Lung tissue eosinophils may be cleared through luminal entry rather than apoptosis: effects of steroid treatment.

Spontaneous or steroid-induced eosinophil apoptosis occurring in vitro has not been demonstrated in lung tissues in vivo. This study examines cell apoptosis in rat lungs using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) technique and transmission electron microscopy (TEM). After establishing sustained lung edema and eosinophilia by challenge with Sephadex beads intratracheally, budesonide treatment was started intratracheally. Sephadex alone increased the total number of apoptotic cells, which were not efficiently engulfed by macrophages or other cells, in vivo. Yet apoptotic tissue eosinophils were exceedingly rare (1 of 360 TEM-analyzed eosinophils). By contrast, approximately 20% of eosinophils in the airway lumen were apoptotic, and unengulfed. Budesonide promptly inhibited edema but 3 d of steroid treatment were required to reduce the established tissue eosinophilia. Not at any time point did budesonide induce eosinophil apoptosis (0 of 318 TEM-analyzed tissue eosinophils). We conclude that (1) eosinophil apoptosis can occur but is a rare event in vivo in respiratory tract tissues; (2) airway tissue eosinophils, rather than undergoing apoptosis, are eliminated by migration into airway lumen followed by apoptosis and mucociliary clearance; (3) anti-inflammatory steroid treatment may not increase eosinophil apoptosis in vivo nor may it affect the luminal entry of eosinophils; (4) steroids permit elimination of eosinophils into airway lumen and slowly resolve established lung eosinophilia.

Role of macrophage migration inhibitory factor (MIF) in allergic and endotoxin-induced airway inflammation in mice.

Macrophage migration inhibitory factor (MIF) has recently been forwarded as a critical regulator of inflammatory conditions, and it has been hypothesized that MIF may have a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). Hence, we examined effects of MIF immunoneutralization on the development of allergen-induced eosinophilic inflammation as well as on lipopolysaccharide (LPS)-induced neutrophilic inflammation in lungs of mice. Anti-MIF serum validated with respect to MIF neutralizing capacity or normal rabbit serum (NRS) was administered i.p. repeatedly during allergen aerosol exposure of ovalbumin (OVA)-immunized mice in an established model of allergic asthma, or once before instillation of a minimal dose of LPS into the airways of mice, a tentative model of COPD. Anti-MIF treatment did not affect the induced lung tissue eosinophilia or the cellular composition of bronchoalveolar lavage fluid (BALF) in the asthma model. Likewise, anti-MIF treatment did not affect the LPS-induced neutrophilia in lung tissue, BALF, or blood, nor did it reduce BALF levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha). The present data suggest that MIF is not critically important for allergen-induced eosinophilic, and LPS-induced neutrophilic responses in lungs of mice. These findings do not support a role of MIF inhibition in the treatment of inflammatory respiratory diseases.

Neurochemical changes in the entopeduncular nucleus and increased oral behavior in rats treated subchronically with clozapine or haloperidol.

The purpose of the present experiment was to test the possibility that atypical antipsychotics and classical antipsychotics differentially regulate specific neurochemical processes within the entopeduncular nucleus. For these experiments, rats were administered clozapine (25 mg/kg), haloperidol (1 mg/kg), or Tween-80 (control) daily for 21 days. Dopamine D(1)-receptor binding was assessed with in vitro receptor autoradiographic methods and the mRNAs corresponding to the two forms of glutamate decarboxylase (glutamate decarboxylase-65 and glutamate decarboxylase-67) were analyzed using in situ hybridization histochemical methods. In addition, vacuous chewing movements (VCM) were measured throughout the drug administration period as a functional indicator of drug action and changes in striatal dopamine D(2)-receptor binding were measured as a positive control for D(2)-receptor antagonist properties of haloperidol and clozapine. In agreement with previous reports, haloperidol increased D(2)-receptor binding throughout the striatum while clozapine had a more limited impact on D(2)-receptors. Behavioral analysis revealed that both haloperidol and clozapine enhanced the display of vacuous chewing movements to a similar extent but with a different postinjection latency. In the entopeduncular nucleus, clozapine increased D(1)-receptor binding compared to controls while haloperidol was without effect. With respect to the regulation of GAD mRNAs, haloperidol increased glutamate decarboxylase-65 and glutamate decarboxylase-67 mRNA levels throughout the entopeduncular nucleus. The effects of clozapine were restricted to increases in glutamate decarboxylase-65 mRNA. These studies show that clozapine and haloperidol, both of which increase the occurrence of VCM, differentially modulate the neurochemistry of the entopeduncular nucleus.

Differential modulation of dopamine D1-receptor binding and mRNA expression in the basal ganglia by the D1-receptor antagonist, SCH-23390.

Dopamine D1-receptor binding in the basal ganglia is differentially regulated by subtype nonspecific dopamine antagonists such as the antipsychotic, Fluphenazine. The purpose of the present study was to determine the relative contributions of D1 and D2 receptor systems in the regulation of basal ganglia D1-receptor binding. Rats were injected twice daily for 21 days with saline, the D1-receptor antagonist, SCH-23390, the D2-receptor antagonist, Raclopride, or both SCH-23390 and Raclopride. Dopamine D1-receptor levels (as indicated by [125I]SCH-23982 binding) and mRNA expression were measured using receptor autoradiographic and in situ hybridization histochemical techniques. [125I]NCQ-298 binding to D2-receptors was also measured as a positive control for the effects of Raclopride. SCH-23390 administration independently increased [125I]SCH-23982 binding in a region-dependent manner with the greatest increases occurring in the entopeduncular nucleus. SCH-23390 also increased D1-receptor mRNA expression in specific striatal subregions suggesting that increases in binding were related to changes in receptor synthesis. In addition, Raclopride independently enhanced D2 binding with comparable increases observed in extrastriatal regions and increases of a lesser magnitude in the striatum. These data show that the modulation of basal ganglia D1-receptor binding observed in animals treated with nonselective antagonists is due primarily to the blockade of D1-receptors. The differential enhancement in basal ganglia D1 binding observed after D1-receptor blockade may be due to anatomical or phenotypic heterogeneity within the population of striatal D1-receptor synthesizing neurons. Similarly, the differential enhancement in striatal and extrastriatal D2-receptor binding may be due to differences in the regulation of striatal and extrastriatal D2-receptor synthesizing neurons.

Modulation of basal ganglia neurotransmission by the classical antipsychotic fluphenazine is due in part to the blockade of dopamine D1-receptors.

Classical antipsychotics, such as fluphenazine, influence neurotransmission by blocking both dopamine D1- and D2-receptors which in turn results in widespread adaptive changes in the neurochemistry of the basal ganglia. The purpose of the present study was to determine the role of D1-receptors in mediating some of these neurochemical events, including changes in D1- and D2-receptor binding, and the expression of preproenkephalin and glutamic acid decarboxylase mRNAs. For these experiments, rats were given a depot injection of fluphenazine decanoate or injected twice daily for 21 days with the D1-receptor antagonist SCH-23390. An additional group received both fluphenazine and SCH-23390 and controls were given saline. Fluphenazine administration decreased D2-receptor binding throughout the basal ganglia while SCH-23390 was without effect. In contrast to the uniform reduction in D2-receptor binding, fluphenazine altered D1-receptor binding in a region-dependent manner. Region-dependent changes were also observed in animals given SCH-23390 which increased binding in the entopeduncular nucleus and posterior caudate-putamen without affecting other brain regions. Both fluphenazine and SCH-23390 significantly enhanced preproenkephalin and glutamic acid decarboxylase (GAD) mRNA expression in the anterior striatum. Fluphenazine also increased GAD mRNA levels in the entopeduncular nucleus. Together, these results indicate that the attenuation of D1-receptor-mediated neurotransmission modulates a number of clinically relevant neurochemical processes in the basal ganglia.

Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the organization of limbic and motor circuits of the entopeduncular nucleus, suggesting that GABAergic transmission through the limbic pathway is regulated predominantly by glutamic acid decarboxylase-65, whereas glutamic acid decarboxylase-67 is of principal importance in the motor pathway. These data provide additional evidence that the neurons of the limbic and motor subregions of the entopeduncular nucleus are neurochemically distinct.

Cholecystokinin, dopamine D2 and N-methyl-D-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior.

Receptor autoradiography was used to investigate the distribution of brainstem binding sites for cholecystokinin, dopamine and N-methyl-D-aspartate with particular reference to the nucleus of the solitary tract of the rat, an area involved in the control of ingestive behavior. Binding sites for the A and B subtypes of the cholecystokinin receptor, labeled with [(125)I]cholecystokinin octapeptide sulfate in the presence or absence of antagonists for the devazepide (A) or L-365,260 (B) receptor, were present throughout the caudal rostral extent of the nucleus of the solitary tract, the A type predominating in the commissural, medial and gelatinous part and the B type in the lateral part. In the most rostral part of the medial nucleus of the solitary tract, both A and B receptors were present. Dopamine D2 receptors, labeled with [(125)I]NCQ-298, were found in all parts of the nucleus of the solitary tract. No binding to the dopamine D1 receptor, labeled with [(125)I]SCH-23982, was found in the brainstem. N-Methyl-D-aspartate receptors, labeled with [(3)H]dizocilpine maleate, were also present in the entire caudorostral extent of the nucleus of the solitary tract. Binding to cholecystokinin A receptors was co-distributed with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the caudal and rostral parts of the nucleus of the solitary tract, and binding to cholecystokinin B receptors overlapped with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the rostral nucleus of the solitary tract. These results are consistent with the hypothesis that cholecystokinin, dopamine and glutamate interact in the nucleus of the solitary tract in the control of ingestive behavior.

Inhibition of nitric oxide synthase reduces Sephadex-induced oedema formation in the rat lung: dependence on intact adrenal function.

In the present study we have investigated the effect of L-nitro arginine mono methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase on Sephadex induced inflammation in the rat lung. Instillation of Sephadex into the airways induced an inflammatory reaction characterized by a long-lasting interstitial oedema, measured as an increase in lung weight, and an influx of inflammatory cells into the airways. L-NAME given s.c. prevented the increase in lung weight following Sephadex instillation. The inactive enantiomer D-NAME had no effect, nor did aminoguanidine which indicates that this effect of L-NAME was mediated by inhibition of the constitutive form of NOS. Treatment with L-NAME did not reduce an established oedema. In contrast, L-NAME tended to enhance the influx of oesinophils into the airways of Sephadex-instilled animals. L-NAME did not have any effect on the development of oedema in adrenalectomized rats or in animals where formation of glucocorticosteroids (GCS) was inhibited with metyrapone. L-NAME did not however, increase plasma levels of corticosterone. The present results indicate that, in this model, inhibition of NO-synthesis has marked anti-inflammatory effects. The underlying mechanism is complex but seems not to involve prevention of overproduction of NO.

Neuroleptics differentially modulate central dopamine D1-receptor binding.

The effect of the classical neuroleptic, fluphenazine, on dopamine D1-receptor binding was examined in different regions of the basal ganglia. Whereas exposure to fluphenazine for 18 months reduced [125I]SCH-23982 binding to D1-receptors in the caudate putamen, nucleus accumbens and olfactory tubercle, binding in the entopeduncular nucleus was enhanced after fluphenazine treatment. Competition studies indicated that the region-dependent changes in [125I]SCH-23982 binding after fluphenazine exposure were not due to differences in the affinity of fluphenazine or other dopamine ligands for D1-binding sites. These data suggest that in addition to modulating striatal function, classical neuroleptics may also alter neurotransmission in the basal ganglia by enhancing dopamine receptor binding in the entopeduncular nucleus.

Characterization of dopamine receptor binding sites in the subthalamic nucleus.

Experiments were undertaken to determine the distribution and binding profile of dopamine (DA) receptors within a key extra-striatal region of the rat basal ganglia, the subthalamic nucleus (STh). Analysis of [125I]NCQ-298 autoradiograms showed that binding sites of the D2-receptor family are abundant in the STh. Competition studies indicated that these sites were specifically of the D2 subtype. However, contrary to previously published data, [125I]SCH-23982 autoradiograms failed to reveal D1 receptor binding in the STh. These data suggest that DA acting at D2 receptors may directly modulate STh neural activity and furthermore that the antagonism of STh D2 receptor binding by neuroleptics may be involved in the expression of extrapyramidal motor disturbances.

Glutamate inhibits ingestive behaviour.

Male rats treated with reserpine were motionless and ingested only a few of ten consecutive intraoral injections of a 1 M solution of sucrose. While injection of apomorphine, a dopamine agonist, stimulated locomotion and stereotyped sniffing in reserpinized rats, it did not reactivate ingestive responses. The non-competitive N-methyl-D-aspartate receptor antagonist MK801, however, stimulated locomotion as well as ingestion suggesting involvement of glutamate in the suppressive effect of resperpine on ingestive responses. A series of experiments was therefore undertaken to investigate the possible physiological role of glutamate in feeding. For this purpose, we used Grill's intraoral intake test, in which the rat is infused intraorally with a sucrose solution and the amount ingested measured. In untreated rats, MK801 dose-dependently facilitated ingestion of the sucrose solution and antagonized inhibition of ingestion by cholecystokinin octapeptide. Administration of cholecystokinin octapeptide or ingestion of sucrose increased the concentration of glutamate in the nucleus of the solitary tract, a brain stem relay transmitting sensory information from the gastrointestinal tract to the forebrain. MK801 was found to bind specifically to this brain area and block the elevation of glutamate and dopamine levels which occurred after treatment with cholecystokinin octapeptide in this neural site. Together these data suggest that dopamine and glutamate may interact within the nucleus of the solitary tract in controlling ingestive behaviour.

Importance of particulate antigen for the induction of dual bronchial reaction in guinea-pigs.

There has been a lack of small animal models for the secondary allergic response (SAR) seen after bronchial challenge in many asthmatic patients. We have found that challenge with particulate instead of soluble antigen will provoke an SAR-like bronchial obstruction in the guinea-pig. The particulate form was obtained by coupling the antigen covalently to Sepharose beads (approximately 100 microns). Different experiments suggest that SAR is obtained only when the challenge is induced via IgE-mediated mechanisms and when the antigen is sufficiently large to provoke frustrated phagocytosis by the invading inflammatory cells. As judged in lungs sections SAR was related to bronchiolitis.

Biotransformation rate (in vitro) and systemic potency (in vivo) of the topical glucocorticoid budesonide in male and female rats.

Budesonide is a glucocorticoid of clinical interest for the topical treatment of skin and respiratory diseases. The in vitro liver biotransformation rate and in vivo systemic potency (thymus involution) of budesonide were studied in male and female rats. The biotransformation rate of [3H]-budesonide was about 4 times slower in the female than in the male rat liver 9000 g supernatant (t 1/2; 230 and 57 min, respectively). The systemic potency of budesonide after peroral or subcutaneous administration was higher (by factors of 6 and 2, respectively) in the female than in the male rat. These results suggest that the liver biotransformation rate of budesonide is of great importance in reducing its systemic action in the male rat.

Influence of 16 alpha, 17 alpha-acetal substitution and steroid nucleus fluorination on the topical to systemic activity ratio of glucocorticoids.

The use of topical glucocorticosteroid therapy on large skin areas or in the lung is sometimes restricted by the occurrence of unwanted, general corticoid actions owing to a profound systemic absorption. To decrease this risk potent glucocorticoids with an enhanced ratio between their topical and their systemic glucocorticoid potencies are wanted. Therefore, structure-activity studies were performed in rat models to investigate what influences the type of substitution in the 16 alpha, 17 alpha-acetal group and the introduction of fluorine in the 9 alpha- or the 6 alpha, 9 alpha-positions have on the topical and the systemic activities, respectively. The introduction of an unsymmetrical 16 alpha, 17 alpha-acetal group (named acetal type B) markedly enhanced the topical anti-inflammatory potency compared with that of the conventional 16 alpha, 17 alpha-acetonide group (named acetal type A). Both acetal types had a similar systemic glucocorticoid potency, however, 9 alpha-Fluoro and especially 6 alpha, 9 alpha-difluoro substitution, on the other hand, enhanced the systemic glucocorticoid activity more than they raised the topical anti-inflammatory potency. Optimal topical to systemic activity ratio was obtained with a nonhalogenated corticoid of acetal type B structure. This compound, budesonide, had at least the same high topical anti-inflammatory potency as fluocinolone acetonide but was about 10 times less potent than this reference to induce systemic glucocorticoid actions. Its lower systemic activity is probably due to a more rapid biotransformation in the liver.

Development of new glucocorticosteroids with a very high ratio between topical and systemic activities.

The very potent topical anti-inflammatory glucocorticosteroids (GCS) most widely used are either 17 alpha-esters of halogenated 16-methyl-17 alpha-hydroxycorticosteroids (e.g. beclomethasone 17 alpha, 21-dipropionate = BDP) or 16 alpha, 17 alpha-acetals of halogenated 16 alpha, 17 alpha-dihydroxy corticosteroids (e.g. triamcinolone acetonide = TA). The purpose of the present investigation was to increase the ratio between the topical anti-inflammatory (TAIP) and the systemic potencies (SP) of GCS 16 alpha, 17 alpha-acetals, as such compounds are not biotransformed in the lung. Structure-activity investigations in rodents showed that fluoro substituents in positions 6 alpha or 9 alpha or both 6 alpha, 9 alpha 9 alpha increased SP more than TAIP. On the other hand, nonsymmetrical 16 alpha, 17 alpha-acetal substitution increased TAIP more than SP. The best TAIP:SP ratio was obtained with budesonide, which contains this new type of acetal substituent, but has no halogen atoms in the steroid nucleus. In the rat and the mouse budesonide has a 5--10 times better TAIP:SP ratio than 16 alpha, 17 alpha-acetonides, like TA, as well as 17 alpha-ester GCS, like BDP. The improved ratio for budesonide is probably due to a high intrinsic GCS activity at the site of application combined with an effective inactivation by biotransformation after systemic absorption. The importance of the inactivation in the liver was verified by experiments in which the biotransformation capacity of the liver was blocked by SKF-525 A.