Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain.

Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry.

Neurotensin receptor down-regulation induced by dexamethasone and forskolin in rat hypothalamic cultures is mediated by endogenous neurotensin.

Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.

Booster-Dependent Alterations of the Subsets of T Lymphocytes and Eosinophils in the Bronchi of Immunized Guinea Pigs.

We investigated whether guinea pigs sensitized to ovalbumin show changes in T cells in the bronchial wall and whether they correlate with eosinophil migration. Animals received two injections of 10 µg ovalbumin in Al(OH)3 with a 2-week interval. Lungs were resected and frozen and cryostat sections stained with monoclonal antibodies recognizing T cells and subsets. Eosinophil peroxidase staining was used to identify this cell type. Sections stained with each marker were read 'blind' and cells enumerated in the bronchial lamina propria. A large number of T cells, mainly of CD4+ subset, were recruited into the bronchi 7 days after the booster injection of the antigen, in contrast to nonimmunized or nonboosted animals. These changes were also accompanied by an infiltration of eosinophils in boosted animals and suggest that T cells and/or their products play an important role in the development of bronchial hyperreactivity in this model.

Emergence of T-lymphocytes, eosinophils and dendritic cells in the bronchi of actively sensitized guinea pigs after antigenic challenge.

Bronchi from guinea pigs actively sensitized to ovalbumin and boosted two weeks later display increased numbers of CD4+ T-lymphocytes and eosinophils. We have further investigated immunopathological changes in sensitized guinea pigs 2 or 24 h after antigenic challenge with ovalbumin. Lungs were resected, frozen and cryostat sections stained with monoclonal antibodies that recognize relevant guinea pig epitopes. Cyanide-resistant peroxidase activity was used to stain eosinophils. No further increase in T-lymphocytes or eosinophils was observed 2 h after challenge. At 24 h, a marked increase in EPO+ eosinophils was found, and this was accompanied by severe mucosal damage characterized by epithelial shedding and ulceration. The numbers of T-lymphocytes remained stable but a novel population of cells with the appearance of dendritic cells was seen in the bronchial wall. They were negative for macrophage markers but were strongly Class II positive. These findings suggest that antigenic challenge results in further recruitment of eosinophils, their activation and release of toxic substances to the epithelium. Furthermore, other cell types, possibly dendritic cells, are attracted to the bronchi and could play a role in maintaining allergic inflammation via antigen presentation.

Distribution and histochemical characterization of pulmonary mast cells in the rat and guinea pig.

The rat and guinea pig are widely used in experimental models dealing with immuno allergic bronchoreactivity. The present study was designed to compare the distribution and heterogeneity of the mast cell population in the respiratory tract of the Sprague-Dawley rat and Hartley guinea pig. Mast cells were identified according to the sequential Alcian blue/safranin O staining method. From the trachea to the peripheral conductive airways, the density of mast cells increased in the guinea pig whereas it decreased in the rat. Although mast cells were observed in the interalveolar septa of the guinea pig, none were found in the rat. In the lung of the rat, three types of mast cells were observed: in the wall of the trachea and large bronchi, safranin-positive, mixed and Alcian-blue-positive mast cells were found, whereas only Alcian-blue-positive mast cells were observed in the small conductive airways. In contrast, only Alcian-blue-positive mast cells were observed in the lung of the guinea pig. These results show that the types and localization of the mast cell populations are distinctly different in these two species. Consequently, experimental models of bronchoreactivity in these species are testing different cellular systems.

Histological study of mast cells in the actively sensitized guinea pig lung and after challenge: effect of a corticosteroid.

Although mast cells are sometimes considered to play a minor role in hypersensitivity reactions, we used a histological method to show the modifications in the guinea pig lung mast cell population in the course of such reactions. We sensitized guinea pigs with ovalbumin and studied the effect of the challenge with and without corticosteroid treatment. We observed that the mast cell count was not modified after sensitization but was decreased after challenge. Twenty-four hours after challenge, the number of mast cells returned to the control value, indicating a renewal of the mast cell pool. A second challenge, 1 week after the first, did not provoke the same mast cell degranulation, suggesting a non-responsiveness to aerosol antigen. Betamethasone dipropionate treatment protected mast cells against challenge: in treated guinea pigs, mast cell degranulation was prevented, and we did not observe any change in mast cell count after challenge. The present study was useful to show an effect of corticosteroids on mast cell degranulation in immediate hypersensitivity reactions in vivo.

Evidence for neurotensin autoreceptors and relationship of neurotensin and its receptors with tyrosine hydroxylase-positive neurons in rat primary hypothalamic cultures.

Neurotensin is present in high quantity in the hypothalamus, where it regulates pituitary hormone secretion. A relationship between dopaminergic and neurotensinergic systems has been suggested in the hypothalamus in studies showing an effect of neurotensin on tuberoinfundibular dopaminergic neurons. In order to determine the anatomical basis of such interactions, primary cultures of rat hypothalamic neurons were used. Tyrosine hydroxylase and neurotensin containing cells were identified by immunocytochemistry and neurotensin binding sites by [125I]Tyr3-neurotensin autoradiography. Colocalization studies showed that neurotensin immunoreactivity was present in 16% of tyrosine hydroxylase-positive cells, and that these neurotensin/tyrosine hydroxylase neurons represented more than half (58%) of the neurotensinergic population. Five percent of the tyrosine hydroxylase-positive cells had neurotensin binding sites, suggesting that only a restricted number of hypothalamic dopaminergic neurons is responsive to neurotensin. Neurotensin binding sites were also found on some neurotensin-positive cells, demonstrating for the first time the presence of autoreceptors for this peptide on neurons. These results in primary cultures provide a cellular basis for direct effects of neurotensin on a subpopulation of hypothalamic dopaminergic cells, and support the possibility of an autocrine action of neurotensin in the hypothalamus.

Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins.

Bordetella pertussis expresses factors such as filamentous hemagglutinin, agglutinogens, pertactin, and pertussis toxin, which participate in bacterial adhesion; pertussis toxin, dermonecrotic toxin, lipopolysaccharide, and tracheal cytotoxin, which are responsible for toxic effects; and adenylate cyclase-hemolysin, which is required to initiate infection. By using a murine respiratory model, we showed that the RGD sequences of filamentous hemagglutinin and pertactin are important for bacterial persistence. However, mutants deficient in filamentous hemagglutinin and agglutinogens or in pertactin and the RGD sequence of filamentous hemagglutinin behaved as did wild-type B. pertussis, i.e., induced bronchopneumonia, alveolitis, and an influx of macrophages, lymphocytes, and polymorphonuclear leukocytes into bronchoalveolar lavage fluids. These results suggest that these adhesins are not involved in the induction of pulmonary lesions following infection. The intensity of inflammation was markedly reduced after infection with mutants deficient in either hemolytic activity or pertussis toxin expression, whereas a mutant devoid of adenylate cyclase activity behaved as did the avirulent mutant. Pertussis toxin and adenylate cyclase-hemolysin may act indirectly by altering immune cell functions and thus allowing other factors, such as filamentous hemagglutinin, agglutinogens, and pertactin, to trigger adhesion and lipopolysaccharide, dermonecrotic toxin, and tracheal cytotoxin to induce their toxic effects. However, it is possible that pertussis toxin is also responsible for the induction of some pulmonary alterations.

Effect of antigen provocation of IL-5 transgenic mice on eosinophil mobilization and bronchial hyperresponsiveness.

We investigated whether allergen-induced eosinophil recruitment into mouse airways modifies the in vivo bronchopulmonary responses to standard agonists, and adaptation of a technique described for larger animals. Swiss, CBA, and IL-5 transgenic mice were immunized with ovalbumin and challenged intranasally after 14 days. Immunization alone was followed by increased eosinophil counts in bone marrow and blood, whereas antigenic challenge induced eosinophil infiltration in lungs and bronchoalveolar lavage fluid, which was suppressed by dexamethasone. Despite the high eosinophil counts, no bronchopulmonary hyperreactivity to methacholine or serotonin was detected 3 to 96 hours after antigenic provocation. Our results demonstrate that immunization augments the production of eosinophils by mice, which is further increased by antigenic challenge, but that eosinophil overproduction and lung infiltration, per se, are not sufficient to induce bronchopulmonary hyperreactivity, even in constitutively hypereosinophilic IL-5 transgenic mice.

Sequential study of pleural peritoneal and blood cells in acute pleurisy induced by calcium pyrophosphate in the rat.

We studied the sequential cellular events that occur during nonspecific pleurisy induced by a nondiffusible, nonantigenic and endotoxin-free irritant, calcium pyrophosphate (CaPP). The study was conducted over 10 days and concerned not only the pleural cavity but also the peritoneal cavity and the blood. After injection of CaPP, the first cellular event in the pleural space was a "leukocyte disappearance reaction," followed by an important increase in the polymorphonuclear leukocytes (PMN) and then by an increase in the macrophages. This phase is also characterized by the appearance of submesothelial nodules composed of macrophages and numerous polymorphonuclear cells. All the cell populations returned to their normal value on day 10, although granulomatous submesothelial nodules were present. In the blood, the principal observation was an increased PMN count associated with a decreased lymphocyte count at 4 h. In the peritoneal cavity, it was remarkable that, although functional modifications have been reported in the same model, the different leukocyte populations did not vary during the reaction.

Immunopathologic alterations in the bronchi of immunized guinea pigs.

Isolated lungs from guinea pigs actively sensitized to ovalbumin and boosted 2 wk later display an enhanced bronchoconstriction and release larger amounts of secondary mediators as compared with lungs from nonimmunized animals when stimulated by platelet-activating factor or other agonists. We have investigated changes in T lymphocytes and eosinophils found in the bronchial wall of immunized and nonimmunized guinea pigs. The animals received two injections of 10 micrograms ovalbumin in Al(OH)3, at a 2-wk interval. Two studies were performed: (1) the animals were killed 7 days after the booster injection of antigen, (2) they were challenged with ovalbumin at this same day and killed after 2 or 24 h. Lungs were resected and frozen, and cryostat sections stained using monoclonal antibodies that recognize T cells, T cell subsets, or other relevant epitopes. Cyanide-resistant peroxidase activity was used to identify eosinophils. A large number of T cells, mainly of the CD4+ subset, and eosinophils were recruited into the bronchi 7 days after the booster injection of the antigen, compared with nonimmunized or nonboosted animals. In antigen-challenged animals, the numbers of T cells did not change but eosinophils were further increased in number at the 24 h time point. Also at this time point, a population of cells with a dendritic appearance was seen in the bronchial wall, which did not express macrophage markers but was strongly class II positive. Class II positivity was also noted in the bronchial epithelium and on many cells infiltrating the mucosa. These findings suggest that activated T cells and/or their products play an important role in the bronchopulmonary immunopathology associated with this model and possibly with the development of bronchial hyperreactivity.

Cell localization and regulation of expression of cytochrome P450 1A1 and 2B1 in rat lung after induction with 3-methylcholanthrene using mRNA hybridization and immunohistochemistry.

In order to characterize the response of various pulmonary cell types to polycyclic aromatic hydrocarbons, the expression of cytochrome P450 (CYP) 1A1 and 2B1 mRNA in the lung of rats, with or without induction by 3-methylcholanthrene (3MC), was analyzed by in situ hybridization using appropriate 35S-labeled riboprobes. The expression of the corresponding proteins was investigated immunohistochemically. Following induction with 3MC, the kinetics of mRNA expression differed considerably between Clara cells and type II pneumocytes and venous endothelial cells. In Clara cells, mRNA expression was detected as early as 1 h after induction, peaked between 2 and 4 h, and was completely undetectable at 14 h. In contrast, venous endothelial cells and type II pneumocytes exhibited permanent mRNA expression of CYP 1A1 in 3MC-pretreated rats. These kinetic results explain the striking absence of correlation between mRNA and protein expression observed in Clara cells 24 h after the end of the induction protocol, as these cells exhibited intense protein expression with no mRNA. In contrast, a good correlation was observed for mRNA and protein expression of CYP 2B1, with similar expressions for Clara cells and type II pneumocytes, but no expression in endothelial cells. This study clearly distinguished the regulation of CYP 1A1 expression in the rat lung from that described in the liver. The differences observed in the various lung cell types, whatever the post-transcriptional mechanisms involved, emphasize that studies must be performed at the cellular level in order to understand the specific response to xenobiotics, not only of this organ as a whole but also of its various anatomic structures.

Emergence of T-lymphocytes, eosinophils and dendritic cells in the bronchi of actively sensitized guinea pigs after antigenic challenge

Bronchi from guinea pigs actively sensitized to ovalbumin and boosted two weeks later display increased numbers of CD4+ T-lymphocytes and eosinophils. We have further investigated immunopathological changes in sensitized guinea pigs 2 or 24 h after antigenic challenge with ovalbumin. Lungs were resected, frozen and cryostat sections stained with monoclonal antibodies that recognize relevant guinea pig epitopes. Cyanide-resistant peroxidase activity was used to stain eosinophils. No further increase in T-lymphocytes or eosinophils was observed 2 h after challenge. At 24 h, a marked increase in EPO+ eosinophils was found, and this was accompanied by severe mucosal damage characterized by epithelial shedding and ulceration. The numbers of T-lymphocytes remained stable but a novel population of cells with the appearance of dendritic cells was seen in the bronchial wall. They were negative for macrophage markers but were strongly Class II positive. These findings suggest that antigenic challenge results in further recruitment of eosinophils, their activation and release of toxic substances to the epithelium. Furthermore, other cell types, possibly dendritic cells, are attracted to the bronchi and could play a role in maintaining allergic inflammation via antigen presentation.