Open Access

Ex vivo effects of flavonoïds extracted from Artemisia herba alba on cytokines and nitric oxide production in Algerian patients with Adamantiades-Behçet's disease

  • Djamel Messaoudene1, 2Email author,
  • Houda Belguendouz1,
  • Mohamed Laid Ahmedi1,
  • Tarek Benabdekader2,
  • Fifi Otmani3,
  • Malika Terahi4,
  • Pierre Youinou5 and
  • Chafia Touil-boukoffa1
Journal of Inflammation20118:35

https://doi.org/10.1186/1476-9255-8-35

Received: 16 March 2011

Accepted: 21 November 2011

Published: 21 November 2011

Abstract

Background

Adamantiades-Behçet's disease (ABD) is a chronic multisystemic inflammation with unknown pathophysiology. This disorder is associated with a dysregulation of the cytokine network that hyperactivates neutrophils and macrophages. In this study, we investigate the modulatory effects of flavonoïd compounds extracted from Algerian medicinal plant Artemisia herba alba on Th1 and Th2 cytokines and nitric oxide production.

Methods

The modulatory effects of flavonoïds extracted from Artemisia herba alba on cytokines and nitric oxide production by peripheral blood mononuclear cells isolated from Algerian ABD patients and healthy controls were respectively measured by means of ELISA assays and Griess modified method.

Results

Our results show that flavonoïds significantly reduce the production of interleukin-12, the key effector of T helper 1 (Th1) cells and nitric oxide in a dose-dependent manner in Adamantiades-Behçet's disease. In contrast, the production of IL-4, the key marker of Th2 cells was increased.

Conclusion

This study suggests that in vitro supplementation with flavonoïds extracted from Artemisia herba alba could have potential immuno-modulatory effects characterised by a down-regulation and up-regulation of Th1 and Th2 cytokines, respectively. Moreover, flavonoïds may prevent nitric oxide induced damages.

Keywords

Adamantiades-Behçet's disease Artemisia herba alba Flavonoïds Immunomodulation IL-4 IL-12 nitric oxide

Background

Adamantiades-Behest's disease (ABD) is an inflammatory multisystemic disorder involving mucocutaneous, ocular, arthritic, vascular and central nervous systems. It is most prevalent in the Mediterranean countries, including Algeria, and along the Silk Route. Various factors have been reported contribute to the development of the lesions associated to the disease such as, the genetic susceptibility, environmental factors, anomalies in the inflammatory responses and immune system dysfunction [1, 2].

In response to antigens, mediators such as cytokines and chemokines are produced by various cell types, either hematopoietic or non hematopoietic, These mediators orchestrate the immune response by recruitment and activation of different cell types. The involvement of cytokines and chemokines in ABD pathogenesis is reflected by the increase of their concentrations in sera of patients with ABD and some of these mediators correlate with the clinical activity of the disease. Many studies have indeed reported high sera levels of tumor-necrosis factor (TNF)-α, TNF receptor, soluble IL-2R and multiple interleukins (IL-1, IL-6, IL-8, IL-12) [3]. Among them, IL-12 is known to play a major role in the polarization of T helper (Th)1-type cells and sera IL-12 and interferon (IFN)-γ levels are elevated in ABD [4, 5]. Moreover, the increase of IL-12 levels in the peripheral blood mononuclear cells (PBMCs) of patients with ABD have been described [6]. This cytokine is responsible for the development of a Th-1 type response and may play a crucial role in the pathogenesis of the disease [7]. However, other investigators have reported increased sera levels of Th2-type cytokines, including IL-4, IL-10, and IL-13 in ABD patients [8], suggesting disturbed cytokines production in ABD. Such dysregulation in cytokine release contributes to the regulation of several enzymes such as the inducible nitric oxide (NO) synthase (iNOS). The function of NO has been delineated in a variety of inflammatory processes. An excess of NO production or peroxynitrite radical could indeed cause oxidative damages through its action on membrane lipids, DNA, proteins and lipoproteins [9, 10]. These reactions have functional consequences which may be deleterious [11, 12]. The large amounts of NO production have been shown to be correlated with pathophysiology in a plethora of diseases and inflammation processes, such as bowel inflammatory disease [13] and Adamantiades-Behçet's disease [14]. Consequently, the development of molecules aimed to prevent the overproduction of NO constitutes an interesting area of research of a new treatment of chronic inflammatory diseases [1518].

In the absence of curative treatments in ABD, some patients adopt alternative medicine to avoid the irreversible effects of corticotherapy. For example, Artemisia herba-alba (Asteraceae) known as "desert wormwood", or "Chih" as it is commonly named in Algeria is largely consumed. Artemisia herba-alba is a plant of the Lamiacaea family, growing in arid and semi-arid climates and it is widely used in folk medicine in different countries. It is characteristic of the steppes and deserts of the Middle East, North Africa, Spain and North western Himalayas [19]. Artemisia has been a productive genus in the search for new biologically active compounds. Phytochemical investigations have proven that this genus is rich in terpenoids, flavonoïds, coumarins, acetylenes, caffeoylquinic acids and sterols and it was shown that Artemisia has multiple beneficial bioactivities: anti-malarial, anti-viral, anti-tumor, anti-pyretic, anti-hemorrhagic, anti-coagulant, anti-anginal, anti-oxidant, anti-hepatitis, anti-ulcerogenic, antispasmodic and anti-complementary activities [2026].

The flavonoïds detected in Artemisia herba alba show also a structural diversity starting from common flavonoïds (flavones glycosides and favonols) to the methyled flavonoïds which is very unusual [27, 28]. Some beneficial bioactivities of flavonoïds have been proved, such as antibacterial, anticarcinogenic, antioxidant, antimutagenic, anti-inflammatory, activities and immunomodulatory activities [2934]

In the present work was investigated the effect of the flavonoïds extracted from the medicinal plant A. herba alba on the production of IL-12 and IL-4 and we examined nitric oxide production as a marker of the inflammatory response in the PBMC of patients with Adamantiades-Behçet's disease (ABD). Artemisia herba alba may represent an alternative therapy for Algerian patients with ABD.

Methods

Patients and controls

Samples from Twenty patients (8 men and 12 women) were obtained from the ophthalmology and internal medicine service, Bab El Oued Hospital and Algiers Medicinal University Hospital (Mustapha Bacha), respectively. Patients with ABD (females and males) were tested during the clinically active stage. The mean age of the active stage was 38.43 years (20-58 years) and the mean duration of the disease was 7.69 years (1-18 years). ABD was diagnosed according to the criteria defined by the international study group for ABD set up in 1990 [35]. All ABD patients were showing the major symptoms including uveitis, aphtosis, articular and neurological manifestations and they had been treated with colchicine and other oral medication (methylprednisolon, cyclophosphamid). Clinical characteristics of ABD patients were given in Table 1. Each patient has given a written informal consent for the study required by the ethic committee of the national agency of research development in health (ANDRS) which supported our project. The healthy controls consisted of 8 males and 12 females (mean age 39.7 years, range 20-59).
Table 1

Characteristics of active stage of Adamantiades-Behçet's disease patients (number mean ± standard deviation, percentage)

Sex (M/F)

8/12

Age at disease onset (mean years +SD)

34 ± 10

Follow-up duration (years)

7.69 ± 8.5 (1-18)

Uveitis

7/20 (35%)

Aphtosis

6/20 (30%)

Articular symptoms

4/20 (20%)

NeuroBehçet

3/20(15%)

Treatments

Colchicine, methylprednisolon, cyclophosphamid

Plant materials and flavonoïds extraction

The flowering aerial parts of A. herba alba were collected from Djalfa region (city of south Algeria). The plant was then identified in the department of botany of the national institute of agronomy in Algeria. Flavonoïds were extracted according to the extraction method described previously by Paris and Nothis [36]. Briefly, 20 g of the pulverized plant material were macerated for 24 hours in methano-containing water (7:3). The filtrate was evaporated at 40°C to get completely rid of the solvent mixture. The solid extract was then submitted three times to 50 ml n-butanol to collect the flavonoïds mixture. The solution was filtrated and evaporated at 40°C and then dissolved in water. The extracts were kept frozen (-20°C) until used.

PBMC cultures

PBMCs were separated by centrifugation on Ficoll-hypaque gradient and washed twice in phosphate-buffered saline, pH 7.2. Cells were then harvested for test viability with trypan blue then resuspended in complete medium consisting of RPMI-1640 supplemented with 10% fetal- calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

To test cytokines and NO production, PBMC of ABD patients were treated with different concentrations of flavonoïds (5, 10, 20, 30, 40 or 50 μg/mL) and incubated at 37°C and 5% CO2 during 20 hours. Cells were then harvested for test viability and cultures supernatants were conserved at -70°C for cytokines and NO measurements.

For healthy controls and ABD control (before flavonoïds treatment), PBMCs were pre-activated with phytohaemagglutinin (PHA) (5 μg/mL) in 5% CO2 at 37°C during 20 hours to mimic the pre-activated stage of ABD cells.

Cytokine analysis

The concentrations of IL-12 and IL-4 were measured using enzyme linked immunosorbent assays (ELISA) according to manufacture's instructions (Amersham Pharmacia, England). Supernatants samples were added to appropriate wells of a microtiter-plate coated with a specific monoclonal antibody (mAb) against distinct epitopes of IL-12 or IL-4. After incubation for 2 hours, 50 μL of anti IL-12 mAb or anti IL-4 mAb conjugated to horseradish-peroxidase were added. The coloration reaction was read at 540 nm. A standard curve was used to quantify supernatants levels of IL-12 and IL-4. The lowest level of sensitivity was 10 pg/mL for IL-12 and 5 pg/mL for IL-4 of the cytokine.

NO production by PBMCs

PBMCs of patients and NCs were cultured at 5 × 106 cells/uL (100 uL/well) with 100 uL of flavonoïds extract (5, 10, 20, 30, 40 or 50 μg/mL) in 96-well microtiter-plates in a humidified incubator at 37°C and 5% CO2 for 20 hours. Then NO production was assessed by the determination of the final products of NO oxidation. After reduction of nitrates (NO-3) by nitrate reductase containing Pseudomonas oleoveorans Bacteria (ATCC, 8062) containing nitrate reductase, total nitrite (nitrite NO-2+ nitrate NO-3) was determined with the spectrophotometrically Griess reaction as described by Amri et al[37]. Griess reagent 2% p-amminobenzene sulphanamide in 5% phosphoric acid and 0.2% N (1-naphhtiyl) ethylene diamine (dihydrochlorid) was added to the sample. The mixture was incubated for 10 minutes at room temperature and the absorbance at 543 nm was read by spectrophotometer. The concentration was determined with reference to a sodium nitrites NaNO2 standard (0-200 μmol/mL) curve. Results were expressed as μM of nitrites in supernatants of PBMC cultures.

Statistical analysis

Results were expressed as the mean ± standard deviation. Statistical differences were assessed using one-way ANOVA with posthoc test of the means according to Tukey's method. In single mean comparisons, Student's t-test was used to test the data and considered statistically significant for P values < 0.05. Results and graphics were performed with STATISTCA v. 5 software under windows.

Results

In vitro production of cytokine during the active stage of ABD

To quantify the spontaneous production of IL-12, IL-4 and NO during the active stage, we measured their levels in cultures supernatants of PBMC of ABD patients compared with NCs. As shown in Figure 1A, IL-12 levels in ABD patients were higher than in NCs: 1134.02 ± 83.70 versus 583.02 ± 98.44 pg/mL, p < 0.05. The stimulation with flavonoïds showed an increased level of IL-12 in both ABD patients and NCs (1358.63 ± 118.41 versus 1143.27 ± 104.73 pg/mL, respectively). However, we did not observe any significant difference (P > 0.05). In the absence of PHA stimulation, PBMC from ABD patients showed similar level of IL-12 (1134.03 ± 83.69) compared to PBMC from controls after stimulation with PHA (p < 0.85). This result prompted us to use for the same plant extract treatment experiment the preactivated PBMC from controls and those from ABD patients without activation with PHA.
Figure 1

Cytokines and nitric oxide concentration in PBMC supernatants cultures. PBMC (5 × 106 cells/ml) of patients with ABD and healthy controls were cultured with or without 5 μg/ml phytohemagglutnin (PHA) for 20 h. Supernatants were collected and the production level of lL-12 (A) and IL-4 (B) was determined by a sandwich ELISA. Values shown are mean ± S.D.*p < 0.001 was significantly different from the control value. C. Concentration of nitric oxide in the supernatants of culture of PBMC from patients with Adamantiades-Behçet's disease and healthy controls. Cells were treated with 5 μg/mL of PHA. Supernatants were collected after 20 h and the nitrite level was determined by modified Griess reaction. The data represent the mean ± S.D. of cultures. *p < 0.05. NO levels were significantly different from the control values.

Quantitative determination of IL-4 in supernatants of ABD patients and normal control's indicated different profiles according to the disease evolution (Figure 1B). Indeed, during the active phase, we observed a higher spontaneous production in ABD patients' PBMC culture supernatants in comparison to the healthy controls (63.1 ± 37 versus 39.7 ± 13.1 pg/mL, P < 0.05). PHA induced a significant increase in the cytokine production in all groups tested. However, IL-4 levels in PBMCs supernatants, after stimulation with PHA (5 μg/mL) were significantly higher in ABD patients compared to the controls (241.8 ± 33.5 versus 131.3 ± 12.6 pg/mL, p < 0.001) (Figure 1B). In contrast, the preactivated PBMC from controls showed a significant modification in IL-4 production after treatment with PHA at 5 μg/mL compared to ABD patients without stimulation (p < 0.001).

In vitro production of NO during the active stage of ABD

NO measurement in culture supernatants showed that the spontaneous production was higher in ABD PBMC cultures compared to NCs (65.39 ± 15.56 versus 22.84 ± 1.40 μM, p < 0.001). Further, NO levels increased significantly in all culture supernatants after treatment with PHA (P < 0.05). We noticed that NO levels in treated PBMC cultures from ABD was higher than in healthy controls (118.48 ± 15.49 versus 78.31 ± 13.41 μM, p < 0.001) (Figure 1C). The preactivated PBMC cultures from NCs treated with PHA did not show any significant difference compared to those from ABD patients without prestimulation (p = 0.054).

Flavonoïds did not affect cells viability

To assess if there is any cytotoxic effect of flavonoïds, we tested cell viability before and after PHA treatment. Viability of cells was about 90% before and about 70% after experiments with no differences between flavonoïds-treated and untreated control cells. So flavonoïds were not cytotoxic which is consistent with the previous observations [38].

Flavonoïds modulate IL-12 and IL-4 production in PBMCs of ABD patients and NCs

To further confirm the enhancement of the production of the cytokines production by flavonoïds and their aptitude to respond to the PHA preactivated PBMC in healthy controls, flavonoïds were added at different doses 5, 10, 20, 30, 40 or 50 μg/mL for 20 hours. The contents of the wells were centrifuged and kept frozen until analyzed. We observed that flavonoïds did not reduce the IL-12 production in the PBMC stimulation by PHA in NCs (Figure 2). No reversal effects were noticed at any flavonoïd concentrations used. (808.57 ± 123.12 pg/mL, 5 μg/mL of flavonoïds) and (1194.87 ± 53.56 pg/mL, 50 μg/mL of flavonoïds) compared to control values in the absence of flavonoïds (599.47 ± 83.56 pg/mL).
Figure 2

Effect of flavonoïds on IL-12 production by PHA pre-activated peripheral blood mononuclear cells. After washing with medium, various concentrations of flavonoïds (5 -50 μg/mL) was added for a period of 20 h. Supernatants were collected and the levels of IL-12 were determined by ELISA. The data represent the mean ± S.D. of triplicate cultures. *p < 0.001, IL-12 levels are significantly different from the control value.

To test if flavonoïds could induce cytokines modulation in patients without PHA, PBMC from patients were cultured in the presence of different concentrations of flavonoïds (5-50 μg/mL). We observed a significant decrease in IL-12 production in a dose-dependent manner (p < 0.001). Interestingly, we have observed that the pre-treatment by flavonoïds inhibited IL-12 production (1048.89 ± 128.93 pg/mL with 10 μg/mL of flavonoïds) and (778.63 ± 115.21 pg/mL with 50 μg/mL of flavonoïds) compared to control values (1221.42 ± 36.01 pg/mL). (Figure 3). There is no statistical differences between the doses of flavonoïds (30, 40, 50 μg/ml) on IL-12 production in PBMC from ABD patients.
Figure 3

Effect of various concentrations of flavonoïds extracted from A. herba alba on IL-12 production in PBMC (5 × 10 6 cells/mL) of patients with ABD. Presence of cytokines in supernatants was assessed by ELISA. Results are mean ± SD of separate experiments performed in triplicate. *p < 0.05 were significantly different from the control values.

Similarly, the amounts of IL-4 released into supernatants of PBMC from controls subjects after pre-stimulation with PHA were determined by ELISA (Figure 4). Treatment of PBMC by different concentrations of flavonoïds inhibited IL-4 production (73.26 ± 10 pg/mL, 30 μg/mL of flavonoïds) and (89.90 ± 13.25 pg/mL, 50 μg/mL of flavonoïds) compared to the control values in the absence of flavonoïds (55.87 ± 7.98 pg/mL).
Figure 4

Effect of flavonoïds on IL-4 production in PHA-stimulated PBMC of healthy controls. Amounts of IL-4 were measured by ELISA. PBMC (5 × 106 cells/mL) were cultured for 20 h in the absence or presence of flavonoïds after stimulation with PHA (5 μg/mL). Data represent the mean ± SD of three independent experiments in each sample compared to controls value and PHA-treated alone value (ANOVA with post-hoc test).

In PBMC from ABD patients, flavonoïds stimulated IL-4 production in a dose-dependent manner and at significantly greater levels compared to the controls (Figure 5). The highest concentration tested (50 μg/mL) exhibited an increased bioactivity. Treatment of flavonoïds induced IL-4 production (1.116 ± 0.207 pg/mL with 10 μg/mL of flavonoïds) and (0.24 ± 0.060 pg/mL with 40 μg/mL of flavonoïds) compared to the control values in the absence of flavonoïds (55. 87 ± 7.98) (Figure 5).
Figure 5

Effect of flavonoïds extract on IL-4 (pg/mL) production in PBMC of ABD patients ( n = 20). Cells (5 × 106 cells/mL) were treated with different concentrations (5, 10,20,30,40 and 50 μg/mL) of flavonoïds during 20 h. Presence of cytokines in supernatants were measured by ELISA test. Results are mean ± SD of seven separate experiments performed in triplicate. *p < 0.05 IL-4 levels were significantly different from the control value.

Flavonoïds inhibited nitric oxide production in PBMC from ABD patients

Next, we examined the effect of flavonoïds on NO production in PBMC from controls subjects stimulated by PHA were tested. NO levels were measured by Griess modified method. We observed that the treatment did not modulate NO production. As shown in Figure 6, flavonoïds had no statistically significant effect (19.21 ± 2.61 μM with 10 μg/mL of flavonoïds and 16.36 ± 4.25 μM with 50 μg/mL of flavonoïds). The control values in the absence of flavonoïds being 21.03 ± 4.31 μM.
Figure 6

Effect of flavonoïds on nitric oxide production in PHA-stimulated PBMC of healthy controls. PBMC (5 × 106 cells/mL) were stimulated with PHA then cultured with or without flavonoïds (5, 10, 30, 40 and 50 μg/mL). The cell-free supernatants were collected and NO concentration was determined by Griess modified method. The data represents the mean ± S.D. of triplicate cultures.* p < 0.05 NO rates was significantly different from the control value (ANOVA with post-hoc test).

We then tested the inhibitory effect of flavonoïds on NO production in PBMC from ABD patients (Figure 7). Interestingly, we observed that the treatment with flavonoïds during 20 h reduced the NO concentration in all cultures supernatants (p < 0.05). This inhibitory effect was in dose-dependent manner (10 μg/mL and 50 μg/mL). The corresponding nitrite concentrations assessed were respectively: 36.13 ± 5.22 μM and 20.47 ± 3.85 μM
Figure 7

Effect of different concentration of flavonoïds on nitric oxide production by PBMC in patients with ABD. Flavonoïds extracts from A. herba alba are used at the indicated concentrations and compared to the controls. Supernatants were collected to determine the amount of NO. The data represents the mean ± S.D. of triplicate cultures. *p < 0.05 was significantly different from the control value (absence of flavonoïds).

Discussion

It is currently recognized that Th cells may be divided into several functional subclasses, Th-1, Th-2, Treg, Th17 cells, based on the production profile of cytokines and their effects on cell mediated and humoral immunity. Th-1 cells produce IL-12, IFN-γ and enhance cell-mediated immunity. Th-1 cells also can inhibit cell-mediated immunologic activities. In our studies, we showed a significant increase of IL12 levels in supernatant of PBMC culture from ABD patients. IL-12 is an immunoregulatory cytokine regulating cell-mediated immune response by inducing the differentiation of uncommitted CD4 Th cells towards type 1 phenotype and a potent cofactor for stimulating the proliferation of differentiated Th1 cells and IFN-γ synthesis [39]. In our study, we confirmed that IL-12 production by PBMC is significantly higher in ABD patients compared to healthy controls suggesting that IL-12 is involved in the pathogenesis of ABD.

Moreover, Th-2 cells produce IL-4, IL-5 and IL-13 and upregulate humoral immunity [40]. In the current study, higher concentrations of IL-4 were also observed in ABD patients. This Th-2 derived cytokine is primarily involved in the activation of B cells, the promotion of growth and the survival of T cells, the inhibition of macrophage and the activation and suppression of Th-1 cells. Recent studies have showed that IL-4 and IL-12 play a significant role in the regulation of the immune responses by their reciprocal antagonistic mechanisms.

We found that the concentration of nitric oxide in the PBMC supernatant were significantly elevated in ABD patients compared to the healthy controls. Here, we postulated that NO could play an important role in the inflammatory process associated with Adamantiades-Behçet's disease [41]. Several studies have suggested that the overexpression of either inducible NO and proinflammatory cytokines might be intimately involved in the pathogenesis and the evolution of ABD [12, 42]. An increase in the concentration of NO during the ABD was reported in several studies and this in both the sera of patients [43] and also in the synovial liquid [44]. The presence of NO was also observed in uveitis associated with ABD in particular in the aqueous humour [45, 46]. The increase of NO levels in all cases was correlated with the active stage of the ABD.

Stimulation of PBMC cultures from ABD patients with PHA induced an increase of IL-12, IL-4 and NO production. We suggest that the increase of the IL-4 levels in ABD patients after PHA stimulation is probably related to the presence of some factors induced by PHA in PBMC cultures acting on Th-2 cells subset. This purpose remains to be clarified in adequate experiment model. Regarding to the comparison between the production of IL-4 by PHA in healthy controls and ABD patients, the difference observed is probably in relation with the difference in the initial activation level of PBMC state in the two groups of subjects.

Moreover, the increase IL-12 levels after stimulation with PHA on PBMC from ABD patients is related to the production of IFN-γ by Th1 cells. This is consistent with the fact that IFN-γ is known to strongly activate the monocyte/Macrophage system which is the major source of IL-12. Several studies have reported that NO is upregulated by IFN-γ. Recently, our group showed the pivotal role of IFN-γ in pathophysiology of ABD particularly via the NO pathway [46].

There is an increasing interest in herbal medications especially for diseases like ABD [47, 48]. The present study demonstrates that flavonoïds extracts from A. herba alba highly inhibited the production of the proinflammatory cytokine IL-12 in ABD patients PBMC. The mechanism involved remains to be clarified. Furthermore, in our study we reported that the inhibitory effect on IL-12 production was not due to the toxicity of flavonoïds on PBMC. In fact, in our culture system the use of a high flavonoïds concentration at 50 pg/ml after 20 h incubation yielded almost 70% viable cells. It has been shown that increased IL-12 levels and Th1 cytokines did occur in patients with ABD and have been associated with the pathogenesis.

In contrast to IL-12, we found that flavonoïds promoted a significant increase in IL-4 produced. IL-4 is one of the Th-2 cytokines which has been associated with an improvement in the inflammatory diseases [49]. In the study reported by Koteswara Rao et al., [50], flavonoïds have been shown to inhibit extensively the proinflammatory cytokines like TNF- α, IL-12 in a dose-dependent manner. These authors suggested that flavonoïds mediate differentiation from Th-1 to Th-2 cell types and our results are consistent with this study. We also suggest the role of other cytokines or immunoregulatory mediators in the differential regulation of IL-4 (upregulated) and IL-12 (downregulated). These suggestions remain to be clarified in an adequate experimental model. However, it is possible that the inhibition of IL-12 production may be partially mediated by the action of flavonoids through IL-4 induction as both IL-4 and IL-12 have shown to have antagonism effects. IL-4 exerts strong inhibition on Th1-mediated inflammatory processes involving the regulation of the synthesis of inflammatory cytokines (IL-2 TNF-α, IL-1β) and chemokines (CXCL8, CXCL10, CCL2). The effect of flavonoïds on cytokine modulation constitutes a very exciting finding for their possible therapeutic applications.

For the role of NO, we suggest that flavonoïds regulate not only the balance Th1/Th2 towards Th-2 but also NO production. The results presented here show that flavonoïds isolated from A. herba halba, affect also NO production in PBMC isolated from patients with ABD in a dose-dependent manner. The inhibitory activity could be resulted from the inhibition of iNOS expression and/or its activity.

Conclusion

We report here the evidence that the Th-1 cytokines (IL-12) and NO are involved in the pathogenesis of ABD. Our limited follow-up study also suggests that flavonoïds extracts from A. herba alba have an effect on the inhibition and the stimulation of the production of IL-12 and of IL-4, respectively. This constitutes a way to switch the immune response from Th-1 to Th2. Further investigations will focus on the assessment of the biological activity of this extract in vivo and on the chemical identification of the active components responsible for the anti-inflammatory activity. The knowledge of the role of flavonoïds in the immunomodulatory mechanisms in ABD is a promising area for the development of new natural's agents for the treatment of the disease and other immune-mediated diseases.

Abbreviations

ABD: 

Adamantiades-Behçet's disease

Th: 

T helper cell

IL: 

interleukin: IFN-γ: interferon- γ

NO: 

Nitric oxide

PBMC: 

peripheral blood mononuclear cells

NO3-

nitrate

NO2-

nitrite

NOS2: 

nitric oxide synthase-2.

Declarations

Acknowledgements

The authors would like to thank all patients and voluntary participants at this study. This work was supported by a grant form the ANDRS (National Agency for Scientific Development and Research in Health) Project No.1601/09/2009).

We are grateful to Dr. M.A. Ayoube, Western Australian Institute for Medical Research, for expert preparation of the manuscript.

Authors’ Affiliations

(1)
Laboratoire de Biologie Cellulaire et Moléculaire (LBCM), FSB, USTHB, Université de Bab-Ezzouar
(2)
Département de Biologie, Faculté des sciences, université de Boumerdes
(3)
Service de médecine Interne, CHU Mustapha Bacha
(4)
Service d'ophtalmologie, CHU Bab El Oued
(5)
Laboratoire d'immunologie, Centre Hospitalier Universitaire

References

  1. Amoura Z, Guillaume M, Caillat-Zucman S, Wechsler B, Piette JC: Pathophysiology of Behçet's disease. La revue de medicine interne. 2006, 27: 843-853. 10.1016/j.revmed.2006.02.014.View ArticleGoogle Scholar
  2. Yazici H, Fresko I, Yurdakul S: Behcet's syndrome: disease manifestations, management, and advances in treatment. Nature clinical practice. 2007, 3: 148-155. 10.1038/ncprheum0436.PubMedGoogle Scholar
  3. Hamzaoui K, Hamzaoul A, Guemira F, Bessioul M, Hamza M, Ayed K: Cytokine profile in Behçet's disease patients. Scand J Rheumatol. 2002, 31: 205-210. 10.1080/030097402320318387.PubMedView ArticleGoogle Scholar
  4. Sugi-Ikai N, Nakazawa M, Nakamura S, Ohno S, Minami M: Increased frequencies of interleukin-2- and interferon-gamma producing T cells in patients with active Behçet's disease. Invest Ophthalmol Vis Sci. 1998, 39: 996-1004.PubMedGoogle Scholar
  5. Saruhan-Direskeneli G, Yentür SP, Akman-Demir G, Işik N, Serdaroğlu P: Cytokines and chemokines in neuro-Behçet's disease compared to multiple sclerosis and other neurological diseases. J Neuroimmunol. 2003, 145: 127-134. 10.1016/j.jneuroim.2003.08.040.PubMedView ArticleGoogle Scholar
  6. Imamura Y, Kurokawa MS, Yoshikawa H, Nara K, Takada E, Masuda C, Tsukikawa S, Ozaki S, Matsuda T, Suzuki N: Involvement of Th1 cells and heat shock protein 60 in the pathogenesis of intestinal Behçet's disease. Clin Exp Immunol. 2005, 139: 371-378. 10.1111/j.1365-2249.2005.02695.x.PubMedPubMed CentralView ArticleGoogle Scholar
  7. Frassanito MA, Dammacco R, Cafforio P, Dammacco F: Th1 polarization of the immune response in Behçet's disease: a putative pathogenetic role of interleukin-12. Arthritis Rheum. 1999, 42: 1967-1974. 10.1002/1529-0131(199909)42:9<1967::AID-ANR24>3.0.CO;2-Z.PubMedView ArticleGoogle Scholar
  8. Raziuddin S, Al-Dalaan A, Bahabri S, Siraj AK, Al-Sedairy S: Divergent cytokine production profile in Behçet's disease. Altered Th1/Th2 cell cytokine pattern. J Rheumatol. 1998, 25: 329-33.PubMedGoogle Scholar
  9. Buney S, Caufield JL, Niles JC, Wishnok JS, Tannbaum SR: The Chemistry of DNA damage from nitric oxide and peroxynitrite. Mutation Res. 1999, 424: 37-49. 10.1016/S0027-5107(99)00006-8.View ArticleGoogle Scholar
  10. Steffen Y, Jung T, Klotz LO, Schewe T, Grune T, Sies H: Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells. Protection by (-)-epicatechin. Free Radic Biol Med. 2007, 42: 955-70. 10.1016/j.freeradbiomed.2006.12.024.PubMedView ArticleGoogle Scholar
  11. Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Amer J Physiol. 1996, 271: C1424-C1437.PubMedGoogle Scholar
  12. Yu L, Gengro PE, Niedrberger M, Burk TJ, Schrier RW: Nitric oxide: A mediator in rat tubular hypoxia/reoxygenation injury. Proceeding of the Natural Academy of Science USA. 1994, 91: 1691-1695. 10.1073/pnas.91.5.1691.View ArticleGoogle Scholar
  13. Rafa H, Abdelouaheb K, Belkhelfa M, Medjeber O, Saoula H, Zeriguine R, Nakmouche M, Touil-Boukoffa C: Nitric oxide modulation by retinoic acid, IL-17A and TNF-α in PBMC and colonic mucosa cultures from Algerian patients with inflammatory bowel disease. Cytokine. 2010, 52: 17-34.View ArticleGoogle Scholar
  14. Guenane H, Hartani D, Chachoua L, Lahlou-Boukoffa OS, Mazari F, Touil-Boukoffa C: Production des cytokines Th1/Th2 et du monoxyde d'azote au cours de l'uvéite Behçet et de l'uvéite idiopathique. J Fr Ophtalmol. 2006, 29: 146-152. 10.1016/S0181-5512(06)73762-7.PubMedView ArticleGoogle Scholar
  15. Nathan C: NO as a secretory product of mammalian cells. J FASEB. 1992, 6: 3051-3064.Google Scholar
  16. Hobbs AJ, Higgs A, Moncada S: Inhibition of nitric oxide synthase as a potential, therapeutic target. Ann Res Pharmacol Toxicol. 1999, 39: 191-220. 10.1146/annurev.pharmtox.39.1.191.View ArticleGoogle Scholar
  17. Nakagawa T, Yokozawa T: Direct scavenging of nitric oxide by green tea. Food Chem Toxicol. 2002, 40: 1745-1750. 10.1016/S0278-6915(02)00169-2.PubMedView ArticleGoogle Scholar
  18. Shen SC, Lee WR, Lin HY, Huang HC, Ko Ch, Yang LL, Chen YC: In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production. Eur J Pharmacol. 2002, 446: 187-194. 10.1016/S0014-2999(02)01792-2.PubMedView ArticleGoogle Scholar
  19. Vernin G, Merad O, Vernin GMF, Zamkotsian RM, Parkanyi C: GC-MS analysis of Artemisia herba-alba Asso essential oils from Algeria. Dev Food Sci. 1995, 3A: 147-205.View ArticleGoogle Scholar
  20. Ahmed AA, Abou El-Ela M, Jakupovic J, Seif El-Din AA, Sabri N: Eudesmanolides and other constituents from Atemisia herba alba. Phytochem. 1990, 29: 3661-3663. 10.1016/0031-9422(90)85297-S.View ArticleGoogle Scholar
  21. Tang HQ, Hu J, Yang L, Tan RX: Terpenoids and flavonoids from Artemisia species. Planta Med. 2000, 66: 391-393. 10.1055/s-2000-8538.PubMedView ArticleGoogle Scholar
  22. Tan RX, Tang HQ, Hu J, Shuai B: Lignans and sesquiterpene lactones from Artemisia Sieversiana and Inula racemosa. Phytochem. 1998, 49: 157-161. 10.1016/S0031-9422(97)00889-3.View ArticleGoogle Scholar
  23. Jiangsu New Medicine College: A Comprehensive Dictionary of Traditional Chinese Medicine. Shanghai Science and Technology Press, Shanghai. 1977, 627-Google Scholar
  24. El-Thaher TS, Matalka KZ, Taha HA, Badwan AA: Ferula harmonis 'zallouh' and enhancing erectile function in rats: efficacy and toxicity study. Int J Impot Res. 2001, 13: 247-251. 10.1038/sj.ijir.3900706.PubMedView ArticleGoogle Scholar
  25. Davis SR, Dalais FS, Simpson ER, Murkies AL: Phytoestrogens in health and disease. Recent Prog Horm Res. 1999, 54: 185-211.PubMedGoogle Scholar
  26. Singh MM, Agnihotri A, Garg SN, Agarwal SK, Gupta DN, Keshri G, Kamboj VP: Antifertility and hormonal properties of certain carotane sesquiterpenes of Ferula jaeschkeana. Planta Med. 1988, 54: 492-494. 10.1055/s-2006-962526.PubMedView ArticleGoogle Scholar
  27. Saleh NAM, El- Negoumy SI, Abd-All MF, Abou- Zaid MM, Dellamonica G, Chopin J: Flavonoid glycosides of Artemisia monosperma and A. herba alba. Phytochem. 1985, 24: 201-203. 10.1016/S0031-9422(00)80845-6.View ArticleGoogle Scholar
  28. Saleh NAM, El-Negoumy SI, Abou-Zaid MM: Flavonoids of Artemisia judaica, A. monosperma and Artemisia herba-alba. Phytochem. 1987, 26: 3059-3064. 10.1016/S0031-9422(00)84593-8.View ArticleGoogle Scholar
  29. Erlund I: Review of the flavonoïds quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability and epidemiology. Nutr Res. 2004, 24: 851-874. 10.1016/j.nutres.2004.07.005.View ArticleGoogle Scholar
  30. Nair MP, Mahajan S, Reynolds JL, Aalinkeel R, Nair H, Schwartz SA, Kandaswami C: The flavonoid quercetin inhibits proinflammatory cytokine (Tumor necrosis factor alpha) gene expression in normal peripheral mononuclear cells via modulation of the NF-κB system. Clin Vaccine Immunol. 2006, 13: 319-328. 10.1128/CVI.13.3.319-328.2006.PubMedPubMed CentralView ArticleGoogle Scholar
  31. Harborne JB, Williams CA: Advances in flavonoids research since 1992. Phytochem. 2000, 55: 481-504. 10.1016/S0031-9422(00)00235-1.View ArticleGoogle Scholar
  32. Hsu CL, Yen GC: Phenolic compounds: Evidence for inhibitory effects against obesity and their underlying molecular signalling mechanisms. Mol Nutr Food Res. 2008, 52: 53-61. 10.1002/mnfr.200700393.PubMedView ArticleGoogle Scholar
  33. Peterson J, Dwyer J: Flavonoids: Dietary occurrence and biochemical activity. Nutrition Res. 1998, 18: 1995-2018. 10.1016/S0271-5317(98)00169-9.View ArticleGoogle Scholar
  34. Zhang J, Shen Q, Lu JC, Li JY, Liu WY, Yang JJ, Li J, Xiao K: Phenolic compounds from the leaves of Cyclocarya paliurus (Batal.) Iljinskaja and their inhibitory activity against PTP1B. Food Chem. 2010, 119: 1491-1496. 10.1016/j.foodchem.2009.09.031.View ArticleGoogle Scholar
  35. International Study Group for Behçet's disease: Criteria for diagnosis of Behçet's disease. Lancet. 1990, 335: 1078-1080.Google Scholar
  36. Paris R, Nothis A: Plantes médicinales de la nouvelle Calédonie./2, Etude particulière de plantes à dérivés polyphénoliques. Plant Med Phytothérapie. 1970, 4: 63-74.Google Scholar
  37. Amri M, Aït Aïssa S, Belguendouz H, Mezioug D, Touil-Boukoffa C: In Vitro Antihydatic Action of Gamma Interferon (IFN) is Dependent of Nitric Oxide (NO) pathway. J Interferon Cytokine Res. 2007, 22: 781-787.View ArticleGoogle Scholar
  38. Krakauer T, Li BQ, Young HA: The favonoid baicalin inhibits superantigen-induced inflammatory cytokines and chemokines. FEBS Letters. 2001, 500: 52-55. 10.1016/S0014-5793(01)02584-4.PubMedView ArticleGoogle Scholar
  39. Trinchieri G: Interleukin 12 and its role in the generation of TH1 cells. Immunol Today. 1993, 14: 335-338. 10.1016/0167-5699(93)90230-I.PubMedView ArticleGoogle Scholar
  40. Elenkov IJ, Chrousos GP: Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines and susceptibility to disease. Trends Endocrinol Metab. 1999, 10: 359-68. 10.1016/S1043-2760(99)00188-5.PubMedView ArticleGoogle Scholar
  41. Evereklioglu C, Turkoz Y, Er H, Inaloz HS, Ozbek E, Cekmen M: Increased serum nitric oxide production in patients with Behçet's disease: is it a new activity marker?. J Am Acad Dermatol. 2002, 46: 50-54. 10.1067/mjd.2002.118338.PubMedView ArticleGoogle Scholar
  42. Belguendouz H, Messaoudene D, Hartani D, Chachoua L, Ahmedi ML, Lahmar-Belguendouz K, Lahlou-Boukoffa OS, Touil-Boukoffa C: Effet de la corticothérapie sur la production des interleukines 8, 12 et du monoxyde d'azote au cours des uvéites « Behçet » et « idiopathique ». J Fr Ophtalmol. 2008, 31: 387-395. 10.1016/S0181-5512(08)71433-5.PubMedView ArticleGoogle Scholar
  43. Evereklioglu C, Er H, Turkoz Y, Cekmen M: Serum levels of TNF-alpha, sIL-2R, IL-6, and IL-8 are increased and associated with elevated lipid peroxidation in patients with Behçet's disease. Mediators Inflamm. 2002, 11: 87-93. 10.1080/09629350220131935.PubMedPubMed CentralView ArticleGoogle Scholar
  44. Duygulu F, Evereklioglu C, Calis M, Borlu M, Cekmen M, Ascioglu O: Synovial nitric oxide concentrations are increased and correlated with serum levels in patients with active Behcet's disease: a pilot study. Clin Rheumatol. 2005, 24: 324-30. 10.1007/s10067-004-1015-3.PubMedView ArticleGoogle Scholar
  45. Yilmaz G, Sizmaz S, Yilmaz ED, Duman S, Aydin P: Aqueous humor nitric oxide levels in patients with Behçet disease. Retina. 2002, 22: 330-5. 10.1097/00006982-200206000-00012.PubMedView ArticleGoogle Scholar
  46. Belguendouz H, Messaoudene D, Lahmar K, Ahmedi L, Medjeber O, Hartani D, Lahlou-Boukoffa O, Touil-Boukoffa C: Interferon-γ and Nitric Oxide Production During Behçet Uveitis: Immunomodulatory Effect of Interleukin-10. J interferon Cytokine Res. 2011, 31: 643-51. 10.1089/jir.2010.0148.PubMedView ArticleGoogle Scholar
  47. Mi-Sun K, Hwan-Suck C, Jun-Gyoung L, Woon Ki L, Chung-Yeon H, Eon-Jeong L, Kwang-Ho C, Dac-Han W, Hyung-Min K: Inhibition of cytokine production by the traditional oriental medicine, 'Gamcho-Sasim-Tang' in mitogen-stimulated peripheral blood mononuclear cells from Adamantiades-Behçet's patients. J Ethnopharmacol. 2002, 83: 123/-128. 10.1016/S0378-8741(02)00221-0.View ArticleGoogle Scholar
  48. Seonghyang S, Dongsik B, Seung Ihm L, Young Ae K, Eun-So L, Jee Yong H, Jang Hyun K, Suh Young C, Sungnack L: Combined treatment with colchicine and Herba Taraxaci (arazacum mongolicum Hand.-Mazz.) attenuates Behçet's disease-like symptoms in mice and influences the expressions of cytokines. Interl Immunopharmacol. 2003, 3: 713-721. 10.1016/S1567-5769(03)00071-7.View ArticleGoogle Scholar
  49. Noben-Trauth N, Hu-Li J, Paul WE: IL-4 secreted from individual naive CD4+ T cells acts in an autocrine manner to induce Th2 differentiation. Eur J Immunol. 2002, 32: 1428-33. 10.1002/1521-4141(200205)32:5<1428::AID-IMMU1428>3.0.CO;2-0.PubMedView ArticleGoogle Scholar
  50. Koteswara Rao Y, Fang SH, Tzeng YM: Anti-inflammatory activities of flavonoids isolated from Caesalpinia pulcherrima. J Ethnopharmacol. 2005, 100: 249-253. 10.1016/j.jep.2005.02.039.View ArticleGoogle Scholar

Copyright

© Messaoudene et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement