Selective blockade of lymphopoiesis induced by kalanchosine dimalate: inhibition of IL-7-dependent proliferation.

Lymphopoiesis and myelopoiesis continuously generate mature cells from hematopoietic cell progenitors during the lifetime of the organism. The identification of new endogenous or exogenous substances that can act specifically on the differentiation of distinct cell lineages is of relevance and has potential therapeutical use. Kalanchoe brasiliensis (Kb) is a medicinal plant from the Crassulaceae family, used in folk medicine to treat inflammatory and infectious diseases. Here, we show that short-term treatment of naïve mice with Kb led to a strong and selective inhibition of lymphopoiesis, affecting B and T cell lineages without reduction of the myeloid lineage development. Similar effects were observed after treatment with the highly purified compound kalanchosine dimalate (KMC), obtained from Kb. Numbers of mature lymphocytes in secondary lymphoid organs were preserved in Kb(KMC)-treated mice. The effect of Kb(KMC) was not a result of secondary augmentation of plasma levels of endogenous corticoids; neither involves TNF-alpha, type-I IFN, or TLR2/TLR4 ligands, which have all been described as selective inhibitors of lymphopoiesis. Flow cytometry analysis of the phenotypes of T and B cell precursors indicate a blockade of maturation on IL-7-dependent, proliferative stages. In vitro, Kb(KMC) inhibited the IL-7-dependent proliferation of pre-B cells and does not induce massive apoptosis of B and T cell precursors. These results suggest that Kb(KMC) is selectively blocking lymphopoiesis through a mechanism that does not involve the previously characterized substances, possibly acting on the IL-7 signaling pathway, opening new perspectives for a potential therapeutic use of Kb-derived drugs.

Inhibition of B cell development by kalanchosine dimalate.

Kalanchoe brasiliensis (Kb) is a medicinal plant from the Crassulaceae family, used in folk medicine to treat inflammatory and infectious diseases. Here we show that short-term treatment of mice with a highly purified compound named kalanchosine dimalate (KMC), obtained from Kb, led to a strong and selective inhibition of B cell development in the bone marrow, without affecting the myeloid lineage development. Numbers of mature B lymphocytes in bone marrow or peripheral lymphoid organs were preserved in KMC treated mice. The inhibitory effect of KMC was acute and rapidly reverted with the interruption of the treatment. In vitro, KMC, inhibited the interleukin-7 dependent proliferation of B cell precursors and do not induce cell death. Also in vitro, the maturation of B cell precursors was not affected by KMC. KMC does not inhibit the proliferative response to IL-3 or IL-2. These results suggest that KMC is selectively affecting B cell lymphopoiesis, possibly acting on the IL-7 signaling pathway, opening new perspectives for a potential therapeutic usage of Kb derived drugs.

Lysophosphatidylcholine produced by the phospholipase A2 isolated from Lachesis muta snake venom modulates natural killer activity as a protein kinase C effector.

We have showed that a phospholipase A(2) isolated from Lachesis muta snake venom, denoted LM-PLA(2)-I, had some biological effects. Here, we examined its effects on lymphocytes. Pre-incubation of human peripheral blood lymphocytes with LM-PLA(2)-I plus phosphatidylcholine (PC) stimulated the natural killer (NK) activity. This was accompanied by DNA binding of nuclear transcription factor kappaB and the increase in PKC activity with translocation of the enzyme from the cytoplasma into the plasma membrane. These effects were reproduced when lymphocytes were pre-incubated with commercial lysophosphatidylcholine (LPC) and abolished by stausrosporin or p-bromophenacyl bromide. Evaluation of phosphorylated PKC isoforms showed that pre-incubation with LPC activated the autophosphorylation of the PKCzeta isoform. Taken together, these results confirm that the enzymatic activity of the phospholipase A(2) present in L. muta venom is for the biological activity of the snake venom, and strongly suggest that the LPC produced may be acting as a modulator of PKC isoforms.

Mechanisms of increased survival after lipopolysaccharide-induced endotoxic shock in mice consuming olive oil-enriched diet.

We examined the impact of dietary fatty acid intake on lipopolysaccharide (LPS)-induced endotoxic shock. C57Bl/6J mice were fed for 6 weeks with a commercial laboratory chow (CC) or with test chows containing 7% (w/w) canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). The increase in body weight and energy consumption were similar for all diets tested. In the sixth week, mice were injected intraperitoneally with 400 microg of bacterial LPS to induce endotoxic shock. LPS induced a massive neutrophil infiltration into the peritoneal cavity and an increase in lipid body (LB) formation in leukocytes recovered from the peritoneal fluid of mice fed with CC, CO, SeO, or SO. In addition, there were increases in prostaglandin E(2) (PGE(2)), leukotriene B4 (LTB(4)), and cytokines IL-6, IL-10, and MCP-1 in peritoneal lavage, as well as in plasma TNF-alpha. In contrast, mice fed with OO exhibited reduced neutrophil accumulation and LB formation, and also had lower levels of PGE(2), LTB(4), MCP-1, and TNF-alpha. All mice fed with CC, CO, SeO, or SO died within 48 to 72 h after LPS injection. Interestingly, mice fed with the OO diet were resistant to endotoxic shock, with 60% survival at 168 h. These data indicate that intake of OO may have a beneficial role, reducing the magnitude of the inflammatory process triggered by endotoxic shock through modulation of LB formation and of the production of inflammatory mediators.

In vivo anti-inflammatory action of eugenol on lipopolysaccharide-induced lung injury.

Eugenol, a methoxyphenol component of clove oil, suppresses cyclooxygenase-2 expression, while eugenol dimers prevent nuclear factor-kappaB (NF-kappaB) activation and inflammatory cytokine expression in lipopolysaccharide-stimulated macrophages. Our aim was to examine the in vivo anti-inflammatory effects of eugenol. BALB/c mice were divided into four groups. Mice received saline [0.05 ml intratracheally (it), control (Ctrl) and eugenol (Eug) groups] or Escherichia coli LPS (10 microg it, LPS and LPSEug groups). After 6 h, mice received saline (0.2 ml ip, Ctrl and LPS groups) or eugenol (160 mg/kg ip, Eug and LPSEug groups). Twenty-four hours after LPS injection, pulmonary resistive (DeltaP1) and viscoelastic (DeltaP2) pressures, static elastance (E(st)), and viscoelastic component of elastance (DeltaE) were measured. Lungs were prepared for histology. In parallel mice, bronchoalveolar lavage fluid was collected 24 h after LPS injection. TNF-alpha was determined by ELISA. Lung tissue expression of NF-kappaB was determined by EMSA. DeltaP1, DeltaP2, E(st), and DeltaE were significantly higher in the LPS group than in the other groups. LPS mice also showed significantly more alveolar collapse, collagen fibers, and neutrophil influx and higher TNF-alpha levels and NF-kappaB expression than the other groups. Eugenol treatment reduced LPS-induced lung inflammation, improving lung function. Our results suggest that eugenol exhibits in vivo anti-inflammatory action in LPS-induced lung injury.

Aspirin and indomethacin reduce lung inflammation of mice exposed to cigarette smoke.

Neutrophil accumulation response to cigarette smoke (CS) in humans and animal models is believed to play an important role in pathogenesis of many tobacco-related lung diseases. Here we evaluated the lung anti-inflammatory effect of aspirin and indomethacin in mice exposed to CS. C57BL/6 mice were exposed to four cigarettes per day during 4 days and were treated i.p. with aspirin or indomethacin, administered each day 1h before CS exposure. Twenty four hours after the last exposure, cells and inflammatory mediators were assessed in bronchoalveolar lavage (BAL) fluid and the lungs used for evaluation of lipid peroxidation, p38 mitogen-activated protein kinase (MAPK) phosphorylation and nuclear transcription factor kappaB (NF-kappaB) activation. Exposure to CS resulted in a marked lung neutrophilia. Moreover, the levels of oxidative stress-related lipid peroxidation, prostaglandin E(2) (PGE(2)), interleukin 1beta (IL-1beta), monocyte chemotactic protein 1 (MCP-1), and activated NF-kappaB and p38 MAPK were greatly increased in CS group. Aspirin or indomethacin treatment led to a significant reduction of neutrophil influx, but only aspirin resulted in dramatic decrease of inflammatory mediators. Moreover, both drugs reduced lung p38 MAPK and NF-kappaB activation induced by CS. These results demonstrate that short-term CS exposure has profound airway inflammatory effects counteracted by the anti-inflammatory agents aspirin and indomethacin, probably through COX-dependent and -independent mechanisms.

Nitric oxide levels and the severity of juvenile idiopathic arthritis.

In this study, we evaluate the distribution of nitric oxide (NO) in the serum of juvenile idiopathic arthritis (JIA) patients, correlating it with parameters of the severity of the disease. Ninety-seven patients with mean age 11.7 years and disease duration 4.8 years, showing active disease or not, grouped as oligoarticular (n = 34), polyarticular (n = 29) and systemic (n = 34) group, presenting uveitis and positive RF with erosive arthritis or active disease and erosions had significantly high levels of NO than the inactive ones. NO correlated with TNF-alpha in the oligoarticular subtype (P < 0.03), with pain in the polyarticular subtype with active disease (P < 0.04) and with ESR in the systemic subtype with active disease (P < 0.03). TNF-alpha concentration was high in all patients with active disease, accompanying NO production. The data confirm the production of NO in JIA patients, indicating a possible positive correlation between the production of NO and severity of the disease.

Kalanchosine dimalate, an anti-inflammatory salt from Kalanchoe brasiliensis.

This report describes the isolation and characterization of kalanchosine dimalate (KMC), an anti-inflammatory salt from the fresh juice of the aerial parts of Kalanchoe brasiliensis. KMC comprises the new metabolite kalanchosine (1) and malic acid (2) in a 1:2 stoichiometric ratio. Kalanchosine (1), 3,6-diamino-4,5-dihydroxyoctanedioic acid, is the first naturally occurring dimeric bis(gamma-hydroxy-beta-amino acid) and is at least partially responsible for the anti-inflammatory properties of K. brasiliensis.

C-glycosylflavones from the aerial parts of Eleusine indica inhibit LPS-induced mouse lung inflammation.

The infusion of aerial parts (EI) of Eleusine indica Gaertn (Poaceae) is used in Brazil against airway inflammatory processes like influenza and pneumonia. Pre-treatment with 400 mg/kg of crude extract inhibited 98% of lung neutrophil recruitment in mice exposed to aerosols of lipopolysaccharide (LPS) from Gram-negative bacteria, in a dose-dependent manner. At 400 microg/kg, schaftoside (6-C-beta-glucopyranosyl-8-C-alpha-arabinopyranosylapigenin) and vitexin (8-C-beta-glucopyranosylapigenin), isolated from EI, inhibited 62% and 80% of lung neutrophil influx, respectively. These results may justify the popular use of E. indica against airway inflammatory processes.