cells obtained from spleen (SPL), mesenteric lymph nodes (MLN) and pulmonary hilar lymph nodes (PLN). The highest levels of IgE were detected in the culture supernatants of MLN cells after infection: IgE levels were modest in PLN and negligible in SPL. In contrast, the highest levels of IgG2a were produced by PLN cells, followed by MLN and SPL cells. These results indicate that the MLN is the most significant site for IgE production in nematode infection, while IgG2a production is more marked in PLN. In naive rats, the spontaneous secretion of IFN-gamma was highest in PLN cells, followed by MLN and SPL cells. After the infection, IFN-gamma levels were significantly decreased in MLN and PLN. Suppression of IFN-gamma secretion was also observed in concanavalin A (ConA)-stimulated MLN and PLN cells from infected rats. In MLN, the ratio of CD4+ to CD8+ T cells was increased after the infection. Stimulation with an allergen-rich, excretory-secretory (ES) substance of the nematode enhanced ongoing IgE production, and suppressed IFN-gamma secretion by MLN and PLN cells. In contrast, an allergen-poor, adult worm extract potentiated IFN-gamma secretion. These results show that nematode-induced IgE antibody response is associated with the suppressed production and/or secretion of IFN-gamma, particularly in the MLN, and that some molecules in the ES substance may trigger these immune responses.
Asunto(s)
Inmunoglobulina E/biosíntesis , Interferón gamma/biosíntesis , Ganglios Linfáticos/inmunología , Nippostrongylus , Infecciones por Strongylida/inmunología , Animales , Antígenos Helmínticos/inmunología , Células Cultivadas , Inmunoglobulina G/biosíntesis , Leucocitos Mononucleares/inmunología , Pulmón , Masculino , Mesenterio , Nippostrongylus/inmunología , Ratas , Ratas Sprague-Dawley , Bazo/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
In order to examine the effective site of sensitization for IgE responses, we transplanted 2000 adult-stage worms of the nematode Nippostrongylus brasiliensis into the duodenum or the peritoneal cavity of naive rats. Total serum IgE began to increase 1 week after the nematode inoculations and reached a peak at week 2. Living worms inoculated into the duodenum induced the highest serum IgE, this being 800 times the level in control animals. Intraperitoneal inoculations of living and dead worms resulted in increases of the serum IgE levels to 120 and 13 times the control level, respectively. The intraduodenal inoculation of living adult worms also induced a significant increase in specific IgE against the excretory-secretory (ES) product of adult N. brasiliensis 1 week later than the rise in total IgE, whereas intraperitoneal inoculations did not induce such an increase. These results suggest that sensitization through the intestinal mucosa with adult N. brasiliensis might be important for the effective induction of both specific and non-specific IgE responses. Since these findings also indicated that factors secreted by living worms play an important role in the induction of total IgE response, the ES product was injected to naive rats for 6 consecutive days (total 2.7-4.4 mg). Intraperitoneal injection of the ES product alone induced a 14.7-fold increase in total IgE without any specific IgE response. This indicates that some constituents of the ES product have the potential to trigger a non-specific IgE response.
Asunto(s)
Anticuerpos Antihelmínticos/biosíntesis , Inmunoglobulina E/biosíntesis , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Alérgenos/inmunología , Animales , Antígenos Helmínticos/inmunología , Inmunoglobulina G/biosíntesis , Cinética , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
IgE, IgG and mast cell responses were studied in rats infected weekly with 10 larvae of Nippostrongylus brasiliensis (NB). Worm recovery at 8 weeks of repeated infections was six-fold greater than that of a single infection with 10 larvae, suggesting the accumulation of worms during the repeated infections. Total serum IgE was increased after 2 weeks of infection, and further increased after repeated infections: at 6 weeks of infection the level was four to six times higher than that after a single infection. Anti-NB IgG1 levels were also significantly higher after repeated infections than after a single infection. On the other hand, there was no significant difference in the level of anti-NB IgE between single and repeated infections, as determined by ELISA, as well as by passive cutaneous anaphylaxis (PCA) reaction. Mastocytosis was induced in the small intestine after both single and repeated infections, but the levels did not differ between the two. These results indicate that total IgE and specific IgG1 production are augmented by repeated helminth infections, but specific IgE and mast cell responses are not. This pattern of response may minimize the development of IgE-dependent hypersensitivity reactions with repeated helminth infections.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Mastocitos/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Ensayo de Inmunoadsorción Enzimática , Recuento de Leucocitos , Masculino , Mastocitosis/inmunología , Nippostrongylus/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos EspecíficosRESUMEN
All basophils, mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC) are derived from the multipotential hematopoietic stem cell. Mutations at the c-kit locus resulted in deficiency of MMC and CTMC in both mice and rats. To investigate the role of the c-kit receptor tyrosine kinase for production of basophils, we used white spotting/white spotting (Ws/Ws) mutant rats that have a small deletion at the tyrosine kinase domain of the c-kit gene. When Ws/Ws, nude athymic, and normal (+/+) rats were infected with Nippostrongylus brasiliensis (NB), the number of basophils increased greater than 50-fold in the peripheral blood of Ws/Ws and +/+ rats but did not increase in that of nude rats. Blood histamine concentration increased significantly in Ws/Ws and +/+ rats but did not increase in nude rats. Immature basophils increased greater than 10-fold in the bone marrow of Ws/Ws and +/+ rats but did not increase in that of nude rats. Mature and immature basophils that developed after the NB infection were identified by electron microscopy. The present result confirms that T-cell-derived cytokines are indispensable for the augmented production of basophils and suggests that stimulation via the c-kit receptor may not be necessary for the augmented production.
Asunto(s)
Basófilos/patología , Médula Ósea/patología , Células Madre Hematopoyéticas/patología , Mastocitos/fisiología , Nippostrongylus , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Receptores de Superficie Celular/genética , Eliminación de Secuencia , Infecciones por Strongylida/sangre , Animales , Basófilos/fisiología , Basófilos/ultraestructura , Células Madre Hematopoyéticas/fisiología , Recuento de Leucocitos , Proteínas Proto-Oncogénicas c-kit , Ratas , Ratas Endogámicas BN , Ratas Mutantes , Ratas Desnudas , Infecciones por Strongylida/patologíaRESUMEN
Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c-kit gene and are deficient in both mucosal mast cells (MMC) and connective tissue-type mast cells (CTMC). The role of the c-kit receptor in the development of MMC and CTMC was investigated by infecting Ws/Ws and control +/+ rats with Nippostrongylus brasiliensis (NB), which induces T-cell-dependent mast cell proliferation. Although mast cells did not develop in the skin of Ws/Ws rats, a significant number of mast cells developed in the jejunum after NB infection. These mast cells had the MMC protease phenotype (rat mast cell protease [RMCP] I-/II+) and lacked heparin because they were not stained with berberine sulfate. Globule leukocytes were also detected in the mucosal epithelium of these rats. However, the number of MMC and the serum concentration of RMCP II in NB-infected Ws/Ws rats were only 13% and 7% of those of NB-infected +/+ rats, respectively. A small number of mast cells also developed in the lung, liver, and mesenteric lymph nodes of Ws/Ws rats after NB infection. Although mast cells in these tissues had the MMC phenotype throughout the observation period, the increased mast cells in the lung and liver of +/+ rats acquired a CTMC-like phenotype and were RMCP I+/II+, berberine sulfate+, and formalin resistant. These results indicate that the need for the stimulus through the c-kit receptor appears to be greater in the development of CTMC in the skin as well as for CTMC-like mast cells in the lung and liver than for the development of MMC.