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IMMUNOMODULATORY ACTIVITY OFTHE WATER EXTRACT OF Thymusvulgaris, Thymus daenensis, AND Zatariamultiflora ON DENDRITIC CELLS AND TCELLS RESPONSESZahra Amirghofran PhD a b , Hossein Ahmadi a & Mohammad HosseinKarimi ca Department of Immunology, Medical School, Shiraz University ofMedical Sciences, Shiraz, Iranb Medicinal and Natural Products Chemistry Research Center, ShirazUniversity of Medical Sciences, Shiraz, Iranc Shiraz Transplant Research Center, Nemazee Hospital, ShirazUniversity of Medical Sciences, Shiraz, IranAccepted author version posted online: 20 Jan 2012.
To cite this article: Zahra Amirghofran PhD , Hossein Ahmadi & Mohammad Hossein Karimi (2012)IMMUNOMODULATORY ACTIVITY OF THE WATER EXTRACT OF Thymus vulgaris, Thymus daenensis,AND Zataria multiflora ON DENDRITIC CELLS AND T CELLS RESPONSES, Journal of Immunoassay andImmunochemistry, 33:4, 388-402, DOI: 10.1080/15321819.2012.655822
To link to this article: http://dx.doi.org/10.1080/15321819.2012.655822
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IMMUNOMODULATORY ACTIVITY OF THE WATER EXTRACT OFThymus vulgaris, Thymus daenensis, AND Zataria multiflora ON
Zahra Amirghofran,1,2 Hossein Ahmadi,1 andMohammad Hossein Karimi3
1Department of Immunology, Medical School, Shiraz University of Medical Sciences,Shiraz, Iran2Medicinal and Natural Products Chemistry Research Center, Shiraz University ofMedical Sciences, Shiraz, Iran3Shiraz Transplant Research Center, Nemazee Hospital, Shiraz University of MedicalSciences, Shiraz, Iran
& Thymus vulgaris (thyme), Thymus daenensis, and Zataria multiflora are medicinal plantsbeing used widely for infections and inflammatory diseases in folk medicine. In this study, the effects ofthe water extract of these plants on the activation of dendritic cells (DCs) and T cells was investigated.Both T. vulgaris and Z. multiflora decreased the proliferation of mitogen-stimulated lymphocytes,whereas T. daenensis induced cell proliferation in a dose-dependent manner (p< 0.001). All the threeplants increased the CD40 expression on DCs (p< 0.04). The extent of allogenic T cell proliferation inthe presence of T. vulgaris and Z. multiflora extracts was significantly decreased (p< 0.02). Theeffect of the extracts on secretion of IFN-c and IL-4 cytokines showed that none of the extracts influencedthe pattern of cytokine production by T helper (Th) cells toward a Thl or Th2 profile. In conclusion, allthe extracts had the ability to activate DCs. Whereas Z. multiflora and T. vulgaris extracts showedimmunoihibitory effects on allogenic T cell proliferation, the main effect of T. daenensis was on mito-genic T cell response. These data may partly explain the mechanisms underlying the beneficial immu-nomodulatory effects of these extracts in infections and immune-related diseases.
Keywords dendritic cells, medicinal plants, Thymus daenensis, Thymus vulgaris, Zatariamultiflora
INTRODUCTION
Dendritic cells (DCs) are the most potent antigen-presenting cells(APC) that play a central role in the initiation of T-cell responses to tumors,microbial pathogens, and inflammation.[1] These cells can conduct the
Address correspondence to Zahra Amirghofran, PhD, Immunology Department, Medical School,Shiraz University of Medical Science, Shiraz 71345-1798, Iran. E-mail: [email protected]
Journal of Immunoassay and Immunochemistry, 33:388–402, 2012Copyright # Taylor & Francis Group, LLCISSN: 1532-1819 print/1532-4230 onlineDOI: 10.1080/15321819.2012.655822
DENDRITIC CELLS AND T CELLS RESPONSES
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naive T cells into either immunogenic (T helper, Th) or immunotolero-genic (regulatory T cells) after exposure to a particular antigen.[2] DCsare scattered throughout the peripheral tissues as immature cells withthe capability to capture and process antigens. After capturing the antigens,these cells migrate to secondary lymphoid organs and locate in the T-cellareas and present antigens to naive T cells.[3] During migration, they differ-entiate into mature DCs and lose their ability to capture antigens andacquire the ability to interact and stimulate T cells with the help of themajor histocompatibility complex (MHC) class II molecule.[4] MatureDCs are initial producers of cytokines that promote T helper cell differen-tiation, thus controlling the quality of the immune responses.[5,6]
Medicinal plants have been widely investigated for their possible effectson the immune response to find out their therapeutic applications inimmune-related illnesses.[7–11] In various studies, the immunomodulatoryeffects of several plants on DCs have been investigated, e.g., Scutellaria barbata,a traditional herbal medicine that is widely used for its anti-inflammatoryactivity in China, has revealed a down-regulatory effect on the function ofDCs.[12] Mucuna (Mucuna pruviens var. utilis) has been reported to possessa DC differentiation- and maturation-inducing activity. This plant specificallycan stimulate differentiation of bone marrow cells to immature DCs.[13] Theextract of Chrysanthemum coronarium has also caused DC maturation andup-regulation of surface expression levels of MHC and CD86 molecules.[14]
During of our course of study on several plants, we examined the immu-nomodulatory effects of the extracts of three plants belonging to the Labiateafamily on human lymphocyte activation. The plants were Thymus vulgaris(thyme), an important medicinal plant native to the Mediterranean regionof Europe; and Thymus daenensis and Zataria multiflora, which are two nativeplants to Iran. T. vulgaris is currently used for treatment of infectious diseases,asthma, and various other inflammatory diseases.[15] The essential oil ofT. vulgaris has shown antibacterial, antifungal, and antioxidant effects.[16]
T. daenensis is an endemic plant that is used for treatment of infectious dis-eases in folkmedicine.[17] Z. multiflora, an important medicinal plant growingin Iran, Pakistan, and Afghanistan, is also currently used for the treatment ofinfections, asthma, sore throat, and edema in folk medicine.[18] All of thesethree plants are known as avishan in Persian, and are very similar in theirmajor constituents and pharmacological properties. In a previous study,the in vivo anti-inflammatory effect of the water extract of one of these plants,Z. multiflora, on acute and chronic inflammation has been reported.[19] Inour preliminary study, the water extract of these plants showed the abilityto activate (T. daenensis) and inhibit (Thymus vulgaris and Zataria multiflora)proliferation of human lymphocytes. In view of the central role of DCs inactivation of human T lymphocytes, we decided to study the possible immuo-modulatory effects of these extracts on the expression of DC differentiation
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markers, as well as their ability to modulate T cell proliferation. Study of theeffect of these plants, currently used for various medicinal purposes includ-ing infections and inflammatory diseases, on activity of DCs may explainone of the mechanisms underlying their beneficial effects in these diseases.
EXPERIMENTAL
Preparation of the Extracts
The dried aerial parts of the plants T. daenensis, T. vulgaris, and Z. multi-flora were collected at the time of flowering ( June) from Dena Mountains,Estahban, and Shiraz in the south of Iran, respectively, and identified byDr. Mojtaba Asadollahi from the Medicinal and Natural Products ChemistryResearch Center, Shiraz University of Medical Sciences, Shiraz, Iran. Vou-cher specimens with numbers 12-1-61, 14828, and 85-16 for T. daenensis,T. vulgaris, and Z. multiflora, respectively, were deposited in the herbariumof the Shiraz University. Plants were air dried in shade, powdered, anddefatted with petroleum ether for 4 h. The defatted aerial parts of theplants were suspended in water for three period of 24 hours each, and aftercollection of the supernatants, they were dried on rotary evaporator. Theyields (w=w) of the extracts were 4.6% for T. vulgaris, 14% for Z. multiflora,and 13.8% for T. daenensis. Dried extracts were later dissolved in RPMI 1640medium (Sigma, St. Louis, USA) to obtain 2mg=mL solution.
Animals
The study was conducted on eight- to ten-week-old male BALB=c andC57BL=6 mice obtained from Razes Institute, Shiraz, Iran. Animal experi-ments were conducted in accordance with current ethical regulations onanimal research. All protocols were approved by the ethical committee ofthe Shiraz University of Medical Sciences for handling of the animals.The animals were maintained under standard laboratory conditions andreceived standard mouse chow and water ad libitum.
Preparation of DCs From Mouse Spleen
DCs were prepared from spleen of BALB=c mice as previouslydescribed,[20] with some modifications. Briefly, after cervical dislocation,spleens were removed from mice, cut into small fragments, and thendigested with 1mg=mL collagenase D (Roche, Molecular Biochemicals,Mannheim, Germany) and 0.02mg=mL DNase (Roche) in RPMI 1640medium (Sigma). They were incubated for 30min at 37�C under 5%
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CO2, then dissociated in RPMI containing 5mM EDTA. After washing withphosphate-buffered saline (PBS) and centrifugation at 300�g and 4�C for10min, the cell pellet was resuspended in culture medium, and the lowdensity and dead cells were depleted by density gradient centrifugationon Nycodenz 12.5% (w=v), d¼ 1.068 (Axis-Shied, Norway) at 600�g for15min at 4�C. Splenic DCs were collected at the interface and washed twicewith RPMI. Cells were cultured in complete medium containing 5%heat-inactivated fetal calf serum, L-glutamine, pyruvate, penicillin, andstreptomycin (all from Gibco, Germany) and incubated for 2 h at 37�C ina 5% CO2 atmosphere. Culture plates were then washed gently with warmRPMI medium, and the non-adherent cells were discarded. The adherentcells were maintained in the complete culture medium in the presenceor in the absence of extracts and incubated at 37�C in a 5% CO2 atmos-phere for 18h. After that, DCs become non-adherent and float in themedium. The DCs were collected and after adjusting the cell count to1� 105=mL immediately used in the assays. Overnight-matured DCsisolated using this method were 90% viable. To verify the purity of DC pre-parations, they were stained with anti-CD11c monoclonal antibody. Thecells were routinely more than 90% CD11cþ as assessed by flow cytometry.
Cell Viability Assay
The cytotoxic effects of the extracts on DCs were examined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayas described previously.[21] Briefly, after 2 h incubation of DCs as men-tioned above, they were seeded in flat-bottom 96-well plates and treatedwith 0.01–200mg=mL of the extracts for 18 h. 10mL of MTT (5mg=ml,Sigma) was added to each well and incubated for an additional 4 h at37�C, followed by treatment with 100mL of dimethyl sulfoxide (DMSO)for 4h. The optical density (OD) of each well was determined by spectro-photometry at dual wavelengths of 570 and 630nm on a microplate reader(Pharmacia, Sweden). Viability percentage was calculated by the followingformula: (OD of treated cells=OD of control)� 100. The control wasextract-untreated cells. All experiments were plated in triplicate wells andwere performed at least three times.
In Vitro Cell Proliferation Assay
The effects of extracts on cell proliferation was measured using a5-bromo-20-deoxy-uridine (BrdU) cell proliferation kit (Roche) accordingto the manufacturer’s instructions. Splenocytes were separated fromBALB=c mice and grown at concentration of 1� 105=well in flat-bottom
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96-well plates for 48 h in the presence or absence of different concentrationof the extracts. Concanavalin (Con) A (Pharmacia, Sweden) at a subopti-mal concentration (1 mg=mL) was added to the culture of splenocytes. Afterlabeling with BrdU for the final 18 h of the incubation period, DNA wasdenatured and cells were incubated with anti-BrdU monoclonal antibodyfor detecting incorporated BrdU. The OD related to the BrdU level wasmeasured with a microplate reader at 450nm. The proliferation indexwas calculated by dividing the OD of treated cells to OD of control. Thecontrol was extract-untreated cells containing Con A.
Flow Cytometry Analysis
Phenotypic analysis of DCs was performed by flow cytometry. IsolatedDCs were treated with the extracts for 18h and then transferred to flowcytometry tubes. Cells were washed at 300g for 7min at 4�C, then werestained with 1mL of monoclonal antibodies including phycoerythrin (PE)-conjugated anti CD11c, fluorescine isothiocyanate (FITC)-conjugatedMHC II, FITC-conjugated CD86, FITC-conjugated CD40, and FITC- andPE-conjugated isotype controls, all from Becton Dickinson (BD, San Jose,CA). Cell samples were incubated with antibodies at 4�C for 30min, thenwere washed twice with PBS and fixed with 1% paraformaldehyde. Cells wereanalyzed on a FACSCalibur flow cytometer (BD) that was equipped with Cell-Quest software (BD). Data were analyzed using WinMidi software (Scripps,LaHoya, CA). Forward- and side-scatter parameters were used to gate livecells. The mean fluorescence intensity (MFI) for different markers was com-pared with that of negative control (extract-untreated cells containingmedium alone). Tumor necrosis factor (TNF)-a, the known DC maturationcytokine, at concentration of 50ng=mL was used as the positive control.
Allogenic Mixed Lymphocyte Reaction (MLR)
T-cells were co-cultured at different ratios with allogenic DCs fromBALB=c mice. DCs were treated with 25mg=mL mitomycin (Sigma) at37�C for 20min and washed three times with culture medium and thenadjusted to 1� 104 cells=mL. Responder T cells were isolated from spleenof C57BL=6 mice by a nylon wool-enrichment technique, and adjusted to2� 105 cells=mL. The purity of T cells was determined using anti CD3monoclonal antibody (more than 90%) by flow cytometry analysis. T cellsuspension (100mL) was added to 100 mL of DCs cultured in 96-wellround-bottomed culture plates in the presence or absence of the extracts.Cells were incubated for 4 days at 37�C and 5% CO2, after which BrdU wasadded into the wells. Cell proliferation after a further 24h was measured by
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BrdU proliferation assay as mentioned before. Controls were a series oftriplicate wells containing DCs and T cells without the extracts. The pro-liferation index was calculated by dividing the OD of treated cells to theOD of the controls. In a similar experiment after 3 days of co-culture,the supernatant of cells were collected for cytokine assay.
Cytokine Assay
The supernatants fromMLR culture were collected and stored at�20�Cuntil assayed for cytokines. The levels of mouse interleukin (IL)-4 and inter-feron (IFN)-c in culture supernatants were determined in duplicate by theenzyme-linked immunosorbent assay (ELISA, R&D System, Germany)according to the manufacturer’s protocol. Microtiter plates were coatedwith specific antibody to capture the cytokine in MLR culture supernatants.A second layer antibody was then added. Cytokine concentrations weredetermined by using a standard curve resulting from known amounts ofthe relevant cytokine using absorbance readings at 450 nm. The IL-4 andIFN-c assays both had a lower sensitivity limit of 2 pg=mL.
Statistical Analysis
Data were presented as mean� standard deviation, and the differencesbetween groups were assessed using Graph Pad Prism software (Graph-PadSoftware Inc., San Diego, CA), the Student’s t-test, and one-way analysis ofvariance (ANOVA). P values less than 0.05 were considered as significant.
RESULTS
Effects of the Extracts on Proliferation of Lymphocytes
To study the general effect of the extracts on immune cells, their effectson the proliferation of activated-mouse spleen cells were investigated. Asshown in Figure 1, extracts demonstrated different effects on the prolifer-ation of lymphocytes. Both T. vulgaris and Z. multiflora decreased cell pro-liferation; the proliferation indices for these extracts at 50mg=mL were0.78� 0.007 (p¼ 0.002) and 0.65� 0.11 (P¼ 0.02), respectively. T. daenensisinduced cell proliferation at different concentrations ranging from1.98� 0.19 at 0.1mg=mL to 1.44� 0.31 mg=mL at 100mg=mL (p< 0.001).
Effects of the Extracts on Viability of DCs
Various concentrations of the extracts were examined for their cyto-toxicity on DCs by MTT colorimetric assay. According to the results
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obtained, concentrations of �50mg=mL of T. vulgaris and Z. multifloraand �200mg=mL of T. daenensis, which did not show any significant cyto-toxic effects on DCs, were selected for the next experiments (data notshown).
FIGURE 1 Effects of the extracts on the proliferation of activated lymphocytes determined by BrdU cellproliferation assay. Mice spleen cells were stimulated with concanavalin (Con) A and then treated withdifferent concentrations of the extracts. Control was extract-untreated cells stimulated with Con A. Theproliferation index was calculated by dividing the optical density (OD) of treated cells to OD of control.Data represent mean� standard error of at least three independent experiments. �p< 0.02.
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Effects of the Extracts on Phenotype of DCs
DCs isolated from the spleen of BALB=c mice were treated with differ-ent concentrations of the extracts and the changes in the percentageexpression and fluorescent intensity of MHC II, CD86 and CD40 moleculeswere determined by flow cytometry. As the results presented in Figure 2show, all the extracts increased the intensity of expression of CD40 onDCs. The MFI of CD40 expression in cells treated with 50mg=mL T. vulgariswas 45.8� 6.2 compared to untreated cells (20.7� 4.6), p¼ 0.04. Thecorresponding data for MFI of CD40 expression on DCs treated with Z. mul-tiflora was 34.5� 1.6 compared to 15.65� 1.75 in untreated control(p< 0.003). Treatment of DCs with 50mg=mL T. daenensis also increasedCD40 intensity of expression (MFI, 40.9� 1.2) compared to that ofuntreated control (20.7� 4.6), p¼ 0.035. T. vulgaris was the only extractthat at 50mg=mL significantly increased the percentage expression ofCD40 from 81.5� 0.5 in untreated control to 89.5� 0.4 in treated cells
FIGURE 2 The mean fluorescence intensity (MFI) of CD40, CD86, and MHC II molecule expressionon dendritic cells treated with various concentrations of the extracts determined by flow cytometeranalysis. Control (negative) was extract-untreated cells. Tumor necrosis factor (TNF)-a (50ng=ml)was used as positive control. Data represent mean� standard error of at least three independent experi-ments. �p< 0.04.
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(p¼ 0.004). The mean intensity of MHC II molecule expression at 1 mg=mLof T. vulgaris and T. daenensis extracts was increased, but in comparison tothe negative control, the difference was not significant. Z. multiflora also did
FIGURE 3 Expression of CD40 molecule on dendritic cells (DCs) treated with 50 mg=mL of the extractsanalyzed by flow cytometer. Negative control was extract-untreated cells. Tumor necrosis factor (TNF)-a(50 ng=mL) was used as positive control. DCs were gated on CD11cþ cells. The result shown is one rep-resentative experiment out of three independent experiments carried out in triplicate. Data representthe mean fluorescence intensity (MFI) of CD40 expression in cells treated with the extracts or mediumalone (M2) and the relevant isotype control for CD40 (M1) (color figure available online).
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not affect the expression of MHC II molecule on mouse DCs comparedwith that of the control cells (Figure 2). None of the extracts increasedthe percentage expression of CD86 molecule on DCs. The mean intensityof expression of this molecule showed an increase at concentration of50 mg=mL of all the extracts [e.g., T. vulgaris, MFI, 112� 32 (in treatedcells) vs. 79� 22 (in untreated cells)], but the result was not significant.In Figure 3, the flow cytometry histograms representing the MFI of CD40expression in DCs treated with 50mg=mL of each extract as an exampleare demonstrated.
Effects of the Extracts on Allogenic MLR
To study the effects of the extracts on allogenic immune response andstimulation of T cells, allogenic T cells from C57BL=6 were co-cultured withDCs isolated from BALB=c mice. As shown in Figure 4, Z. multiflora inhib-ited the proliferation of T cells in allogenic MLR with proliferation indicesranging from 0.71� 0.05 at 1 mg=mL to 0.49� 0.02 at 50 mg=mL, p< 0.02.The extent of T cell proliferation in the presence of T. vulgaris extractwas significantly decreased at 50mg=mL (proliferation index, 0.43� 0.03,p¼ 0.02), whereas T. daenensis showed no significant effect on MLR.
Effects of the Extracts on Cytokine Production
To determine whether the extracts could affect the production of cyto-kines by T cells, the effect of extracts on secretion of IFN-c and IL-4, twomajor cytokines involved in Th1 and Th2 mediated responses by CD4þ
helper T cells, were measured in the supernatant of mixed lymphocyte cul-ture. As shown in Figure 5, treatment of cells with T. vulgaris at 1 mg=mL
FIGURE 4 Proliferation of T cells in a mixed leukocyte reaction. Dendritic cells were cultured with allo-genic Tcells at 1:10 ratio in the presence of various concentrations of the extracts for 4 days. Control wasextract-untreated cells. Cell proliferation was measured using BrdU assay. The proliferation index wascalculated by dividing the optical density (OD) of treated cells to OD of control. Data represent mean -� standard error of at least three independent experiments. �p< 0.02.
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decreased IFN-c production (1116� 10 pg=mL compared to the control;1381� 166 pg=mL) but increased IL-4 secretion (107� 38 compared tothe control; 75� 26 pg=mL); however, the result was not significant. Z. mul-tiflora and T. daenensis also showed no significant effect on the secretion ofcytokines.
DISCUSSION
In the present study, the effect of three plants of the Labiatea familywere investigated on the mouse DC phenotype and activity. These plants,in view of their usefulness in folk medicine, are important species of theLabiatae family with a high similarity in their major constituents and a widerelated spectrum of pharmacological properties. There is some evidenceindicating that these plants may possess abilities to modulate immuneresponses. A considerable stimulation of leucopoiesis, an elevation ofthrombocyte counts in blood, anti-inflammatory effects against acute andchronic inflammation in the rat, and immunoinhibitory effects against lym-phocytes have been reported for these plants.[19,22,23] In this study, we firstexamined the effect of the extract of these plants on the T cell mitogenicresponse. Con A, which is a polyclonal activator of T cells, was used asthe mitogen. The interaction of T cells with this mitogen can initiate acascade of biochemical events and gene expression that induces the resting
FIGURE 5 Cytokine production in mixed leukocyte reaction. Dendritic cells were cultured with allo-genic T cells at 1:10 ratio in the presence of various concentrations of the extracts for 3 days. Cytokineswere measured in the culture supernatants by enzyme-linked immunosorbent assay. Control wasextract-untreated cells. Data represent mean� standard error of two independent experiments. No sig-nificant data were obtained.
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T cells to enter the cell cycle, then proliferate and differentiate.[24] Asresults of our study showed Z. multiflora and T. vulgaris extracts similarlydecreased the proliferation of Con A-stimulated mouse lymphocytes,whereas T. daenensis increased this activity, indicating the ability of theextracts to modulate the mitogenic activation of T cells.
The activation of T cells mainly is dependent on the state and the num-ber of adhesion and co-stimulatory molecules on DCs.[25] In the next step,the effects of the extracts on the expression of important co-stimulatorymolecules on DCs were examined. DCs express constitutively MHC II mole-cules that present antigens to CD4þ T cells; however the number of thesemolecules in immature DCs is low.[26] Mature DCs export a large numberof peptide-loaded MHC II to the surface. Simultaneously, co-stimulatorymolecules such as CD40 are up-regulated.[27] CD40 has been identifiedas an important co-stimulatory protein that is involved in up-regulationof other stimulatory and co-stimulatory molecules. This molecule is criticalfor DC maturation and function,[27] and plays a central role in adaptiveimmune responses. As results of this study showed, Z. multiflora, T. vulgaris,and T. daenensis extracts enhanced CD40 expression on the DCs, indicatingtheir ability to modulate immune responses. Despite this capability, theextracts failed to significantly up-regulate MHC II molecule expression.This was not unexpected since DCs in our study were isolated from spleentissue, and it has been reported that DC subsets from spleen have highMHC II expression.[28] During DC maturation, the expression of importantco-stimulatory molecules such as CD80 and CD86 is also up-regulated.[27]
This expression provides a lower activation threshold to render Tcells moresensitive to antigen stimulation.[29] In the present study, although CD86expression on DCs increased with increasing concentrations of all theextracts, the result was not significant, showing the limited capability ofthe extracts to induce DC activation.
A major role for DCs has been suggested in allogenic immuneresponse.[30] The effect of the extracts on this type of immune responsewas studied in MLR assay. Both T. vulgaris and Z. multiflora extracts signifi-cantly suppressed T cell proliferation in MLR. In this activity, Z. multifolrawas stronger than T. vulgaris as it inhibited the proliferation at lowerconcentrations. Lymphocyte proliferation is a critical event leading to theinitiation and development of inflammation. Therefore this result couldbe in agreement with a previous study showing the in vivo anti-inflammatoryeffect of the water extract of Z. multiflora on acute and chronic inflam-mation.[19] Despite the inhibitory effect of these two extracts on MLR, theyboth increased CD40 expression on DCs. CD40 expression is important forDC maturation and function. It is possible that activation of DCs by theseextracts has led to release of inhibitory cytokines such as IL-10. This cytokinecan inhibit the proliferative responses as well as the production of IFN-c in
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MLR between purified Tcells and DCs.[30] Moreover, the fact should also betaken into account that plants contain various compounds, and at everydose of the extract, a diverse set of compounds with different effects maydominate. With regard to T. daenensis, no significant effect on allogenic Tcell proliferation was detected. Considering the CD40 up-regulation inDCs exposed to the extracts, it is reasonable to assume that while the extractof T. daenensis induced some degree of DC activation, this level was insuf-ficient to induce significant influences on T cells.
Given the inhibitory effects of the extracts of T. vulgaris and Z. multifloraon the allogenic immune response, we tried to find the effect of theextracts on the pattern of production of IFN-c and IL-4, the two major cyto-kines involved in Th1- and Th2-mediated responses by Th cells. As theresults of this study showed, despite a decrease in IFN-c and an increasein IL-4 secretion in MLR culture in the presence of T. vulgaris rather thanthe control, the differences observed did not reach significance. Thechanges in the cytokine production in other extract-treated cells were alsonot significant, indicating that none of the extracts influence the pattern ofcytokine production by CD4þ Tcells toward a Th1 or Th2 profile. A furtherexplanation for obtaining nonsignificant results on cytokine secretioncould be the presence of various compounds, possibly with different modesof actions in the extracts. A variety of phenolic and other chemicalcompounds has been reported to be present in these plants, includingcarvacrol, cymol, linalool, thymol, tannin, flavonoids, saponins, borneol,and triterpenic acids (in T. vulgaris)[18]; thymol, carvacrol, and geraniol(in T. daenensis)[22]; and carvacrol, zatrinal, oleanolic acid, thymol, betulicacid, rosmarinic acid, monoterpenoids, sesquiterpenoids, p-cymene, andy-terpinene (in Z. multiflora).[31] Whether the phenolic compounds in theextracts of these plants or other constituents are responsible for theobserved effects on DCs and T cells needs further investigation.
In conclusion, the results of this study showed that the extracts of all theplants significantly increased CD40 expression on DCs, suggesting their abil-ity to provide an activation signal to these cells. Whereas T. vulgaris andZ. multiflora inhibited both the mitogenic and allogenic T cell responses,T. daenensis only affected the mitogenic Tcell proliferation. These data indi-cated the immunomodulatory effects of the plants studied and showed theirvalue for further studies in order to identify the responsible compounds andinvestigate for other activities to modulate immune responses.
ACKNOWLEDGMENTS
This study was supported by grant no. 4927 from Shiraz University ofMedical Sciences. We would like to thank Hamidreza Zare for helping withflow cytometry.
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