Renin-angiotensin system contributes to naive T-cell migration in vivo.

Angiotensin II (Ang II) plays an important role in the regulation of the T-cell response during inflammation. However, the cellular mechanisms underlying the regulation of lymphocytes under physiologic conditions have not yet been studied. Here, we tested the influence of Ang II on T-cell migration using T cells from BALB/c mice. The results obtained in vivo showed that when Ang II production or the AT1 receptor were blocked, T-cell counts were enhanced in blood but decreased in the spleen. The significance of these effects was confirmed by observing that these cells migrate, through fibronectin to Ang II via the AT1 receptor. We also observed a gradient of Ang II from peripheral blood to the spleen, which explains its chemotactic effect on this organ. The following cellular mechanisms were identified to mediate the Ang II effect: upregulation of the chemokine receptor CCR9; upregulation of the adhesion molecule CD62L; increased production of the chemokines CCL19 and CCL25 in the spleen. These results indicate that the higher levels of Ang II in the spleen and AT1 receptor activation contribute to migration of naive T cells to the spleen, which expands our understanding on how the Ang II/AT1 receptor axis contributes to adaptive immunity.

Synthesis and antimycobacterial evaluation of N'-(E)-heteroaromaticpyrazine-2-carbohydrazide derivatives.

A series of nine N'-(E)-heteroaromatic-pyrazine-2-carbohydrazide derivatives (5a-f and 6a-c) have been synthesized and evaluated against M. tuberculosis ATCC 27294 using the micro plate Alamar Blue assay (MABA), being the activities expressed as the minimum inhibitory concentration (MIC) in µg/ml. Compounds 5a and 5f exhibited potent activities (3.12 and 50µg/mL, respectively) when compared to the first line drug pyrazinamide (MIC>100 µg/mL). Afterwards, these compounds were evaluated for their cell viabilities in non-infected and infected macrophages with Mycobaterium bovis Bacillus Calmette-Guerin (BCG) and 5f was not cytotoxic to host cells in the effective concentration to inhibit the growth of M. tuberculosis.

Lipoxin A(4) attenuates zymosan-induced arthritis by modulating endothelin-1 and its effects.

BACKGROUND AND PURPOSE: Lipoxin A(4) (LXA(4)) is a lipid mediator involved in the resolution of inflammation. Increased levels of LXA(4) in synovial fluid and enhanced expression of the formyl peptide receptor 2/lipoxin A(4) receptor (FPR2/ALX) in the synovial tissues of rheumatoid arthritis patients have been reported. Endothelins (ETs) play a pivotal pro-inflammatory role in acute articular inflammatory responses. Here, we evaluated the anti-inflammatory role of LXA(4), during the acute phase of zymosan-induced arthritis, focusing on the modulation of ET-1 expression and its effects. EXPERIMENTAL APPROACH: The anti-inflammatory effects of LXA(4), BML-111 (agonist of FPR2/ALX receptors) and acetylsalicylic acid (ASA) pre- and post-treatments were investigated in a murine model of zymosan-induced arthritis. Articular inflammation was assessed by examining knee joint oedema; neutrophil accumulation in synovial cavities; and levels of prepro-ET-1 mRNA, leukotriene (LT)B(4), tumour necrosis factor (TNF)-α and the chemokine KC/CXCL1, after stimulation. The direct effect of LXA(4) on ET-1-induced neutrophil activation and chemotaxis was evaluated by shape change and Boyden chamber assays respectively. KEY RESULTS: LXA(4), BML-111 and ASA administered as pre- or post-treatment inhibited oedema and neutrophil influx induced by zymosan stimulation. Zymosan-induced preproET-1 mRNA, KC/CXCL1, LTB(4) and TNF-α levels were also decreased after LXA(4) pretreatment. In vitro, ET-1-induced neutrophil chemotaxis was inhibited by LXA(4) pretreatment. LXA(4) treatment also inhibited ET-1-induced oedema formation and neutrophil influx into mouse knee joints. CONCLUSION AND IMPLICATION: LXA(4) exerted anti-inflammatory effects on articular inflammation through a mechanism that involved the inhibition of ET-1 expression and its effects.

Chemical composition and anti-inflammatory activity of copaiba oils from Copaifera cearensis Huber ex Ducke, Copaifera reticulata Ducke and Copaifera multijuga Hayne--a comparative study.

Copaiba oil is an oleoresin obtained from the Copaifera L. genus (Leguminoseae) commonly featured in anti-inflammatory recipe prescribed by Amazonian traditional medical practitioners and featured in Europe and North America pharmacopeias of the past. Chemical and anti-inflammatory activity investigations from the copaiba oils obtained from Copaifera multijuga Hayne, Copaifera cearensis Huber ex Ducke and Copaifera reticulata Ducke species have proved that, although similar, these oleoresins possess varied composition and anti-inflammatory activity. Chromatographic studies showed that the main compound among sesquiterpenes was beta-caryophyllene (57.5, 19.7 and 40.9%, respectively), followed by alpha-humulene, alpha-copaene, alpha-bergamotene, delta-cadinene, with different amounts in each oleoresin. Among the diterpenes, copalic acid was the main component from Copaifera multijuga Hayne (6.2%) and was found in all the oleoresins studied. In Copaifera cearensis Huber ex Ducke, clorechinic (11.3%) and hardwickiic acids (6.2%) were the major diterpenes while kaurenoic (3.9%) and kolavenic acids (3.4%) predominated in Copaifera reticulata Ducke. The pharmacologic effects of the three oleoresins were evaluated in vitro by measuring the NO production by murine macrophages and in vivo using the zymosan induced pleurisy model in mice. The Copaiba Oil from Copaifera multijuga Hayne (100 mg/kg) was the most potent, inhibiting both NO production and the pleurisy induced by zymosan. The oleoresins from Copaifera cearensis Huber ex Ducke and Copaifera reticulata Ducke were also able to inhibit NO production and the pleurisy but with less intensity.

Pharmacological study of anti-allergic activity of Syzygium cumini (L.) Skeels.

Myrtaceae is a plant family widely used in folk medicine and Syzygium and Eugenia are among the most important genera. We investigated the anti-allergic properties of an aqueous leaf extract of Syzygium cumini (L.) Skeels (SC). HPLC analysis revealed that hydrolyzable tannins and flavonoids are the major components of the extract. Oral administration of SC (25-100 mg/kg) in Swiss mice (20-25 g; N = 7/group) inhibited paw edema induced by compound 48/80 (50% inhibition, 100 mg/kg; P

Pharmacological study of anti-allergic activity of Syzygium cumini (L) Skeels

Myrtaceae is a plant family widely used in folk medicine and Syzygium and Eugenia are among the most important genera. We investigated the anti-allergic properties of an aqueous leaf extract of Syzygium cumini (L.) Skeels (SC). HPLC analysis revealed that hydrolyzable tannins and flavonoids are the major components of the extract. Oral administration of SC (25-100 mg/kg) in Swiss mice (20-25 g; N = 7/group) inhibited paw edema induced by compound 48/80 (50 percent inhibition, 100 mg/kg; P <= 0.05) and, to a lesser extent, the allergic paw edema (23 percent inhibition, 100 mg/kg; P <= 0.05). SC treatment also inhibited the edema induced by histamine (58 percent inhibition; P <= 0.05) and 5-HT (52 percent inhibition; P <= 0.05) but had no effect on platelet-aggregating factor-induced paw edema. SC prevented mast cell degranulation and the consequent histamine release in Wistar rat (180-200 g; N = 7/group) peritoneal mast cells (50 percent inhibition, 1 æg/mL; P <= 0.05) induced by compound 48/80. Pre-treatment of BALB/c mice (18-20 g; N = 7/group) with 100 mg/kg of the extract significantly inhibited eosinophil accumulation in allergic pleurisy (from 7.662 ± 1.524 to 1.89 ± 0.336 x 10(6)/cavity; P <= 0.001). This effect was related to the inhibition of IL-5 (from 70.9 ± 25.2 to 12.05 ± 7.165 pg/mL) and CCL11/eotaxin levels (from 60.4 ± 8.54 to 32.8 ± 8.4 ng/mL) in pleural lavage fluid, using ELISA. These findings demonstrate an anti-allergic effect of SC, and indicate that its anti-edematogenic effect is due to the inhibition of mast cell degranulation and of histamine and serotonin effects, whereas the inhibition of eosinophil accumulation in the allergic pleurisy model is probably due to an impairment of CCL11/eotaxin and IL-5 production.

Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice.

OBJECTIVE: We investigated the antiinflammatory properties of a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. MATERIAL AND METHODS: Zymosan-induced arthritis and pleurisy in Swiss and C57/Bl6 mice (n = 10 per group). Western blot analysis was performed to analyze nuclear factor-kappaB (NFkappaB) translocation in mice peritoneal macrophages stimulated in vitro with zymosan (500 microg/ml). ELISA was performed to evaluate cytokine levels in knee joints. Values of p

Investigations on the anti-inflammatory and anti-allergic activities of the leaves of Uncaria guianensis (Aublet) J. F. Gmelin.

Uncaria guianensis (Aublet) J. F. Gmelin is an herbal medicine from tropical areas of South and Central America. We investigated the anti-inflammatory and anti-allergic properties of an ethanolic extract of U. guianensis leaves, containing alkaloids, flavonoids and phenol carboxylic acids, as revealed by thin layer chromatography (TLC). Oral pre-treatment with U. guianensis inhibited zymosan-induced paw oedema (500 mg/paw) and pleural exudation (100 mg/kg) within 4 h (25-200 mg/kg). U. guianensis (100 mg/kg) inhibited total leukocyte and neutrophil numbers in the pleural cavity 4 h after zymosan stimulation. Pre-treatment with U. guianensis (100 mg/kg, p.o.) inhibited total leukocyte, neutrophil and eosinophil recruitment into the pleural cavity 24 h after LPS (250 ng/cavity, i.t.). Pre-treatment with U. guianensis inhibited paw oedema (25-200 mg/kg) induced by ovalbumin (OVA) within 1 h, and neutrophil and eosinophil recruitment into the mice pleural cavity 24 h after OVA (100 mg/kg). In vitro data revealed that U. guianensis impaired LPS-induced nitric oxide and CXCL8 generation by murine peritoneal macrophages, as well as OVA-induced interleukin-5 synthesis by previously sensitized spleen cells. These results demonstrate that U. guianensis leaves provide effective anti-inflammatory and anti-allergic activities.

Anti-inflammatory and anti-ulcerogenic properties of Stachytarpheta cayennensis (L.C. Rich) Vahl.

In the present work, the anti-inflammatory and gastroprotective properties of ethanolic extracts of Stachytarpheta cayennesis (L.C. Rich) Vahl (Verbenaceae) were assessed. Chromatographic analysis of the crude ethanolic extract, SC01, revealed high concentrations of the iridoid ipolamiide, whereas the SC02, the second ethanolic extract, presented the arylpropanoid verbacoside as a major constituent. The oral administration of SC01 (100 mg/kg) into Swiss mice failed to inhibit paw oedema and pleural exudation induced by carrageenan and zymosan, whereas SC02 (100 mg/kg, p.o.) inhibited oedema and protein extravasation in all instances. Both extracts inhibited total leukocyte accumulation into the pleural cavity 4 and 24h after the intrathoracic (i.t.) injection of carrageenan, due to the inhibition of neutrophil and mononuclear cell influx, whereas only SC02 was able to inhibit leukocyte mobilization induced by zymosan (100 microg/cavity, i.t.). SC02 inhibited LPS (250 ng/cavity)-induced total leukocyte, neutrophil and eosinophil accumulation in the pleural cavity, whereas SC01 selectively inhibited neutrophil influx. In addition, our data indicates that the extract SC02 presents an important anti-ulcerogenic activity, since it inhibited diclofenac-induced (100 mg/kg, p.o.) gastric ulcera. Overall, these data provide evidence for the anti-inflammatory and gastroprotective properties of Stachytarpheta cayennensis, supporting its use in folk medicine for such purposes.

Anti-allergic effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on allergen-induced vascular permeability and hyperalgesia.

OBJECTIVE: We investigated the anti-allergic and analgesic properties of an oil and a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. MATERIALS AND METHODS: Pleurisy, paw and ear edema were induced in Swiss and C57/Bl10 mice mice, whereas thermal hyperalgesia was assessed in Wistar rats (n = 6-10 per group). Values of p < 0.05 were regarded as significant. RESULTS: C. guianensis oil (100 to 400 mg/kg, p.o.) and TNTP (12.5 to 100 mg/kg, p.o.) inhibited pleural exudation, paw and ear edema induced by ovalbumin (OVA) in sensitized mice. TNTP (12.5 to 100 mg/kg, p.o.) also inhibited paw edema induced by histamine, PAF and bradykinin. TNTP (100 mg/kg, p.o.) inhibited prostaglandin E(2) generation in the pleural cavity in response to antigenic challenge. Moreover, C. guianensis oil (100 to 400 mg/kg) and TNTP (12.5 to 100 mg/kg) decreased OVA- and histamine-induced hyperalgesia. CONCLUSION: Taken together, these findings demonstrate the anti-edematogenic and analgesic effects of C. guianensis oil, and points out TNTP as the responsible bioactive compounds.

Effect of Mycobacterium leprae lipids on BCG- and carrageenan-induced cellular recruitment in mouse pleurisy.

Pathogenic mycobacteria survive inside macrophages and deactivate these cells, using a mechanism that is still poorly understood. Mycobacterial cell wall lipids constitute the first contact with the host cell. Although Mycobaterium leprae and M. bovis BCG share common antigens, they induce opposite inflammatory responses. Apolar M. leprae lipids have been shown to be anti-inflammatory by down-regulating macrophage activation and T-cell functions. We wonder if these lipids would influence cellular migration to BCG or to other inflammatory agent. We investigated the effect of M. leprae, its lipids or delipidated bacteria on acute and chronic BCG- or carrageenan-induced pleurisy. Previous injection of intact or delipidated M. leprae did not alter either the BCG- or carrageenan-induced pleural inflammatory reaction. However, M. leprae lipids enhanced carrageenan-induced acute cellular migration without impairing BCG inflow; moreover, they reduced BCG chronic response. Together these data suggest distinct mechanisms for intracellular deactivation and pleural cell recruitment exerted by mycobacterial structures.

Evaluation of anti-inflammatory activity of essential oils from two Asteraceae species.

The essential oils from two Asteraceae species, Porophyllum ruderale (PR) and Conyza bonariensis (CB) were screened for anti-inflammatory activity in the mouse model of pleurisy induced by zymosan (500 microg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity). The main monoterpene constituents of each oil, beta-myrcene (in PR) and limonene (in CB), were tested in the LPS-induced pleurisy model and assayed also for immunoregulatory activity by measurement of the inhibition of NO and production of the cytokines, gamma-interferon and IL-4. The oils, when administered orally, were able to inhibit the LPS-induced inflammation including cell migration; with a similar effect being observed for pure limonene. Pure beta-myrcene and limonene were also effective in inhibiting production of nitric oxide at doses below the cytotoxicity of these monoterpenes. A significant inhibition of gamma-interferon and IL-4 production by limonene and beta-myrcene was also observed.

Mechanisms of allergen- and LPS-induced bone marrow eosinophil mobilization and eosinophil accumulation into the pleural cavity: a role for CD11b/CD18 complex.

OBJECTIVE: The mechanisms involved in bone marrow eosinophil emigration and recruitment to inflammatory sites are not fully understood. The involvement of CD11b/CD18 in marrow eosinophil release induced by lipopolysaccharide (LPS) or allergen was investigated in mice. METHODS: Eosinophil and neutrophil counts in the pleural cavity, blood and bone marrow were performed at different time intervals after the intrathoracic injection of LPS (250 ng/cavity) or ovalbumin (OVA, 12 microg/cavity; into actively sensitized mice) and compared to anti-CD11b/CD 18 (5C6, 1 mg/mouse) or anti-IL-5 (TRFK-5, 500 microg/kg) treated mice. RESULTS: LPS induced local eosinophil influx, that peaked within 24 h and that was preceded by a decrease in marrow eosinophils at 4 h. Antigenic challenge induced a decrease in marrow eosinophils within 4 h, followed by a long lasting pleural eosinophil accumulation and a persistent increase in marrow eosinophil numbers. Pretreatment with anti-CD11b/CD18 abolished LPS-induced neutrophil and eosinophil accumulation in the pleural cavity at 4 and 24 h, respectively. This pretreatment failed to modify neutrophil emigration from bone marrow, but significantly inhibited marrow eosinophil release at 4 h post-LPS or OVA challenge. Anti-IL-5 pretreatment failed to inhibit LPS-induced pleural eosinophil accumulation and mobilization from bone marrow, but it abolished allergen-induced effects, indicating a role for IL-5 in marrow eosinophil mobilization induced by antigen, but not by LPS challenge. CONCLUSIONS: Our results suggest that eosinophil migration induced by antigen or LPS into the pleural cavity is preceded by bone marrow eosinophil release through a mechanism that depends on CD11b/CD18.

Distinct ability to accumulate eosinophils during the inflammatory cellular response to M. bovis BCG in the mouse pleural cavity.

OBJECTIVE AND DESIGN: The host response to Mycobacteria focuses on the development of cell-mediated immunity and granuloma formation. Here, we investigated the onset of cellular responses to mycobacteria in murine pleurisy. MATERIAL: Distinct mouse strains previously described as Bcg susceptible or resistant were inoculated intrathoracically with different doses of live M. bovis BCG. METHODS: At various time intervals, cells harvested from the inflammatory site were identified and ultra-structurally analysed. RESULTS: BCG-induced pleurisy had two peaks of cellular influx at 1 and 15 days after infection. At the first half hour, macrophages were found to be heavily infected. Neutrophil arrival started after 2 h of infection and peaked at 4 h. At this time, neutrophils were found ingesting mycobacteria exclusively with a high infecting dose. BCG was potently more eosinophilotactic in Bcg susceptible mice than in the resistant ones and to other well known eosinophilia inducers: IL-5, PAF-acether or LPS. CONCLUSIONS: Mycobacterial load and mouse susceptibility seem to determine the early granulocyte dynamics in the lesion.

Participation of endogenous endothelins in delayed eosinophil and neutrophil recruitment in mouse pleurisy.

OBJECTIVE: Investigate the role of endothelins in leukocyte recruitment in allergic and non allergic inflammation. METHODS: Pleurisy was induced in mice by intrathoracic injection of ovalbumin (OVA; in sensitized animals), E. coli LPS, carrageenan, Mycobacterium bovis (BCG) or zymosan. Animals were treated with BQ-123 or BQ-788 (1.5-150 pmol/cavity), or intravenously with bosentan (30 mg/kg). RESULTS: None of the ET receptor antagonists modified early neutrophil recruitment (at 4 h) induced by OVA, LPS, carrageenan, BCG or zymosan or plasma leakage caused by carrageenan or zymosan. Mononuclear and eosinophil accumulation triggered by OVA were reduced by BQ-123 (150 pmol/cavity) or bosentan (68 and 43% inhibition of eosinophilia), but unaffected by BQ-788. BQ-123 and bosentan also inhibited LPS increases in neutrophil (by 67 and 40%) and eosinophil (by 63 and 74%) at 24 h. CONCLUSIONS: Endothelins, acting via ETA receptors, play a role in late eosinophil and neutrophil accumulation (24 h), but not in the acute (4 h) neutrophilic response.

Opposite effects of M. leprae or M. bovis BCG delipidation on cellular accumulation into mouse pleural cavity. Distinct accomplishment of mycobacterial lipids in vivo.

OBJECTIVES AND DESIGN: The effect of mycobacterial lipids on the onset of the early acute inflammatory response in BALB/c mice pleurisy was investigated. MATERIALS AND METHODS: Intact Mycobacterium leprae and Mycobacterium bovis BCG (BCG), their lipids, and delipidated mycobacteria were used to evaluate total leukocytes and cell types migrated to the pleural cavity (8 animals/experimental group). RESULTS: BCG Moreau (x10(-6)/cavity), delipidated BCG and its lipids gradually recruited cells leading to arrival, respectively, of neutrophils (7.8+/-1.9, 4.7+/-0.9, 1.8+/-0.25) followed by mononuclear cells (4.8+/-0.8, 3.7+/-0.7, 2.45+/-0.22) and eosinophils (0.39+/-0.08, 0.32+/-0.11, 0.41+/-0.65). BCG delipidation decreased the number of migrated total leukocytes (ANOVA, and Newman-Keuls-Student-test), whereas M. leprae delipidation accumulated neutrophils (0.85+/-0.01) and eosinophils (1.65+/-0.18). CONCLUSIONS: Intact M. leprae and its lipids did not incite any cell recruitment. Apolar external cell wall lipids from M. leprae and BCG induce different cellular responses. They seem to have a crucial importance at the first contact of mycobacteria with the host cell, modulating the influx of neutrophils/macrophages in the early (4/24 h) onset of the inflammatory reaction.

Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium.

The resins and leaves of species of Protium are commonly used by folk medicine. In the present study, we analyse the pharmacological effects of essential oils obtained by steam distillation (leaves and resin) from Protium species. Analysis by gas chromatography (GC) coupled to mass spectrometry and retention indices calculations demonstrate that the resin oil is constituted mainly of monoterpenes and phenylpropanoids: alpha-terpinolene (22%), p-cymene (11%), p-cimen-8-ol (11%), limonene (5%) and dillapiol (16%), whereas sesquiterpenes predominate as the volatile constituents of the leaves. The resin of Protium heptaphyllum (PHP) and leaves of P. strumosum (PS), P. grandifolium (PG), P. lewellyni (PL) and P. hebetatum (PHT) were screened for anti-inflammatory activity by the use of mouse pleurisy model induced by zymosan (500 microg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity), for antinociceptive effect (by means of preventing mice abdominal writhings), as well as NO production from stimulated macrophages and proliferation of neoplasic cell lines: Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma) and J774 (mouse monocytic cell line). The oils from PHP, PS and PL were able to inhibit protein extravasation but no sample inhibited total or differential leucocyte counts after administrating p.o. (100 mg/kg) 1 h before stimulation with zymosan. The oils from PG, PL and PHT inhibited neutrophil accumulation whereas PHP and specially PL inhibited LPS-induced eosinophil accumulation in mouse pleural cavity. PHT was also able to inhibit mononuclear cells accumulation. Antinociceptive effect was not observed, when animals received oral administration of the essential oils (100 mg/kg). In vitro treatment with essential oils (100 microg/well) changed the NO production from stimulated mouse macrophages. PHP inhibited in 74% and PS in 46% the LPS-induced NO production. In contrast, treatment with PL was able to increase in 49% the NO production. Cell lines proliferation was affected by the oils assayed in the range of 60-100% for Neuro-2a, 65-95% for SP2/0 and 70-90% for J774. Taken together these results showed that essential oils could be useful as efficient pharmacological tools.

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins.

1. The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS-induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. 2. Intrathoracic (i.t.) injection of LPS (250 ng cavity(-1)) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des-Arg9-[leu-8]BK (0.025 and 0.25 nmol cavity(-1)) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity(-1)) or HOE 140 (2.5 nmol cavity(-1)), increased LPS-induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg(-1) i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down-regulating LPS-induced eosinophil accumulation. 3. BK administered at 15 and 25 nmol cavity(-1), i.t. or i.p. also inhibited LPS-induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity(-1). Nevertheless, when injected at doses of 50 and 100 nmol cavity(-1) BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. 4. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity(-1) i.p., significantly inhibited the eosinophil influx induced by LPS. 5. The mechanism by which B2 receptor agonists inhibit LPS-induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase-2 inhibitor, NS-398. Pretreatment with either indomethacin or NS-398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS-induced eosinophil influx in Tyr8BK (0.25 nmol cavity(-1)) injected mice. 6. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinphil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

Lymphocyte activation and cytokine production by Pisum sativum agglutinin (PSA) in vivo and in vitro.

Mice spleen cells were incubated in vitro for 24 h with Pisum sativum agglutinin (PSA). The addition of these supernatants (SN) to macrophage cultures induced the production of nitric oxide (NO) by these cells in a dose-dependent manner. NO release was blocked in the presence of IFN gamma antibodies and partially inhibited by TNF alpha antibodies. The ability of PSA in inducing the production of IFN gamma and TNF alpha by spleen lymphocytes was confirmed assaying these cytokine levels in the SN. Spleen cells stimulated in vitro with PSA were highly activated showing an increased expression of the earlier activation marker, CD69, and a great proliferative response. On the other hand, spleen cells obtained from mice treated with PSA 24 h earlier, did not produce significant levels of IFN gamma or TNF alpha when incubated in vitro and showed a significantly lower proliferation rate when pulsed in vitro with PSA or Concanavalin A (ConA). The lower responsiveness to mitogens was also evident after 48 and 72 h after the treatment in vivo with the lectin. Nevertheless, the flow cytometric analysis of spleen lymphocytes obtained from PSA-treated animals showed a high degree of activation in cells CD3+. There was a decrease in the expression of L-selectin and VLA-4, when compared to controls, in parallel with a significant increase in the expression of CD69 and CD122 (IL-2R) in lymphocytes recovered from PSA-injected animals. The data point to evidence that PSA induces immunomodulatory effects, activating spleen lymphocytes in vivo, which become unresponsive to a second stimulation in vitro.

Opposite cellular accumulation and nitric oxide production in vivo after pleural immunization with M. leprae or M. bovis BCG.

Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.