Lidocaine-derivative JMF2-1 prevents ovalbumin-induced airway inflammation by regulating the function and survival of T cells.

BACKGROUND: Inhalation of the local anaesthetic lidocaine has been suggested to be beneficial for asthmatics, but airway anaesthesia is unpleasant and may exacerbate bronchoconstriction. Our previous study showed that inhalation of the lidocaine analogue JMF2-1 can elicit the anti-inflammatory properties of lidocaine without anaesthesia. This prompted further research on the mechanism of action and putative therapeutic application of JMF2-1. OBJECTIVE: We tested the hypothesis that JMF2-1 would prevent allergen-induced lung inflammation and airway hyperresponsiveness (AHR) by modulating T cell function in vivo and in vitro. Methods Local and systemic changes in leucocyte levels, cytokine production and lung mechanics were examined in a murine model of lung inflammation. JMF2-1 (0.05-2%) or saline was aerosolized twice a day during the ovalbumin (OVA)-provocation period (19-21 days post-sensitization). Analyses were performed 24 h after the final challenge. Primary cultured lymph node cells were used to assess the effects of JMF2-1 (100-600 µm) at the cellular level. RESULTS: OVA challenge resulted in lung recruitment of CD4(+) T cells and eosinophils, increased generation of inflammatory cytokines and AHR to inhaled methacholine within 24 h. These changes were prevented by JMF2-1 nebulization, and occurred in parallel with an increase in the number of apoptotic cells in the lung. JMF2-1 treatment did not alter levels of CD4(+) or CD8(+) T cells in the thymus or lymph nodes of naïve mice, although it inhibited OVA-induced IL-13 production and the lymphocyte proliferative response in vitro. It also induced apoptosis of OVA-activated lymphocytes in a mechanism sensitive to z-VAD, indicating that JMF2-1 mediates caspase-dependent apoptosis. CONCLUSION: Inhalation of JMF2-1 prevents the cardinal features of asthma by reducing T(H) 2 cytokine generation and lung eosinophilic inflammatory infiltrates via local inhibition of T cell function and survival. JMF2-1 may represent a novel therapeutic alternative for asthma control with distinct advantages over local anaesthetics.

Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme.

1. This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti-eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the beta2-agonist salbutamol was also assessed. 2. Sensitized guinea-pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg(-1), i.p.) and RP 73401 (5 mg kg(-1), i.p.) treatments. In contrast, only rolipram inhibited eotaxin production. 3. Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 microg cavity(-1)) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti-mouse eotaxin antibody (0.5 mg kg(-1)) significantly reduced allergen-evoked eosinophilia in this model. 4. Local pretreatment with rolipram (40 microg cavity(-1)) or RP 73401 (40 microg cavity(-1)) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen-evoked eosinophil recruitment in rats. 5. Local injection of beta2-agonist salbutamol (20 microg cavity(-1)) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination. 6. Treatment with rolipram and RP 73401 dose-dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone. 7. Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen-induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.

Selective inhibition of phosphodiesterase type IV suppresses the chemotactic responsiveness of rat eosinophils in vitro.

Previous studies demonstrated that the selective inhibition of phosphodiesterase type IV suppresses antigen-induced eosinophil infiltration and also downregulates certain eosinophil functions assessed in vitro. In the current study, we compared the effect of selective inhibitors of phosphodiesterase IV with the effect of phosphodiesterase III and V inhibitors, focusing on eosinophil chemotaxis stimulated by platelet-activating factor (PAF) and leukotriene B4 in a modified Boyden chamber. The effect of beta 2-adrenoceptor agonists and forskolin as well as the analogue N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2 cyclic AMP) was also determined. For this purpose eosinophils were obtained by lavage of the peritoneal cavity of normal Wistar rats and purified on Percoll gradients to 85-95% purity. Our results showed that PAF and leukotriene B4 (0.001-10 microM) elicited a concentration-dependent increase in eosinophil migration with maximal responses observed at 1 microM and 0.1 microM respectively. Pre-incubation with the type IV phosphodiesterase inhibitor, rolipram (1-100 microM), suppressed the chemotactic response triggered by PAF and leukotriene B4, in association with elevation of eosinophil cyclic AMP, whereas the compounds milrinone and SK&F 94836 (type III selective) as well as zaprinast (type V selective) were ineffective. The beta 2-adrenoceptor agonists salbutamol and salmeterol (1-100 microM) did not alter the intracellular levels of cyclic AMP and also failed to inhibit the eosinophil response. Moreover, incubation of eosinophils with the adenylate cyclase activator forskolin (1-100 microM), while inducing a discrete increase in cyclic AMP, markedly inhibited PAF- and leukotriene B4-induced eosinophil chemotaxis. Eosinophils treated with a combination of individually inactive amounts of forskolin plus rolipram significantly inhibited the eosinophil migration elicited by PAF and leukotriene B4, but did not change cyclic AMP baseline levels. Though only at the highest concentration tested (100 microM), the analogue Bt2 cyclic AMP abolished the eosinophil chemotaxis. Thus we conclude that the direct inhibitory effect of phosphodiesterase IV inhibitors on eosinophil chemotaxis may account for their suppressive activity on tissue eosinophil accumulation following antigen challenge.

Local exposure to salbutamol or Bt2 cyclic AMP inhibits pleural exudation and leukocyte influx caused by antigen in rats.

The local effect of salbutamol and N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2 cyclic AMP) on the rat pleural inflammation caused by allergen was investigated. Antigen (ovalbumin, 12 micrograms/cavity) intrathoracically administered to immunized rats led to a marked pleural protein extravasation and leukocyte infiltration, as attested by the quantification of protein and enumeration of leukocytes recovered from the pleural cavity. Salbutamol (10-40 micrograms/cavity) and the cell-permeable cyclic AMP analogue, Bt2 cyclic AMP (20-160 micrograms/cavity), injected 1 h and 5 min before the antigen, respectively, inhibited the exudation occurring within 30 min, and neutrophil and eosinophil accumulation occurring 4 and 24 h, respectively. The late eosinophilia was also markedly attenuated by salbutamol administered 10 min post-challenge, when mast cells had already been degranulated. Pretreatment with the beta-adrenoceptor antagonist propranolol (1 mg/kg, i.v.) failed to modify the inhibitory effect of Bt2 cyclic AMP, but abolished the blockade caused by salbutamol of leukocyte infiltration under conditions where the salbutamol anti-exudatory activity was impaired to about 80%. In another set of experiments, salbutamol (20 and 40 micrograms/cavity) markedly inhibited the exudation caused by histamine and 5-hydroxytryptamine (5-HT) which, though to a lesser extent, was also sensitive to Bt2 cyclic AMP (80 micrograms/cavity). As observed with allergic pleurisy, propranolol impaired the inhibition by salbutamol of histamine- and 5-HT-induced exudation, whereas the Bt2 cyclic AMP inhibition was not affected. We conclude that salbutamol and Bt2 cyclic AMP share the ability to inhibit pleural exudation and leukocyte recruitment caused by allergen in immunized rats, suggesting that the anti-inflammatory effect of salbutamol may be mediated by a cyclic AMP signaling pathway, probably via beta 2-adrenoceptor activation.

Association of leprosy with HLA-DR2 in a Southern Brazilian population.

The association between HLA specificities and leprosy was investigated in a southern Brazilian population. One hundred and twenty-one patients and 147 controls were typed for HLA-A, B, Cw, DR and DQ. Patients were subdivided into the following subgroups, according to clinical, histological and immunological criteria: lepromatous (N = 55), tuberculoid (N = 32), dimorphous (N = 20), and indeterminate (N = 14). The frequencies of HLA specificities were compared between the total group of patients and controls, and between the same controls and each subgroup of patients. After correction of the probabilities, deviations, were not significant, except for the DR2 specificity, which presented a frequency of 44.2% in the total group of patients and 56.3% in the subgroup of individuals with the tuberculoid form of the disease, compared to 23.3% in the controls. Stratified analysis showed that the increased DR2 frequency in the total group of patients was due to the subgroups with tuberculoid and dimorphous forms. The relative risk of tuberculoid leprosy for DR2-positive individuals was 4.2, and the etiologic fraction of DR2 was 0.429. In conclusion, a positive association of the DR2 specificity with the tuberculoid form of leprosy, but not with the lepromatous, dimorphous, or indeterminate forms, was demonstrated in this Southern Brazilian population.

Antigen-induced pleural eosinophilia is suppressed in diabetic rats: role of corticosteroid hormones.

Previous studies have evidenced for the existence of interactive regulatory mechanisms between insulin and steroid hormones in different systems. In this study, we have investigated whether endogenous corticosteroids could be implicated in the hyporeactivity to antigen challenge observed in sensitized diabetic rats. Alloxinated rats showed a long-lasting increase in the blood glucose levels and a reduction in the number of pleural mast cells at 48 and 72 hr, but not at 24 hr after alloxan administration. In parallel, they also showed a significant elevation in the plasma levels of corticosterone together with an increase in the adrenal/body weight ratio. Antigen-evoked eosinophil accumulation appeared significantly reduced in rats pretreated with dexamethasone as well as in those rendered diabetic 72 hr after alloxan. In the same way, naive animals treated with dexamethasone also responded with a significant decrease in the number of pleural mast cells. Interestingly, when sensitized diabetic rats were pretreated with the steroid antagonist RU 38486 a reversion of the reduction in the allergen-induced eosinophil accumulation was noted. We conclude that the down-regulation of the allergic inflammatory response in diabetic rats is close-related to reduction in mast cell numbers and over expression of endogenous corticosteroids.

Mechanism underlying acute resident leukocyte disappearance induced by immunological and non-immunological stimuli in rats: evidence for a role for the coagulation system.

OBJECTIVE: To investigate the involvement of the fibrinogen-fibrin system in the acute reduction of the resident leukocyte population following pleural inflammation. METHODS: Sensitized and naive rats were injected intrapleurally (i.pl.) with antigen (ovalbumin) and platelet-activating factor (PAF) or bradykinin, respectively. Heparin (0.25 U/cavity), EDTA (80 microg/cavity) and hirudin (1 U/cavity) were injected locally 5 min before challenge, whereas fucoidin was injected intraperitoneally 30 min before stimulation. RESULTS: Antigen challenge led to a rapid reduction in the number of resident leukocytes 30 min post-challenge (from 7.7 +/- 0.4 x 10(6) cells/cavity to 2.3 +/- 0.2 x 106 cells/cavity, n = 6, p < 0.001). The pleural stimulation of naive rats with either PAF or bradykinin also led to a significant decrease in the pleural leukocyte population, which occurred in parallel with the formation of a fibrin meshwork containing captured cells, as attested by electron microscopy. Heparin prevented the drop in the total leukocyte numbers, without modifying either plasma leakage or histamine release at 30 min or the subsequent neutrophil and eosinophil infiltration noted 4 and 24 h post-challenge, respectively. Similarly, hirudin and EDTA prevented the antigen-induced leukocyte disappearance reaction. Heparin also impaired the drop in the pleural leukocyte numbers evoked by PAF and bradykinin. CONCLUSION: Our data show that the pleural resident cell disappearance phenomenon noted early after inflammatory provocation depends on the activation of the fibrinogen-fibrin system, and is not required for the subsequent leukocyte recruitment.

Alloxan diabetes reduces pleural mast cell numbers and the subsequent eosinophil influx induced by allergen in sensitized rats.

Alloxan damages insulin-producing cells and has been used as an inducer of experimental diabetes in several animal species. In this study, administration of alloxan (40 mg/kg, i.v.) to rats was followed by a selective and time-dependent reduction in the number of pleural mast cells (50 +/- 2.2%, p < 0.01; mean +/- SEM), while mononuclear cell and eosinophil counts were not altered. As compared to naive rats, the reduction in mast cell numbers was first noted 48 h following alloxan administration and remained unaltered for at least 60 days. It is noteworthy, that the depletion in the mast cell population was not accompanied by alterations in the total amount of histamine stored per cell. Sensitized rats turned diabetic by alloxan treatment performed 72 h before challenge showed a less pronounced antigen-induced mast cell degranulation compared to nondiabetic rats. Moreover, rats injected with alloxan 72 and 48 but not 24 h before challenge, reacted to allergenic challenge with 50% reduction in the number of eosinophils recruited to the pleural cavity within 24 h. We found that the less pronounced eosinophil accumulation did not relate to an intrinsic cell locomotor abnormality since eosinophils from diabetic rats presented similar chemotactic responses to LTB4 and PAF in vitro as compared to matching controls. Insulin (3 IU/rat) restored basal levels of mast cells and reversed the subsequent inhibition of allergen-induced pleural eosinophilia, suggesting a causative relationship between these phenomena. Treatment with insulin also significantly increased the number of mast cells in the pleural cavity of naive rats (from 637 +/- 57 to 978 +/- 79 x 10(3) cells/cavity, p < 0.001). Consistently, previous depletion of mast cells by means of local treatment with compound 48/80 significantly reduced the antigen-induced eosinophil recruitment in sensitized animals. We conclude that the reduction in the pleural mast cell population noted in alloxan-treated rats could be directly implicated in the diminished pleural eosinophil influx following allergen challenge. This hyporesponsiveness is independent of an intrinsic abnormality of cell chemotaxis, but can be imitated by local mast cell depletion.

Cyclooxygenase-2-derived prostaglandin E2 and lipoxin A4 accelerate resolution of allergic edema in Angiostrongylus costaricensis-infected rats: relationship with concurrent eosinophilia.

In noninfected rats, challenge with allergen following local IgE sensitization induced a pleurisy marked by intense protein exudation that plateaued from 30 min to 4 h after challenge, reducing thereafter. Infection of rats with Angiostrongylus costaricensis induced a 5-fold increase in blood eosinophil numbers by 25 days postinfection, whereas the numbers of eosinophils in the pleural cavity ranged from normal to a weak increase. In infected rats, identically sensitized, challenge with Ag induced a much shorter duration of pleural edema with complete resolution by 4 h, but no change in the early edema response. In parallel, infection increased the number of eosinophils recovered from the pleural cavity at 4 h, but not at 30 min, following allergen challenge. Pretreatment with IL-5 (100 IU/kg, i.v.) also increased eosinophil numbers in blood and, after allergen challenge, shortened the duration of the pleural edema and increased pleural eosinophil numbers. There were increases in the levels of both PGE2 and lipoxin A4 (LXA4) in pleural exudate. Selective cyclooxygenase (COX)-2 inhibitors, NS-398, meloxicam, and SC-236, did not alter pleural eosinophilia, but reversed the curtailment of the edema in either infected or IL-5-pretreated rats. Pretreatment of noninfected animals with the PGE analogue, misoprostol, or two stable LXA4 analogues did not alter the magnitude of pleural exudation response, but clearly shortened its duration. These results indicate that the early resolution of allergic pleural edema observed during A. costaricensis infection coincided with a selective local eosinophilia and seemed to be mediated by COX-2-derived PGE2 and LXA4.

Anti-allergic properties of the natural PAF antagonist yangambin.

In this study we examined the ability of the furofuran lignan yangambin to influence the local and systemic responses induced by antigen or PAF in actively sensitized or normal rats. Given intraperitoneally 1 h before stimulation, yangambin inhibited the pleural neutrophil and eosinophil infiltration evoked by the i.pl. injection of PAF or antigen into normal or 14 daysensitized rats whereas plasma exudation evoked by both stimuli was unaffected. The pleural neutrophil influx (6 h) after LTB4 stimulation was also significantly inhibited by yangambin. We also evidenced that the hemoconcentration, thrombocytopenia, and leucocytosis noted after i.v. PAF were all attenuated by yangambin. In actively sensitized rats, pretreatment with yangambin failed to modify the antigen-induced hemoconcentration and leucocytosis, but dose-dependently abrogated the thrombocytopenia noted 1 h post-stimulation. In vitro, the anaphylactic contraction of longitudinal jejunal segments to antigen challenge was significantly inhibited by yangambin (10(-5)-10(-4) M). Likewise, the contraction of jejunal segments from normal rats to PAF was markedly blocked by yangambin under conditions where the response to 5-hydroxytryptamine (5-HT) was not altered. In conclusion, our results show that antigen- and PAF-induced pleural neutrophil and eosinophil accumulation, but not exudation, is sensitive to treatment with yangambin. In addition, yangambin also suppressed the pleural neutrophil infiltration triggered by LTB4 as well as the blood thrombocytopenia and intestinal anaphylaxis elicited by antigen in rats. Thus, our findings indicate that yangambin shows an antagonistic action on receptors other than those of PAF, i.e., LTB4, and strongly suggest that it may be a useful drug in the treatment of some allergic inflammatory responses.

Kinetics of eosinophil and IgE-mast cell changes following infection with Angiostrongylus costaricensis in Wistar rats.

Human abdominal angiostrongyliasis is a severe eosinophilic disease caused by Angiostrongylus costaricensis. Previous studies have demonstrated that wild rodents are critically involved as definitive hosts to this nematode in nature. In this study, we have evaluated the susceptibility of Wistar rats (Rattus norvegicus) to A. costaricensis infection. Kinetics of parasitological and pathological changes, including the number of adult worms recovered from mesenteric arteries, and of IgE, mast cell and eosinophil levels in several compartments have been assessed. The oral inoculation of third-stage larvae (L3) into adult Wistar rats led to a marked accumulation of worms in the branches of the mesenteric arteries 25 and 50 days post-inoculation. Intense bone marrow eosinophilia ranging from 7 to 50 days was accompanied by marked accumulation of eosinophils in the blood, peritoneal and bronchoalveolar spaces. Eosinophilic periarteritis, oedema and granuloma in the intestinal and lung tissues were also histologically evident. Total serum IgE and specific anti-parasite IgE peaked at 25 days post-infection, as measured by ELISA and by the passive cutaneous anaphylaxis test, respectively. At that time point, there was a drastic reduction in the number of intact mast cells in the peritoneal effluent. These findings indicate that Wistar rats are permissive to A. costaricensis infection. IgE-mast cell activation and massive tissue eosinophil infiltration are marked features in the process and are likely to play a crucial role in the immune-response evoked by this parasite.

Association of leprosy with HLA-DR2 in a Southern Brazilian population

The association between HLA specificities and leprosy was investigated in a Southern Brazilian population. One hundred and twenty- one patients and 147 controls were typed for HLA-A, B, Cw, DR and DQ. Patients were subdivided into the following subgroups, according to clinical, histological and immunological criteria: lepromatous (N = 55), tuberculoid (N = 32), dimorphous (N = 20), and indeterminate (N = 14). The frequencies of HLA specificities were compared between the total group of patients and controls, and between the same controls and each subgroup of patients. After correction of the probabilities, deviations were not significant, except for the DR2 specificity, which presented a frequency of 44.2 per cent in the total group of patients and 56.3 per cent in the subgroup of individuals with the tuberculoid form of the disease, compared to 23.3 per cent in the controls. Stratified analysis showed that the increased DR2 frequency in the total group of patients was due to the subgroups with the tuberculoid and dimorphous forms. The relative risk of tuberculoid leprosy for DR2-positive individuals was 4.2, and the etiologic fraction of DR2 was 0.429. In conclusion, a positive association of the DR2 specificity with the tuberculoid form of leprosy, but not with the lepromatous, dimorphous, or indeteterminate forms, was demonstrated in this Southern Brazilian population.