Effects of budlein A on human neutrophils and lymphocytes

ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.


INTRODUCTION
The ability of the immune system to respond to a variety of stimuli is central to its function in pathogen clearance and tissue repair 4 . The in different pathological settings is maintained by signaling 6 , and most research has focused on understanding the mechanisms that regulate the immune response 4,6,8,9 . New therapeutic agents are currently under investigation for pathological conditions in which the balance between activation and suppression of the immune response has failed 6 . Studies have shown that plant-derived compounds have immunomodulatory effects without direct immunosuppressive action such as clinically used corticosteroids 2, 3,16,27 . However, the mere isolation of new naturally-occurring antidrug until the exact mechanism of action and their possible toxic effects are completely determined 9 . Moreover, many biologically active compounds that do not afford a new drug or even a principal compound can be used as prototypes for the study of the mechanisms involved in a certain biological effect. Budlein A, a sesquiterpene lactone (SL) formerly isolated from the Central American species Aldama buddlejiformis (DC) 1 and more recently from the South American A. robusta Gardner (Asteraceae), has been described as presenting 1,7,27 . Recent studies have activities in mice 22,27 . Although this information is important for determining its properties, it provides restricted parameters about how this compound affects other cells, adaptive immune response and T cell compartment. An immunomodulatory substance might show antibacterial activity, but the induced cell death could also be harmful to immune responses, pathogen clearance and tissue repair 11,18 . In this context, research to develop new by lymphocyte activation, has been stimulated. Given the broad information relating SLs and immunomodulation, we should evaluate whether budlein A modulates the activation of innate and adaptive immune cells.

Budlein A
Budlein A (MW=374.1365) was isolated from the leaves of Viguiera robusta, as previously described 27 . Its chemical structure was determined by means of spectrometric analysis, i.e., IR, 1H, and 13C nuclear magnetic resonance (NMR) spectrometry as well as comparison with an authentic sample and data reported in literature 5 . Analytical procedures using spectroscopy and chromatographic techniques indicated that the purity of budlein A used in this study ranged between 95-98%. Prior to the bioassays, budlein A was dissolved in dimethylsulfoxide (DMSO) (SIGMA -St. Louis, MO, USA) (0.1% in RPMI 1640 medium -GIBCO -Grand Island, NY, USA).

Healthy volunteers
We used peripheral blood from 30 healthy volunteers (17 men and 13 women; age ranged between 27-50 years). All subjects had no active diseases at the time of phlebotomy. All subjects signed an informed consent releasing the use of blood for research purposes approved by the Bauru School of Dentistry, University of São Paulo.

Isolation of leukocytes
Peripheral blood was harvested with heparin (50 U/ml) from healthy subjects. Both groups of leukocytes were layered over two different density Histopaque gradients (SIGMA) and centrifuged for 30 min at 450X g. Peripheral blood mononuclear Histopaque 1077) and neutrophils were isolated from the second buffy coat layer (onto Histopaque 1119). Cells were then washed twice with Roswell Park Memorial Institute medium (RPMI) at 200 X g cells was >99% viable when assessed by Trypan blue exclusion.

Myeloperoxidase activity
Myeloperoxidase (MPO) activity was determined by enzymatic reaction, as previously described 26 . Neutrophils were harvested after culture and centrifuged at 350X g for 15 min, and the pellet and centrifuged twice at 10,000X g for 15 min at 4°C. The MPO activity in the suspended pellet was assayed by measuring the change in absorbance at 450 nm using tetramethylbenzidine (1.6 mM) and H 2 O 2 (0.5 mM) (BD Biosciences).

Apoptosis
Cells were harvested after culture and the viability was analyzed by flow cytometry, as previously described 26

RESULTS
Budlein A inhibits MPO activity, cytokine production, and induces neutrophil apoptosis budlein A on neutrophils, cells were stimulated with fMLP or LPS in presence or absence of budlein A (1, 10, or 100 μM) or dexamethasone (positive control), and the myeloperoxidase (MPO) activity, IL-6, CXCL8, IL-10, and IL-12 production and apoptosis were analyzed. Consistent with prior observation 27 , budlein A inhibited in a concentration-dependent manner the MPO activity by LPS-stimulatedneutrophils (Fig. 1A). Our results also indicate that at a concentration of 10 and 100μM, budlein A reduced MPO activity by fMLP-stimulated neutrophils ( Figure 1A).
Because neutrophils appear to be important producers of cytokines during innate immune response and cytokines are important mediators of the inflammatory process, we assessed whether budlein A affects cytokine and chemokine production by neutrophils ( Figure 1B). Unstimulated neutrophils produced basal levels of IL-6, IL-10, CXCL8, and IL-12. Budlein A decreased CXCL8 production, induced IL-10 production and did not alter IL-12 and IL-6 production by unstimulated neutrophils. After 21 h of culture, LPS and fMLP enhanced IL-6, CXCL8, IL-12, and IL-10 production by human neutrophils. In contrast, budlein A inhibited in a concentration-dependent manner IL-6 and CXCL8 production by fMLP and LPSstimulated neutrophils. Moreover, budlein A, at only 100 μM concentration, inhibited IL-10 and IL-12 production by fMLP and LPS-stimulated-neutrophils, while at 10 μM it inhibited IL-12 production by LPS-activated neutrophils ( Figure 1B). To verify whether the results were the consequence of cell death, we analyzed the apoptosis of neutrophils. Our data showed that only budlein A at 100 μM concentration significantly induced neutrophil apoptosis (33.19±5.70%), similar to that observed after culture with dexamethasone (22.83±0.51%) ( Figure 1C). Similar results were observed when fMLP or LPS-stimulated neutrophils were treated with budlein A (100 μM) ( Figure 1C).

Budlein A inhibited T cell proliferation
To determine the anti-inflammatory effects of budlein A on lymphocytes, cells were cultured with PHA in presence or absence of budlein A (1, 10, or 100 μM) or dexamethasone (positive Lymphocytes exhibit proliferative response after PHA stimulation, and the addition of budlein A (100 2A). These results indicate that budlein A interferes with PHA-induced T cell proliferation. Regarding cytokine production, the results evidenced that lymphocytes, with similar effects to dexamethasone ( Figure 2B).
Since budlein A negatively modulates the secretion of cytokines by PHA-stimulated lymphocytes, and this could be a consequence of cell death, their apoptosis was evaluated ( Figure  2C). We found that budlein A (1, 10, or 100 μM) does not induce lymphocyte apoptosis ( Figure  2C). These data indicate that budlein A exerts an important role against lymphocytes' activation, but it did not induce cell death.

DISCUSSION
Although budlein A has been described as activity 1,7,27 immune cells are lacking. In the present study, we demonstrated that budlein A inhibit neutrophils activation and induce cell death, but, despite also inhibiting lymphocyte activation, it did not induce lymphocyte death.
Budlein A inhibited in a concentration-dependent manner the release of biochemical mediators by neutrophils, and this activity could possibly reduce tissue damage caused by the enzyme 24,28 . The MPO activity can be deleterious to the host, and it has been implicated in the pathogenesis of many 21 . Myeloperoxidase has also been established as a risk factor for many forms of cancer via its ability to damage/mutate cellular 9,21 . via the inhibition of nuclear factor-kappa B (NFthe p65 subunit 18 , impairing its activation and the consequent production of inflammatory mediators 15,17 .
Another important effect of budlein A on neutrophils shown in this study was the inhibition of IL-6, CXCL-8, IL-10, and IL-12 production. Promodulate the late events to control the immune response 23 . The imbalance between pro-and antiinflammation and autoimmune diseases 8,20,23 . Imbalance in the production of inflammatory Because low cytokine production might have occurred as a consequence of cytotoxic effects exerted by budlein A, we analyzed the apoptosis of human neutrophils. Only budlein A, at the 100 μM concentration, induces neutrophils apoptosis.
The death of neutrophils observed in this study may be due to the absence of growth factors, particularly CXCL8, since budlein A, as well as other sesquiterpenes, inhibits activation of NFneutrophil survival 1,23  proliferation via other sesquiterpenes; however, the viability rate of the cells had not been evaluated 23 . Here, inhibition of lymphocyte proliferation might be a consequence of undetectable levels of IL-2, an important cytokine inducing lymphocyte proliferation. Thus, budlein A might be an important inhibitor of uncontrolled lymphocyte proliferation without inducing their apoptosis. Such situation would facilitate their use in diseases without directly affecting their survival.
Therefore, our results also show that budlein A induced apoptosis of neutrophils but not lymphocytes, and indicate that budlein A shows distinct immunomodulatory effects on immune cells (Figure 3).

CONCLUSIONS
budlein A reveal an important potential to the study of mechanisms of action and therapeutics 1,5 . In the present study, we demonstrated that budlein A inhibits neutrophil activation and induces cell death, but, despite also inhibiting lymphocyte activation, it did not induce lymphocyte death. Further studies diseases. It will be of interest to evaluate how the balance between these positive and negative effects could be regulated during the course of the immune response.