Phytomedicine
Volume 19, Issue 2 , Pages 115-121, 15 January 2012

Kalanchoe pinnata inhibits mast cell activation and prevents allergic airway disease

  • E.A. Cruz

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • Instituto de Biofísica Carlos Chagas Filho, Brazil
    • ,
  • S. Reuter

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • ,
  • H. Martin

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • ,
  • N. Dehzad

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • ,
  • M.F. Muzitano

      Affiliations

    • Núcleo de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro, Brazil
    • ,
  • S.S. Costa

      Affiliations

    • Núcleo de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro, Brazil
    • ,
  • B. Rossi-Bergmann

      Affiliations

    • Instituto de Biofísica Carlos Chagas Filho, Brazil
    • ,
  • R. Buhl

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • ,
  • M. Stassen

      Affiliations

    • Institute of Immunology, University of Mainz, Germany
    • ,
  • C. Taube

      Affiliations

    • III. Medical Clinic, Dept. of Pulmonary Medicine, University of Mainz, Langenbeckstr. 1, 55101 Maniz, Germany
    • Corresponding Author InformationCorresponding author. Tel.: +49 6131 17 6848; fax: +49 6131 17 6668.

published online 01 August 2011.

Article Outline

Abstract 

Aqueous extract of Kalanchoe pinnata (Kp) have been found effective in models to reduce acute anaphylactic reactions. In the present study, we investigate the effect of Kp and the flavonoid quercetin (QE) and quercitrin (QI) on mast cell activation in vitro and in a model of allergic airway disease in vivo. Treatment with Kp and QE in vitro inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/FcɛRI crosslinking, whereas treatment with QI had no effect. Similarly, in vivo treatment with Kp and QE decreased development of airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and production of IL-5, IL-13 and TNF. In contrast, treatment with QI had no effect on these parameters. These findings demonstrate that treatment with Kp or QE is effective in treatment of allergic airway disease, providing new insights to the immunomodulatory functions of this plant.

Keywords: Asthma, Inflammation, Mast cells, Therapy, Murine model

 

Back to Article Outline

Introduction 

Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) is a medicinal plant largely used in the folk medicine for the treatment of gastric ulcer, pulmonary infections, and rheumatoid arthritis (Perry and Metzger 1980). In experimental studies Kalanchoe pinnata (Kp) has been tested in different disease models. Indeed, Kp was effective in treatment of inflammation and gastric ulcer but also hepatoprotective and anti-tumor promoting activities have been described (Nassis et al., 1992, Pal and Chaudhuri, 1990, Pal and Chaudhuri, 1991, Supratman et al., 2001, Yadav and Dixit, 2003). Especially in models of cutaneous and visceral leishmaniasis, diseases driven by exacerbated T helper 2 (TH2) responses, treatment with the aqueous leaf extract of Kp demonstrated immunosuppressive properties (Rossi-Bergmann et al., 1994, Da-Silva et al., 1995, Gomes et al., 2009). In addition, we recently reported effects of oral Kp treatment in a murine model of acute anaphylaxis, associated with a reduced production of antigen-specific IgE antibodies, reduced eosinophilia, and impaired capacity to produce IL-5, IL-10 and TNF (Cruz et al. 2008).

Comparable to anaphylaxis allergic asthma is also based on IgE-mediated inflammatory responses. Asthma is characterized by airway hyperresponsiveness (AHR), airway inflammation and airway obstruction (Busse and Lemanske 2001). Following sensitization, re-exposure to an allergen leads to mast cell activation by crosslinking of high affinity IgE receptors (FcɛRI) via allergen specific IgE. Although there are substantial differences between human asthma and murine models of allergic airway disease, diverse studies in mice have demonstrated that mast cell activation and their produced mediators are important for the subsequent development of allergic airway inflammation and AHR (Williams and Galli, 2000, Yu et al., 2006, Nakae et al., 2007, Reuter et al., 2008). Human studies have demonstrated increased numbers of mast cell in the airway in asthma and have detected mediators like histamine, PGD2, and tryptase in bronchoalveolar lavage fluid both in symptomatic asthma and after allergen inhalation challenge. Mast cell numbers in bronchoalveolar lavage and airway biopsies could be related to AHR in some studies (Wardlaw et al. 1988). Terapeutical strategies for asthma treatment have specific effects on mast cell function, including β2 agonists, leukotriene antagonists and antihistamines. However, treatment approaches of asthma with drugs targeting the mast cell produced mediator histamine, e.g. blockade of the histamine receptor 1 or 2, have been disappointing, particularly when compared to responses achieved by inhaled corticosteroids (Robinson 2004). Given the many different mast cell produced mediators (Galli et al. 2008), an approach to prevent mast cell activation might be more effective compared to inhibiting single mediators. Indeed, in a previous study we have demonstrated that Kp prevented antigen-induced mast cell degranulation and histamine release in vitro (Cruz et al. 2008). So far the effect of Kp treatment on development of allergic airway disease is not known.

Recent studies show that quercetin glycosides flavonoids, like quercitrin (QI), can be metabolized into quercetin aglycone (QE) and quercetin conjugates (Walle, 2004, Wang and Morris, 2005, Spencer et al., 2004). Kp has only trace amounts of QE flavonoid, still previous studies suggested that that QE can be a metabolite of Kp (Muzitano et al. 2008). Indeed, after oral administration, Kp, QE and QI demonstrated similar effectiveness in treating cutaneous leishmaniasis, suggesting that these flavonoids are active components of Kp (Muzitano et al. 2008). Fatty acids and the glycosylated QE flavonoid have been associated with the immunomodulatory and anti-leishmanial activities (Almeida et al., 2000, Muzitano et al., 2006a, Muzitano et al., 2006b). Also the QI flavonoid, a quercetin glycoside isolated from Kp has been described as a effective component of Kp extract to prevent anaphylactic reactions (Cruz et al. 2008).

Based on these previous findings we investigated the effect of treatment with the aqueous extract of Kp and the QE and QI flavonoids using a mast cell dependent murine model of allergic airway disease (Reuter et al. 2008). In the present study we show that treatment with Kp and QE effectively prevent the development of AHR and airway inflammation, whereas treatment with QI has no effect on allergic airway disease.

Back to Article Outline

Material and methods 

Reagents 

Ovalbumin (OVA), Minimum Essential Medium (MEM), pyruvate, antibiotics and Methacholine (MCh) were all purchased from Sigma Chemical Co. (Hamburg, Germany). Iscove's Modified Dulbecco's Medium (IMDM) and heat-inactivated Fetal Calf Serum (FCS) were purchased from Gibco Laboratories (Darmstadt, Germany). IgG2b biotin-conjugated detection antibodies, streptavidin–horseradish peroxidase, tetramethylbenzidine (substrate-reagent), anti-DNP IgE Ab, anti-IgE Ab, anti-CD117 Ab, recombinant murine cytokines and antibodies for ELISA were purchased from BD Pharmingen (Heidelberg, Germany). Anti-FcɛRI biotinylated and anti-CD107a Ab were purchased from R&D Systems (USA) and eBiosciences (Frankfurt, Germany), respectively. Microscopy Hemacolor-Set was purchased from Merck (Darmstadt, Germany). All solutions were freshly prepared immediately before use.

Aqueous extract of Kp and quercitrin isolation 

Kp was prepared as previously described (Cruz et al. 2008). Briefly, Kalanchoe pinnata leaves were collected prior to blooming from the outskirts of Rio de Janeiro, Brazil (voucher specimen #292.697 as deposited at the Rio de Janeiro's Botanical Garden). The leaves were extensively washed and extracted with distilled water at 20% (w/v) for 30min at 50°C. The aqueous extract was filtered, lyophilized and stored at −20°C. The endotoxin content was always less than 10EU/mg, as determined by the Limulus Amebocyte Lysate assay according to manufacturer's instructions (Cambrex Company, USA). Kp was reconstituted to 40mg/ml with phosphate buffered saline (PBS) just prior to use. For quercitrin (QI) isolation, the extract was partitioned with dichloromethane at pH 2 and then pH 11, and the residual aqueous phase was partitioned with ethyl acetate. An aliquot of this organic fraction was re-suspended in distilled water and chromatographed on a RP-2 column affording 3 fractions. The flavonoid-rich fraction was eluted with 3:7 MeOH/H2O and further purified with a H2O/MeOH gradient on a RP-18 column. Two flavonoid fractions were obtained. The first fraction, enriched in one compound, was chromatographed on a Sephadex G-15 column (31.0cm×0.8cm; H2O) affording quercitrin as a yellow powder: Rf 0.83; 1H and 13C NMR (CD3OD) as reported before (Muzitano et al. 2006a); purity of QI was determined by HPLC as 98.1% (Muzitano et al. 2006b). Quercetin (QE) was purchased from Sigma Chemical Co (St. Louis, USA).

Animals 

BALB/c mice were obtained from the Zentrale Tierzuchtanstalt of the Johannes Gutenberg University Medical Center (Mainz, Germany). Mice were used at age 8–12 weeks. Animal procedures were conducted in accordance with current federal, state, and institutional guidelines and performed according to the Helsinki convention for the use and care of animals.

Generation of bone marrow-derived mast cell (BMMC) 

For the generation of BMMC, mice were sacrificed by cervical dislocation, intact femurs and tibias were removed, and bone marrow cells were harvested by repeated flushing with MEM (Muzitano et al. 2006b). Cell cultures were established at a density of 3×106cells/ml in IMDM supplemented with 10% FCS (inactivated at 56°C), 2mM l-glutamine, 1mM pyruvate, 100U/ml penicillin, 100μg/ml streptomycin, 20U/ml mIL-3, 50U/ml mIL-4, and 200ng/ml stem cell factor. Nonadherent cells were transferred to fresh culture plates every 2–3 days for a total of at least 21 days to remove adherent macrophages and fibroblasts. FACS analyses using an anti-CD117 Ab and anti-FcɛRI as well as May-Grünwald-Giemsa and toluidine blue staining revealed that the resulting cell population consisted of about 95% BMMC (data not shown).

In vitro mast cell degranulation 

Bone marrow-derived mast cells (BMMC) were plated at 1×106cells/well and incubated with anti-DNP IgE Ab (2μg/ml) for 48h at 37°C. The cells were washed once with PBS and treated with Kp at 250, 500 or 1000μg/ml or with QE or QI at 25, 50 or 100μg/ml for 1h. After treatment, the cells were washed once and anti-CD107a Ab was added together with anti-IgE Ab (2μg/ml) in 1ml of supplemented IMDM. After 2h, the cells were centrifuged at 1200rpm for 5min, additionally stained with anti-CD117 Ab and FACS analyzed. CD107a (LAMP-1) is an intracellular protein found on granule membranes that becomes exposed on mast cell surface upon degranulation (Grützkau et al. 2004). The double CD117/CD107a positive cells were considered as degranulated mast cells. Inhibition of degranulation was calculated=[1−(number of CD117/CD107a positive cells in treated group/number of CD117/CD107a positive cells in PBS group)]×100 (Stassen et al. 2006).

Antigen sensitization and challenge 

Mice were sensitized by intraperitoneal (i.p.) injection with 100μl of 20μg OVA solution in PBS on days 0 and 14. Mice were then challenged via the airways on days 28, 29 and 30, using nebulised OVA at 1% (weight/volume) in PBS with an ultrasonic nebuliser (NE-U17; Omron, Hoofdorp, the Netherlands). Experimental groups consisted of four mice per group and each experiment was performed at least twice. From days 27 to 30 mice received daily doses of 400mg/kg of aqueous extract of Kp or 30mg/kg of QE or QI, in 200μl of PBS by intragastric gavage. Control mice received 200μl of PBS.

Measurement of airway reactivity 

Measurements of airway resistance (RL) were performed in anaesthetised, intubated and mechanically ventilated (FlexiVent; Scireq, Montreal, QC, Canada) mice in response to increasing doses of inhaled methacholine (MCh; 3.125, 6.25, 12.5, 25 and 50mg/ml) as previously described (Reuter et al. 2008). Measurements of the RL were performed every 15s following the nebulization with each single dose until a plateau phase was reached.

Bronchoalveolar (BAL) cells and fluid 

After assessment of airway function, lungs were lavaged via the tracheal tube with PBS (1ml). Numbers of lavaged cells were counted using trypan blue dye exclusion. The bronchoalveolar fluid was obtained by centrifugation and stored at −20°C until use. Differential cell counts were made from cytocentrifuged preparations that were fixed and stained with a Microscopy Hemacolor-Set.

Histology 

Lungs were fixed by inflation (1ml) and immersion in 10% formalin, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). Slides were examined in a blinded fashion with a microscope (BX40; Olympus, Hamburg, Germany). The presence of inflammatory cell infiltration and the number of goblet cells were analyzed respectively using HE and PAS-stained slides and imaging software (Analysis; Soft Imaging Systems, Stuttgart, Germany) as previously described (Bopp et al. 2009).

Antigen-specific immunoglobulin and cytokine ELISA 

Serum was obtained 48h after the last OVA challenge. OVA specific IgG1 and IgG2b titers were determined using ELISA. Biotin-conjugated detection antibodies, streptavidin–horseradish peroxidase and substrate-reagent were used in concentrations as recommended by the manufacturer (BD-Pharmingen, Heidelberg, Germany). OVA-specific IgE was measured as described previously (Reuter et al. 2008). Briefly, plates were coated with the rat anti-mouse IgE (PharMingen). Following administration of 3% BSA-PBS for 2h, serial dilutions of sera were incubated overnight at 4°C. Then biotin-labeled OVA was added for 2h and absorption was read after adding streptavidin–horseradish peroxidase and o-phenylenediamine. The antibody titer was defined as the reciprocal serum dilution yielding an optical density, measured at 450nm, of 0.2 after linear regression analysis.

The levels of IL-5, IL-13, TNF and IFN-γ were determined in the supernatants of BMMC culture or in the BAL fluid by ELISA using recombinant murine cytokines and antibodies according to the manufacturer's instructions (BD Pharmingen, Heidelberg, Germany).

Statistical analysis 

The data were reported as mean±standard error of the mean (SEM). ANOVA and Tukey post test were used to determine the levels of difference between all groups. Comparisons for all pairs were performed by the Student t-test. A p value ≤0.05 was considered to be significant. The different values for all measurements are expressed as mean±SEM.

Back to Article Outline

Results 

Kp and QE prevent mast cell degranulation and TNF and IL-6 release in vitro 

In previous studies we have shown that Kp can decrease histamine release of mast cells (Cruz et al. 2008). In the present study the effect of QE, QI flavonoids as well as Kp was assessed on mast cell degranulation and cytokine release following activation by IgE crosslinking. Treatment with either QE, QI or Kp had no effect on mast cell apoptosis. Indeed, numbers of annexin V+ propidium iodide+ mast cells were low in BMMCs treated with QE, QI or Kp (<10%, <10%, <11%, respectively) compared to BMMCs treated with PBS (<10%). Interestingly, treatment with QE and Kp significantly (p<0.05) decreased degranulation of BMMCs in vitro following IgE/FcɛRI crosslinking in a dose dependent manner (Fig. 1A and B). In contrast, treatment with QI had no effect on mast cell degranulation (Fig. 1A). Furthermore production of TNF and IL-6 were measured in cell culture supernatants. Similarly to the effect on degranulation treatment with QE and Kp significantly (p<0.01) reduced levels of TNF in supernatant, whereas no effect was detected when incubating cells with QI (Fig. 1C). Also treatment with QE significantly reduced levels of IL-6 in supernatant, whereas QI and Kp did not significantly decrease IL-6 (Fig. 1D).

  • View full-size image.
  • Fig. 1. 

    Effect of Kp, QE and QI on mast cell degranulation and cytokine production in vitro. BMMC were treated as described in ‘Material and methods’. Panel A shows % inhibition of degranulation of BMMCs treated with either QE or QI in different concentrations compared to PBS treated BMMCs. Panel B shows % inhibition of degranulation of BMMCs treated with Kp compared to PBS treated BMMCs. Means are given from 3 independent experiments. Cell culture supernatants were from unstimulated (anti-IgE−) or stimulated (anti-IgE+) BMMCs treated with either PBS, 50 or 100μg QE (QE 50; QE 100), 50 or 100μg QI (QI 50; QI 100) or 500 or 1000μg of Kp (Kp 500; Kp 1000) 2h after stimulation and cytokines were quantified by ELISA. Panel C shows levels of IL-6 and panel D displays levels of TNF. Means±SEM are given from 3 independent experiments. *p<0.01 compared to anti-IgE+ PBS.

Treatment with Kp or QE effectively protects mice against development of AHR 

As QE and Kp effectively decreased mast cell degranulation and cytokine production, we investigated their effect in a murine model of allergic airway disease. Based on previous results, we chose a model which is mast cell dependent (Reuter et al. 2008). Indeed, in the present study mice displayed increased airway responsiveness to MCh following sensitization and challenge. Similarly to the effects on BMMCs observed in vitro, sensitized and challenged mice treated with Kp or QE showed a significant reduction of airway reactivity to MCh. In contrast, treatment with QI had no effect on AHR (Fig. 2).

  • View full-size image.
  • Fig. 2. 

    Treatment with Kp and QE inhibits development of AHR. Airway resistance was assessed in sensitized and challenged mice, 48h after the last challenge. Sensitized and challenged mice (sens/chall, n=8), and sensitized and challenged mice treated with Kp (sens/chall Kp, n=8), QE (sens/chall QE, n=8) or QI (sens/chall, QI n=8) compared to challenged only mice (chall, n=8). Means±SEM are given. *p<0.05.

Treatment with Kp and QE reduces airway inflammation and goblet cell metaplasia 

To assess the effect of Kp and QE on airway inflammation, inflammatory cell accumulation in the BAL fluid and lung tissue was evaluated 48h following the last airway challenge. At this time increased total cell count and numbers of lymphocytes, neutrophils and eosinophils were detected in sensitized mice compared to non-sensitized controls (Fig. 3). Sensitized and challenged mice treated either with Kp or QE before airway challenge showed a significant reduction in total cell count and especially in numbers of lymphocytes and eosinophils (Fig. 3). Treatment with QI had no effect on airway inflammation.

  • View full-size image.
  • Fig. 3. 

    Treatment with KP and QE reduces cellular composition of BAL fluid. Cellular composition in BAL fluid were assessed 48h after the last airway challenge in sensitized and challenged mice (sens/chall, n=8), and sensitized and challenged mice treated with Kp (sens/chall Kp, n=8), QE (sens/chall QE, n=8) or QI (sens/chall, QI n=8) compared to challenged only mice (chall, n=8). Means±SEM are given. *p<0.05 compared to chall, sens/chall Kp and sens/chall QE.

In parallel and preceding the increases in BAL fluid, allergen sensitization and airway challenge lead to an increase in peribronchial inflammation and goblet cell numbers compared to mice that were challenged only (Fig. 4). Similar to results in BAL fluid mice treated either with Kp or QE before airway challenge displayed a reduction in peribronchial inflammation and numbers of goblet cells (Fig. 4). In contrast, treatment with QI had no effect on these parameters.

  • View full-size image.
  • Fig. 4. 

    Treatment with KP and QE reduces tissue inflammation. Tissue inflammation and goblet cell hyperplasia were evaluated 48h following the last challenge using hematoxylin and eosin staining (H&E) and PAS staining (PAS). Panel A shows representative sections from challenged only (chall, n=8), sensitized and challenged mice (sens/chall, n=8), and sensitized and challenged mice treated with Kp (Kp, n=8), QE (QE, n=8) or QI (QI, n=8) compared to challenged only mice (chall, n=8). Panel B: slides were scored for peribronchial inflammation using a semi-quantitative score from 0 to 4. Panel C: the numbers of mucus-positive cells scored per millimetre basement membrane were quantified for each group as described in ‘Material and methods’. Mean±SEM are given. n.d. non detectable. *p<0.05 compared to chall, #p<0.05 compared to chall, Kp and QE.

Treatment with Kp and QE reduces levels of OVA-specific immunoglobulins in serum 

Inhaled allergen binds to preformed IgE and IgG antibodies in the airways and stimulates mast cells to release inflammatory mediators (Galli et al. 2008). To determine if treatment with Kp and its metabolites affect serum immunoglobulin levels, total IgE and OVA-specific IgE and IgG1 were measured 48h following the last airway challenge. Sensitized and challenged mice showed significantly (p<0.05) increased levels of total IgE and OVA-specific IgE and IgG1 compared to challenged only control mice (Table 1). Treatment with Kp and QE before airway challenged resulted in reduced serum levels of these immunoglobulins, whereas treatment with QI had no effect.

Table 1. Serum immunoglobulin titers.
ChallSens/challKpQEQI
OVA-specific IgE (titer)N.D.30.2±5.2*9.3±2.118.1±7.532.3±5.7*
OVA-specific IgG1 (titer) (×103)15±9475±77*232±103319±112525±123*

Mice were sensitized and challenged as described in ‘Methods’. Serum levels of immunoglobulins were assessed 48h after the last challenge. Mean values±SEM are given. Chall: challenged only, sens/chall: sensitized and challenged, Kp: sensitized and challenged treated with Kp, QE: sensitized and challenged treated with QE, QI: sensitized and challenged treated with QI.

*p<0.05 compared to chall, Kp and QE.

Treatment with Kp and QE alters cytokine production in BAL fluid 

TH2 cytokine production by T cells plays a key role in the induction of allergic airway inflammation and AHR. To evaluate the effects of Kp, QE and QI treatment on the development of TH2 cytokine responses, we assessed concentrations of IL-5, IL-13, TNF and IFN-γ levels in the BAL fluid 48h after the last OVA challenge. Sensitization and challenge resulted in significant (p<0.05) increases in IL-5, IL-13 and TNF, whereas levels of IFN-γ were not different from challenged only mice (Table 2). Treatment of sensitized and challenged mice with Kp or QE significantly reduced the levels of IL-5, IL-13 and TNF, but no effect on levels of IFN-γ was detected.

Table 2. Concentrations of cytokines in BAL fluid.
ChallSens/challKpQEQI
IL-5 (pg/ml)162±16280±14*112±17121±14201±24
IL-13 (pg/ml)n.d.86±16*20±621±586±26*
TNF (pg/ml)512±26817±62*519±36472±45621±47*
INF-γ (pg/ml)17±418±420±219±114±4

Mice were sensitized and challenged as described in ‘Methods’. Levels of cytokines in BAL fluid were assessed 48h after the last challenge. Mean values±SEM are given. Chall: challenged only, sens/chall: sensitized and challenged, Kp: sensitized and challenged treated with Kp, QE: sensitized and challenged treated with QE, and QI: sensitized and challenged treated with QI.

*p<0.05 compared to chall, Kp and QE.

Back to Article Outline

Discussion 

Mast cells play a pivotal role for the development of allergic asthma, but so far therapeutic approaches to directly target mast cells have not been very successful. In the present study we show that an extract of the medicinal plant Kp and its flavonoid QE are effective in inhibiting mast cell degranulation and production of TNF in vitro. Furthermore we show in an acute model of allergic airway disease that following sensitization without adjuvant and challenge of the mice, treatment with medicinal plant extracts inhibits the development of allergic inflammation and AHR.

Potent effects of Kp have been described in a murine model of anaphylactic shock (Cruz et al. 2008). Indeed, treatment with Kp prevented mast cell degranulation and histamine release following IgE/FcɛRI crosslinking. In the present study we show that both, treatment with Kp and also QE effectively reduced mast cell degranulation and cytokine production. In contrast, treatment with QI had no effect on these parameters in vitro. Interestingly, when comparing effects of Kp and QE on cytokine production treatment with Kp and QE effectively reduced levels of mast cell produced TNF. In contrast, treatment with QE also effectively reduced levels of IL-6. These findings are comparable to reports from human mast cells, where treatment with QE can also block IL-6 secretion following mast cell activation (Kandere-Grzybowska et al. 2006). Treatment with Kp had no effect on IL-6 secretion, suggesting different mechanisms of Kp and QE in regard of mast cell inhibition. Indeed, different regulatory pathways of IL-6 and TNF have been described, the latter being dependent on transcription factor NFAT (Klein et al. 2006). Further studies are needed to unravel the effects of Kp on mast cell signaling.

In the present study, the potential effect of treatment with Kp on allergic airway disease was evaluated. Furthermore we tried to identify the active substances responsible for this effect of Kp. It is conceivable that when orally administered, active Kp metabolites are produced and are systemically present upon passage through the digestive system. Flavonoids like QI, a QE glycoside are present in Kp (Muzitano et al. 2006a) and have demonstrated anti-allergic activity (Kawai et al., 2007, Lee et al., 1999, Shaik et al., 2006). Therefore it is reasonable that glycosyl flavonoids are strong candidates as active components of Kp. Also QE is found in many medicinal plants and often the therapeutic effects of these plants result from their high QE content (Lee et al., 1999, Mainardi et al., 2009). Indeed, QI can be hydrolyzed to the aglycone quercetin either in the liver (Walle 2004) or in the intestine by bacterial rhamnosidases (Park et al. 2005). Therefore in the present study we chose to assess the effects of Kp, QE and QI. Flavonoids like quercetin and related polyphenolic compounds have significant antiinflammatory and anti-allergic activities (Theoharides et al., 2001, Kimata et al., 2000). Several enzymes as cyclooxygenase, lipoxygenase and cellular types as macrophages, lymphocytes, epithelial cells have been identified as targets modulated by flavonoids. In addition, QE and others flavonoids display significant antioxidant and radical scavenging properties (González et al. 2011). Different flavonoids have been described as effective compounds in attenuation of TH2-driven allergic airway disease, allergic airway inflammation and hyperreactivity of airways in mice (Medeiros et al., 2009, Song et al., 2010, Franova et al., 2009). Recent studies have demonstrated that QE inhibit several processes of inflammation, including the regulation of NF-kB and p38 MAPK pathways, Th1/Th2 cytokine production, T-bet and GATA-3 gene expression in OVA-induced asthma model in mice. These immunopharmacological effects of QE were associated to its protective activity in asthma (Min et al., 2007, Park et al., 2009).

To further analyze the effects of treatment with Kp and its flavonoids, we utilized a model of allergic airway disease. In murine models the role and contribution of mast cells to the development of allergic airway disease appears to be highly dependent on the sensitization and allergen exposure protocol. It has repeatedly been demonstrated that models which use systemic allergen sensitization without an additional adjuvant the development of AHR and airway inflammation is dependent on mast cells (Williams and Galli, 2000, Yu et al., 2006, Nakae et al., 2007, Reuter et al., 2008). In the present study, also animals were sensitized without an additional adjuvant. Similar to previous results (Reuter et al. 2008), allergen sensitization increased levels of allergen specific IgE and IgG1 were detectable and mice developed AHR and airway inflammation following inhaled allergen exposure. In this model, systemic treatment with either Kp or QE reduced AHR and airway inflammation. This goes in line with the in vitro findings where Kp and QE both reduced degranulation and cytokine production of mast cells. Similar to the in vitro findings treatment with QI had no effect on allergic airway disease. These results suggest that Kp and QE, at least partly through their effect on mast cells, inhibit the development of allergic airway disease. Interestingly, treatment with the QI flavonoid, which has previously been found effective against cutaneous leishmaniasis and anaphylactic shock (Muzitano et al., 2006b, Cruz et al., 2008), had no effect on mast cells in vitro and the development of allergic airway disease in vivo. At least in the in vivo models, it cannot be ruled out that the treatment period was not long enough. Indeed, the anti-anaphylactic activity of quercitrin was obtained with an oral treatment during 14 days (Cruz et al. 2008), whereas in the present study the animals were treated with QI only for 4 days. Additional to the effects on inflammation and AHR also goblet cell metaplasia and cytokine production were assessed in these animals. Again, treatment with Kp or QE reduced the numbers of goblet cells in airway epithelium but also levels of IL-5, IL-13 and TNF in BAL supernatants. Especially IL-5 and IL-13 are important cytokines for development of airway eosinophila and AHR (Taube et al. 2004). Plant extracts and secondary metabolites, including QE, can reduce eosinophilia and eosinophil recruitment in different experimental animal models and contribute to the medical potential of these plant-derived compounds for treating eosinophil-mediated inflammation, such as asthma (Rogerio et al. 2010). Our findings suggest that treatment with Kp or QE preferentially target the development of TH2 responses in the lung, which is also reflected by decreased serum levels of OVA-specific IgE and IgG1 following KP or QE treatment. In contrast to other studies (Park et al. 2009) we did not find an increase in TH1 responses following QE treatment as levels of IFN-γ in BAL fluid and serum levels of OVA-specific IgG2b (data not shown) were not changed. Clinical studies using phytotherapy for asthma treatment have demonstrated that relieve of symptoms and decrease of airway hyperreactivity are associated to reduction of IL-4 and IL-5 without changes on IFN-γ levels (Srivastava et al., 2004, Wen et al., 2005). Therefore, this data suggests that Kp and QE act immunosuppressive and not by induction of TH1 response. In contrast, treatment with QI had no effect also on these other outcome parameters of allergic airway disease. In summary, these data show that treatment with Kp and QE, but not QI, decrease allergen-induced development of AHR, TH2 responses in the lung, lung eosinophilia, and goblet cell metaplasia after allergen exposure of the sensitized host. These suggest that further development of these medicinal plants might offer new therapeutic options for patients with allergic diseases including allergic asthma.

Back to Article Outline

Acknowledgements 

This study was funded by DFG TA 275/4-1 (C.T. and M.S.), TA 275/5-1 C.T. and DAAD.

Back to Article Outline

References 

  1. Almeida AP, Da-Silva SA, Souza ML, Lima LM, Rossi-Bergmann B, de Moraes VL, et al. Isolation and chemical analysis of a fatty acid fraction of Kalanchoe pinnata with a potent lymphocyte suppressive activity. Planta Med. 2000;66:134–137
  2. Bopp T, Dehzad N, Reuter S, Klein M, Ullrich N, Stassen M, et al. Inhibition of cAMP degradation improves regulatory T cell-mediated suppression. J. Immunol. 2009;182:4017–4024
  3. Busse WW, Lemanske RF. Asthma. N. Engl. J. Med. 2001;344:350–362
  4. Cruz EA, Da-Silva SAG, Muzitano MF, Silva PMR, Costa SS, Rossi-Bergmann B. Immunomodulatory pretreatment with Kalanchoe pinnata extract and its quercitrin flavonoid effectively protects mice against fatal anaphylactic shock. Int. Immunopharmacol. 2008;8:1616–1621
  5. Da-Silva SA, Costa SS, Mendonça SCF, Silva EM, Moraes VLG, Rossi-Bergmann B. Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis. Acta Trop. 1995;60:201–210
  6. Franova S, Joskova M, Novakova E, Adamicova K, Sutovska M, Nosal S. Effects of flavin7 on allergen induced hyperreactivity of airways. Eur. J. Med. Res. 2009;14(Suppl. 4):78–81
  7. Galli SJ, Grimbaldeston M, Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat. Rev. Immunol. 2008;8:478–486
  8. Gomes DC, Muzitano MF, Costa SS, Rossi-Bergmann B. Effectiveness of the immunomodulatory extract of Kalanchoe pinnata against murine visceral leishmaniasis. Parasitology. 2009;7:1–6
  9. González R, Ballester I, López-Posadas R, Suárez MD, Zarzuelo A, Martínez-Augustin O, et al. Effects of flavonoids and other polyphenols on inflammation. Crit. Rev. Food Sci. Nutr. 2011;51(4):331–362
  10. Grützkau A, Smorodchenko A, Lippert U, Kirchhof L, Artuc M, Henz BM. LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced section of human mast cells. Cytometry A. 2004;61:62–68
  11. Kandere-Grzybowska K, Kempuraj D, Cao J, Cetrulo CL, Theoharides TC. Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin. Br. J. Pharmacol. 2006;148:208–215
  12. Kawai M, Hirano T, Higa S, Arimitsu J, Maruta M, Kuwahara Y, et al. Flavonoids and related compounds as anti-allergic substances. Allergol. Int. 2007;56:113–123
  13. Kimata M, Shichijom M, Miuram T, Serizawam I, Inagakim N, Nagaim H. Effects of luteolin, quercetin and baicalein on immunoglobulin E mediated mediator release from human cultured mast cells. Clin. Exp. Allergy. 2000;30:501–508
  14. Klein M, Klein-Hessling S, Palmetshofer A, Serfling E, Tertilt C, Bopp T, et al. Specific and redundant roles for NFAT transcription factors in the expression of mast cell-derived cytokines. J. Immunol. 2006;177:6667–6674
  15. Lee E, Choi EJ, Cheong H, Kim YR, Ryu SY, Kim KM. Anti-allergic actions of the leaves of Castanea crenata and isolation of an active component responsible for the inhibition of mast cell degranulation. Arch. Pharm. Res. 1999;22:320–323
  16. Mainardi T, Kapoor S, Bielory L. Complementary and alternative medicine: herbs, phytochemicals and vitamins and their immunologic effects. J. Allergy Clin. Immunol. 2009;123:283–294
  17. Medeiros KC, Faustino L, Borduchi E, Nascimento RJ, Silva TM, Gomes E, et al. Preventive and curative glycoside kaempferol treatments attenuate the TH2-driven allergic airway disease. Int. Immunopharmacol. 2009;9(13–14):1540–1548
  18. Min YD, Choi CH, Bark H, Son HY, Park HH, Lee S, et al. Quercetin inhibits expression of inflammatory cytokines through attenuation of NF-kB and p38 MAPK in HMC-1 human mast cell line. Inflamm. Res. 2007;56:210–215
  19. Muzitano MF, Tinoco LW, Guette C, Kaiser CR, Rossi-Bergmann B, Costa SS. Assessment of antileishmanial activity of new and unusual flavonoids from Kalanchoe pinnata. Phytochemistry. 2006;67:2071–2077
  20. Muzitano MF, Cruz EA, Almeida AP, Da-Silva SA, Kaiser CR, Guette C, et al. Quercitrin: an antileishmanial flavonoid glycoside from Kalanchoe pinnata. Planta Med. 2006;72:81–83
  21. Muzitano MF, Falcão CA, Cruz EA, Bergonzi MC, Bilia AR, Vincieri FF, et al. Oral metabolism and efficacy of Kalanchoe pinnata flavonoids in a murine model of cutaneous leishmaniasis. Planta Med. 2008;75:307–311
  22. Nakae S, Ho LH, Yu M, Monteforte R, Iikura M, Suto H, et al. Mast cell-derived TNF contributes to airway hyperreactivity, inflammation, and TH2 cytokine production in an asthma model in mice. J. Allergy Clin. Immunol. 2007;120:48–55
  23. Nassis CZ, Haebisch EM, Giesbrecht AM. Antihistamine activity of Bryophyllum calycinum. Braz. J. Med. Biol. Res. 1992;25:929–936
  24. Pal S, Chaudhuri NAK. Anti-inflammatory action of Bryophyllum pinnatum leaf extract. Fitoterapia. 1990;61:527–533
  25. Pal S, Chaudhuri NAK. Studies on the anti-ulcer activity of a Bryophyllum pinnatum leaf extract in experimental animals. J. Ethnopharmacol. 1991;33:97–102
  26. Park SY, Kim JH, Kim DH. Purification and characterization of quercitrin hydrolyzing alpha-l-rhamnosidase from Fusobacterium K-60, a human intestinal bacterium. J. Microbiol. Biotechnol. 2005;15:519–524
  27. Park HJ, Lee CM, Jung ID, Lee JS, Jeong YI, Chang JH, et al. Quercetin regulates Th1/Th2 balance in a murine model of asthma. Int. Immunopharmacol. 2009;9:261–267
  28. Perry LM, Metzger J. Medicinal Plants of East Asia Attributed Properties and Uses. Cambridge: MIT Press; 1980;pp. 109–110
  29. Reuter S, Heinz A, Sieren M, Wiewrodt R, Gelfand EW, Stassen M, et al. Mast cell-derived tumor necrosis factor is essential for allergic airway disease. Eur. Respir. J. 2008;31:773–782
  30. Robinson DS. The role of the mast cell in asthma: induction of airway hyperresponsiveness by interaction with smooth muscle?. J. Allergy Clin. Immunol. 2004;114:58–65
  31. Rogerio AP, Sá-Nunes A, Faccioli LH. The activity of medicinal plants and secondary metabolites on eosinophilic inflammation. Pharmacol. Res. 2010;62(4):298–307
  32. Rossi-Bergmann B, Costa SS, Borges MBS, Da-Silva SA, Noleto GR, Souza MLM, et al. Immunosuppressive effect of the aqueous extract of Kalanchoe pinnata in mice. Phytother. Res. 1994;8:399–402
  33. Shaik YB, Castellani ML, Perrella A, Salini V, Tete S, Madhappan B, et al. Role of quercetin (a natural herbal compound) in allergy and inflammation. J. Biol. Regul. Homeost. Agents. 2006;20:47–52
  34. Song MY, Jeong GS, Lee HS, Kwon KS, Lee SM, Park JW, et al. Sulfuretin attenuates allergic airway inflammation in mice. Biochem. Biophys. Res. Commun. 2010;400(1):83–88
  35. Spencer JPE, El Mohsen MMA, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch. Biochem. Biophys. 2004;423:148–161
  36. Srivastava K, Teper AA, Zhang TF, Li S, Walsh MJ, Huang CK, et al. Immunomodulatory effect of the antiasthma Chinese herbal formula MSSM-002 on TH2 cells. J. Allergy Clin. Immunol. 2004;113:268–276
  37. Stassen M, Valeva A, Walev I, Schmitt E. Activation of mast cells by streptolysin O and lipopolysaccharide. Methods Mol. Biol. 2006;315:393–403
  38. Supratman U, Fujita T, Akiyama K, Hayashi H, Murakami A, Sakai H, et al. Antitumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana×tubiflora. Biosci. Biotechnol. Biochem. 2001;65:947–949
  39. Taube C, Dakhama A, Gelfand EW. Insights into the pathogenesis of asthma utilizing murine models. Int. Arch. Allergy Immunol. 2004;135:173–186
  40. Theoharides TC, Alexandrakis M, Kempuraj D, Lytinas M. Antiinflammatory actions of flavonoids and structural requirements for new design. Int. J. Immunopathol. Pharmacol. 2001;14:119–127
  41. Walle T. Absorption and metabolism of flavonoids. Free Radic. Biol. Med. 2004;36:829–837
  42. Wang L, Morris ME. Liquid chromatography–tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites I human plasma and urine. J. Chromatogr. B. 2005;821:194–201
  43. Wardlaw AJ, Dunnette S, Gleich GJ, Collins JV, Kay AB. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma: relationship to bronchial hyperreactivity. Am. Rev. Respir. Dis. 1988;137:62–69
  44. Wen MC, Wei CH, Hu ZQ, Srivastava K, Ko J, Xi ST, et al. Efficacy and tolerability of anti-asthma herbal medicine intervention in adult patients with moderate-severe allergic asthma. J. Allergy Clin. Immunol. 2005;116:517–524
  45. Williams CMM, Galli SJ. Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice. J. Exp. Med. 2000;192:455–462
  46. Yadav NP, Dixit VK. Hepatoprotective activity of leaves of Kalanchoe pinnata Pers. J. Ethnopharmacol. 2003;86:197–202
  47. Yu M, Tsai M, Tam SY, Jones C, Zehnder J, Galli SJ. Mast cells can promote the development of multiple features of chronic asthma in mice. J. Clin. Invest. 2006;116:1633–1641

PII: S0944-7113(11)00236-4

doi:10.1016/j.phymed.2011.06.030

Phytomedicine
Volume 19, Issue 2 , Pages 115-121, 15 January 2012

Article Tools

* Image Usage & Resolution