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Effect of Ajuga bracteosa on Systemic T-CellImmunity in Balb/C Mice: Dual Th1/Th2
Immunostimulatory Effects
Albeena Nisar,* Nayeema Akhtar,† Asma Hassan,* Tabish Banday,*
Bilal Wani* and Mohammed Afzal Zargar*
*Department of Biochemistry, University of Kashmir
Srinagar, India-190008
†Indian Institute of Integrative Medicine
Srinagar, India-190001
Abstract: Ajuga bracteosa (AB) has been widely used in folk medicine in Asian countriesagainst gout, hepatitis, pneumonia, rheumatism, and various neuro inflammatory disorders.The aim of this study was to investigate the possible immunoregulatory effects of theethanolic extract of Ajuga bracteosa (ABEE) on systemic Th1/Th2 immunity in SRBCimmunized Balb/C mice. Animals were orally administered with graded doses of ABEE from6.25mg/kg to 100mg/kg. Post sub-cutaneous immunization with SRBCs and circulatingantibody titers, DTH responses and splenocyte proliferation was monitored as markers ofTh2 and Th1 responses. Cyclophosphamide and levamisole were used as controls. Lym-phocyte immunophenotying (CD4/CD8 cell counts) and intracellular Th1/Th2 cytokineconcentrations were determined using flow cytometry. Treatment with ABEE demonstratedsignificant biphasic immunostimulation of effector T-helper immunity. ABEE at 50mg/kgdose resulted in maximal increase in antibody titers, DTH responses and CD4þ/CD8þ T-cellpercentages indicating maximal activation and proliferation of T and B lymphocytes at thisdose. ABEE, at the same dose, also showed maximal up regulation of LPS and CON Astimulated splenocyte proliferation and also maximal up-regulation of both Th1 (IL-2, IFN-�)and Th2 (IL-4) cytokines which suggest its mixed Th1/Th2 immunostimulatory activity.Comparatively at higher doses (100mg/kg), significant down regulation of all these effectorT-helper (Th) immune responses was observed. The study therefore suggests mixed biphasicimmunostimulatory Th1/Th2 activity of ABEE that could support its immunoadjuvantpotential.
Keywords: Ajuga bracteosa; Herbal Medicine; Th1/Th2 Immunity; Flow Cytometry; IL-2;IFN-�; IL-4.
Correspondence to: Dr. Mohammad Afzal Zargar, Department of Biochemistry, University of Kashmir, India-
190008. Tel: (+91) 099-0620-6662, Fax: (+91) 019-4246-3980, E-mail: [email protected]
The American Journal of Chinese Medicine, Vol. 42, No. 2, 375–392© 2014 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and MedicineDOI: 10.1142/S0192415X14500256
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Introduction
Currently there is a huge upsurge in the use of plant derived extracts for the modulation ofimmune response with respect to T-helper (Th) immunity. Th lymphocyte homeostasis isvital in engineering all the diverse parameters of the immune system with the cytokineresponse, B-cell activation at one end and stimulation of macrophages, evasion of extra-cellular toxins, intracellular pathogens at the other end. During the last decade, T-helperlymphocyte regulation has emerged as a potential target for immunomodulation (Roger,2004). Based on their cytokine profiles, T-helper lymphocytes are derived into two distinctphenotype subsets of Th1 and Th2 effector cells. Th1 cells drive the cellular immunitywhile Th2 cells drive the humoral immunity (Mosmann and Coffman, 1989). There areseveral examples in experimental models where in modulation of the Th1/Th2 balance hasregulated the outcome in several disease states, for example cancer, AIDS, etc. The trendsindicate that such agents, which restore a well balanced Th1/Th2 response, are best suitedfor optimal immunotherapy suited and these agents could exhibit stimulatory, suppressiveor regulatory activities on the system (Patwardhan and Gautam, 2005).
Herein we report, mixed immunostimulatory effects of Ajuga barcteosa (AB) on murineimmune response using SRBC as an antigenic stimulus. AB Wall ex Benth. is a perennialherb, belonging to the family of Labitacea of the genus Ajuga. It comprises about50 species distributed in Pakistan, Arabia, East Africa, and North America. It is distributedin subtropical and temperate regions from Kashmir to Nepal in Western Himalayas andalso in Pakistan, Afghanistan, China, and Malaysia (Ali and Nasir, 1990; Kirtikar andBasu, 1918). In Pakistan, it is found in northern hilly areas, where in the local Punjabilanguage it is called koribooti due to its bitter taste. A. bracteosa is found on rock crevicesas an erect or ascending hairy herb. The flowers are white or purplish-violet tinged fromlower surface in distant, axillary whorls in spikes.
Ayurveda describes A. bracteosa as “Neelkanthi” and is a rich medicinal herb used forages in traditional medicine for the treatment of gout, rheumatism, and various neuroinflammatory disorders (Wealth of India, 1985). It also has been used traditionally in avariety of inflammatory disorders (Indian Herbal Pharmacopoeia, 1998). Also, the leavesof A. bracteosa have been used as a stimulant, diuretic, astringent, and febrifuge (Ali andNasir, 1990). The juice of the plant is applied as hot fermentations against carcinomasand also plastered on burns and insect bites. Even the seeds of the plant are used torelieve stomach ache and diarrhea (Perry and Metzger, 1980). In India it has also beenused for a long time as a traditional remedy for malaria (Chandel and Bagai, 2010). Alsoin Taiwan, A. bracteosa has been used traditionally for the treatment of variousinflammatory disorders such as hepatitis, pneumonia, and bone diseases (Chiu and Chang,1992). Various other species of this genus have also been used as analgesics to dissolveblood clots and to relieve fever, diarrhea, eye problems, and diseases of the bladder.Another member of this genus, Ajuga ducumbens, has been used in China for the relief ofjoint pain and has been reported to upregulate the synthesis of collagen in aged modelrats (Ono et al., 2008).
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Ajuga bracteosa contains sphingolides, bractic acid, diterpenoids, and withanoloides,which have been found to have various enzyme inhibiting activities such as that ofLipoxygenase and cholinesterase (Riaz et al., 2007). Previously, AB has been credited withcancer chemopreventive (Ghufran et al., 2009), antiplasmodial (Chandel and Bagai, 2010),cardiostimulant (Patel et al., 1962), and hepatoprotective (Hsieh et al., 2011) properties.
Given the pharmacological profile of A. bracteosa, the present study was undertaken toevaluate the modulating effects of AB on T-helper lymphocyte homeostasis in SRBCimmunized Balb/c mice using flow cytometry. Our results suggested that A. barcteosa hasprofound immunostimulatory effects with dual Th1/Th2 responses which could indicate itsuse as immunoadjuvant in several disease conditions.
Materials and Methods
Chemicals
Fluorescein isothiocyanate (FITC)-labeled anti-CD4 mouse monoclonal antibody; Phy-coerytherin (PE)-labeled anti-CD8, IL-2, IFN-�, and IL-4 mouse monoclonal antibodies;FACS lysing solution (BD Biosciences, USA); Cyclophosphamide, levamisole, E. colipolysaccharide (LPS), Concanavalin A (Con A) (Sigma-Aldrich, India); Gum acacia (HiMedia, India). Unless otherwise specified, all other chemicals were purchased from HiMedia and Merck and all solvents used were of HPLC grade (Ranbaxy Chemicals Ltd.,Mohali, Punjab, India).
Test Materials
Ajuga bracteosa (whole plant) was collected from the Ferozpur and Drang areas inKashmir. The plants were correctly identified by the Centre of Biodiversity and Taxonomy,University of Kashmir, Srinagar, India and authenticated by a botanist, Dr. Irshad Wan-choo. A voucher sample is retained and deposited at Central Herbarium, Department ofBotany, University of Kashmir, India as 1760 KASH (26/06/2010) [Ajuga bracteosa]. Thematerial was ensued to be free from pathogens, aflatoxins, pesticidal residues, and heavymetals according to guidelines of WHO, 1998.
Preparation of Extracts
The authentically identified whole parts of plant material were shade dried and thenpowdered. About 1.2 kg of A. bracteosa was subjected to Soxhlet extraction separatelywith absolute ethanol (EtOH). The solvent was removed under reduced pressure on avacuum rotary evaporator to get a dark brownish extract of A. bracteosa referred as ABEEextract (yield: 39.2%). The crude extract was stored at 4�C for experimental use. Theextract was studied at doses ranging from 6.25mg/kg to 100mg/kg and the test material forexperimentation was prepared as fresh suspension each time using 1% sterile gum acacia.
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Animals
All experimental procedures used in the present study were in accordance with institutionalguidelines for animal research (CPCSEA, 2003). The study was conducted on male Balb/cmice obtained from healthy animal colony at the Department of Pharmacology, IndianInstitute of Integrated Medicine (IIIM), Jammu. Balb/c mice (male, 3–4 weeks old,weighing 18–22 g) were randomly distributed in groups as per experimental protocols(n ¼ 6). The animals were bred and maintained under standard laboratory conditions;temperature (25�C), humidity (55.6� 10%) and regulated photoperiod of 12 h. Standardlaboratory chow (Amrut Mills, Nashik, India) and water were given ad libitum. Insti-tutional Animal Use and Care Committee of IIIM, Jammu approved all the study protocols.Blood samples were collected through retro-orbital bleeding at specified time points, underether anesthesia and subsequently assayed for antibody titers, cell counts, and cytokines.
Effect on General Behavior and Acute Safety
The effect of test extracts on general behavior and safety was evaluated in mice using theup and down procedure (Organization of Economic Co-operation and Development(OECD), Guideline No. 423, 1996). Mice of either sex (three females and three males;weight: 20–25 g; age: 4–6 weeks) were administered graded doses of ABEE (single dose)up to 2500mg/kg orally by gavage. The animals were observed for toxic symptomscontinuously for the first 4 h after dosing. Finally, the number of survivors was noted after24 h and these animals were then maintained for a further 13 days with observations madedaily. The animals were observed for any changes in general behavior, weight, mortality,or other physiological activities.
Drugs
Levamisole (2.5mg/kg) was used as a positive control and cyclophosphamide (100mg/kg)as an immunosuppressive agent. They were administered orally during the study andprepared fresh in 1% gum acacia.
Antigenic Stimulus
Fresh sheep red blood cells (SRBC) were collected aseptically in cold Alsevers solution fromthe jugular vein of animals housed at IIIM, Jammu. The cells were washed thrice with sterilepyrogen free normal saline (0.9% NaCl, w/v). For immunization and challenge each mousereceived 5� 109 cells/ml i.p. at required time schedule, Day 1 and Day 7. Under our assayconditions, this cell count has been reported to induce optimum immune response.
Treatment
The animals were divided into groups of six animals each. Group I, received 1% gumacacia (Sensitized control); Group II received Levamisole (2.5mg/kg) (Positive control);
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Group III; Cyclophosphomide (50mg/kg) (Negative control); Group IV: ABEE(6.25mg/kg); Group V: ABEE (12.5mg/kg); Group VI: ABEE (25mg/kg); Group VII:ABEE (50mg/kg); Group VIII: ABEE (100mg/kg). All the test extracts and standard drugsdissolved in 1% guma acacia were administered orally for 14 days in a dose volume of0.2ml. All the groups were given antigenic treatment using SRBC on Day 1 and Day 7.
SRBC Specific Humoral Immune Responses
The estimation of circulating antibody titres was done using a standard haemaglutinationtest (Nelson and Mildenhall, 1967). Blood samples were collected in micro centrifuge tubesfrom individual animals by retro-orbital plexus on Day 7 for primary antibody titer and forsecondary antibody titer on Day 15. Serum was separated and briefly equal volumes ofindividual serum samples of each group were pooled. Two-fold dilutions of pooled serumsamples were made in 25�l volumes of normal saline in a micro titration plate to whichwere added 25�l of 1% suspension of SRBC in saline. After mixing, the plates wereincubated at room temperature for 1 h and examined for haemagglutination titer under themicroscope. The reciprocal of the highest dilution of the test serum giving agglutinationwas taken as the antibody titer. The mean titer values of the drug and test extracts treatedgroups were compared to those of the control.
SRBC Specific Cellular Immune Responses
To assess SRBC induced delayed type hypersensitivity (DTH) responses in mice, the testextracts ABEE and other agents, as per the protocol, were administered 2 h after SRBCinjection and once daily on consecutive days. On Day 7 SRBC primed mice were chal-lenged and immunized with 20�l of SRBC antigen (5� 109 cells) in the right hind footpadand 50�l of normal saline was given in the left hind footpad. The difference between theleft and right paw thickness/swelling of foot was measured using a speromicrometer(0.01mm pitch) after 24 h and 48 h (Bafna and Mishra, 2010).
Splenocyte Proliferation Assay
On the 14th day, spleens were removed from the mice of the control and the experimentalgroups, under aseptic conditions and then homogenized in complete medium, RPMI 1640medium supplemented with 12mM HEPES-buffered. Splenocytes were sedimented bycentrifugation at 300� 3 g for 7min at 4�C, washed and re-suspended in RPMI 1640medium containing 10% heat inactivated FCS, penicillin G (100U/ml), streptomycin(100�g/ml), amphotericin B (0.25�g/ml), 2-mercaptoethanol (50�M), 2mM L-glutamine(Sigma) and 1mM sodium pyurvate. The cell count was determined with a haemocyt-ometer by the Trypan blue dye exclusion technique. Cell suspensions (1� 107 viable cells/ml/well) were pipetted into 96 well flat bottom plates (Costar, Cambridge, MA) andcultured in presence of Con A and LPS both used at 10�g /ml as B and T cell mitogens,respectively. The plates were incubated for 72 h at 37�C in saturated atmosphere con-taining humid 5% CO2 followed by the addition of 20�l MTT solution (5mg/ml) to each
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well and incubated for 4 h. The plates were then centrifuged (1400� g) (5min). Untrans-formed MTT was removed carefully and to each well 100�l working DMSO solution(192�l DMSO/8�l 1 M HCL) was added. The plates were then read at OD 570 nm after15min to assay the proliferation responses.
Lymphocyte Immunophenotyping
The analysis of T-helper subsets, namely CD4þ (T-helper cells), CD8þ (cytotoxic cells)and B-cell count (CD19þ), was performed using multiparametric flow cytometric analysison peripheral blood. Mouse monoclonal antibodies conjugated to a fluorochrome anddirected against receptors CD3, CD4, CD8, and CD19 were used for the study. Briefly,mice from different experimental groups were bled at required time schedules and 100�l ofwhole blood was added to each tube. Monoclonal abs conjugated to a flurochrome anddirected against CD3, CD4, CD8, and CD19 were added directly to the tubes. After mixingand incubating at room temperature for 30min in the dark, FACS lysing solution wasadded. The samples were then incubated for 10min at 37�C and spun at 300–400 g.Supernatant was aspirated and washed thrice with phosphate buffer saline (7.4). Afterwashing, enumeration of lymphocytes subsets was done using a flow cytometer (BD,Biosciences) using Cell Quest Pro Software (BD, Biosciences). 10,000 events were col-lected to analyze CD3þ T-cells, namely CD4þ, CD8þ T cells and also CD19þ B-cells.
Intracellular Cytokine Estimation
The detection of cytokines was performed in peripheral blood based on BD Biosciencesprotocol end reported method. Briefly, to 100�l of peripheral blood collected from theanimals, CD4þ and CD8þ monoclonal antibody (mabs) was added. The tubes were mixedand incubated at 37�C in the dark followed by the addition of FACS lysing solution.Subsequently the samples were incubated and centrifuged for 10min at 10,000 rpm. Cellswere washed, permeabilized and stained with phycoerythrin (PE) coupled IL-2, IL-4, andIFN-� mabs. The cells stained were then acquired using a flow cytometer (BD, Bios-ciences). Cell quest software (BD, Biosciences) was used for gating and calculation.10,000 cells were determined with at least 100 cells in every gate of lymphocyte sub-populations. The resulting numbers are percentages of cytokine expression of those sub-populations.
Data Analysis and Statistical Considerations
Data is expressed as mean � S.E.M. Statistical significance of differences was assessed byone way ANOVA followed by the Bonferroni test for multiple comparisons. The level ofsignificance was set at p < 0:05. Percent immunomodulatory activity in different exper-imental groups was derived using following method:
% Modulatory activity ¼ ðTest group� Sensitized control groupÞSensitized control group
� 100:
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Results
Effect on Normal Behaviour and Acute Safety
Groups of animals treated with ABEE at all graded doses up to 2500mg/kg showed nosigns of any abnormal behavior and no mortality was observed, which indicates that thetest extracts were well tolerated up to 2500mg/kg and hence are safe to use. A broad rangeof oral dosages of the test extracts (6.25–100mg/kg) was taken up to get dose response andto determine the dose with most significant effect.
Effect of ABEE on Antigen Specific Responses
As a first step to study the effect on Th immunity, the effect of sensitization protocol on thehumoral and cell mediated responses was established.
Humoral Responses
ABEE, when administered orally at 6.25–100mg/kg doses, also resulted in a dosedependent increase in antibody titers. Maximal immunnomodulatory action was observedat 50mg/kg dose with 56.7% increase in primary and 64.2% in secondary antibody syn-thesis. However, when the dose was raised to 100mg/kg primary and secondary antibodytiters decreased to 26.6% and 25%, respectively indicating biphasic modulatory activity forABEE (Table 1). Under similar conditions, levamisole (positive control) and cyclopho-phomide (negative control) resulted in a significant increase (36.7% and p < 0:001 vs.control) and decrease (�20% and p < 0:001 vs. control) of primary anti-body titres,respectively as compared to the control (Group 1).
Table 1. Immunomodulatory Effect of ABEE on SRBC Induced Humoral Immune Response in vivo
Antibody Response Log 2 Titre (Mean � SEM) (%Change)
Treatment Doses (mg/kg) Day 7 Day 15
Control (sensitized) SRBC 6.0 � 0.18 5.6 � 0.22Levamisole 2.5 8.2 � 0.16*** (36.7") 8.0 � 0.24*** (42.8")Cyclophosphomide 100 4.8 � 0.20*** (20.0#) 3.8 � 0.14*** (32.1#)ABEE 6.25 6.4 � 0.18 (6.7") 6.2 � 0.18 (10.71")ABEE 12.5 6.8 � 0.12* (13.4") 6.8 � 0.16** (21.4")ABEE 25 8.0 � 0.14*** (33.4") 8.2 � 0.26*** (46.4")ABEE 50 9.4 � 0.10*** (56.7") 9.2 � 0.18*** (64.2")ABEE 100 7.6 � 0.14** (26.6 ") 7.0 � 0.28*** (25")Note: aHATitres were determined on day 7 for primary antibody synthesis and day 15 for secondaryantibody synthesis, (") stimulaton, (#) suppression. bNumber of mice are six for each group. cData is
expressed as mean � S.E.M. N ¼ 6; Percent modulatory activity was calculated using formulae givenSec. 3.1. *Denotes comparison between control (sensitized) group I and other experimental groups. The p
values were calculated by One Way ANNOVA. *p < 0:05, **p < 0:01, ***p < 0:001, followed by
Bonferroni correction multiple comparison test.
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Cellular Responses: SRBC Specific DTH Responses
Maximal increase in foot pad thickness was observed in treatment with ABEE at 50mg/kgdose with 67.85% and 59.18% stimulation after 24 h and 48 h, respectively. However,when the dose was raised to 100mg/kg, the DTH responses after 24 h comparatively wasreduced as seen in Fig. 1. Levamisole, under similar conditions, resulted in 53.57% upregulation (p < 0:001) of DTH response after 24 h compared to the control.
Effect of ABEE on Lymphocyte Proliferation
We investigated the effects of ABEE on the proliferation of spleen cells in the presence ofCON A (T-cell mitogen) and LPS (B-cell mitogen) (Fig. 2). In the case of group II, thelevamisole group, proliferative responses to CON A and LPS increased by 56.7% and61.9%, respectively. In group III, receiving cyclophosphomide 46.67% and 36.4%immunosuppression was, respectively, observed for CON A and LPS induced proliferationstudies. ABEE (6.25–100mg/kg) resulted in a significant increase in the proliferationindex. In the case of CON A treated cells, maximal increase of 68.05% proliferation ascompared to the control (sensitized) was seen in cells obtained from a group treated withABEE 50mg/kg. The percentage increase, however, decreased in cells from group VIIItreated with 100mg/kg ABEE (38.86%). In the presence of LPS, ABEE enhanced thecellular proliferation to 18.25%, 36.86%, 48.8%, 61.09%, and 36.17% at 6.25, 12.5, 25,50, and 100mg/kg, respectively (Fig. 2).
Paw
oed
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(Mea
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M)
24 h
rs
48hrs
0.0
0.5
1.0
1.5
2.0
2.5
***
***
***
***
*** ***
***
***
*** ***
**
Control (SRBC sensitized) Levamisole
Cyclophosphomide ABEE-6.25mg/kg b.w
ABEE-12.5mg/kg b.w ABEE-25mg/kg b.w
ABEE-50mg/kg b.w ABEE-100mg/kg b.w
Figure 1. Immunomodulatory effect of ABEE on SRBC induced cellular immune response (DTH) in vivo. Data isexpressed as mean� S.E.M. N ¼ 6; *Denotes comparison between control (sensitized) group I and other
experimental groups. The p-values were calculated by One Way ANNOVA. *p < 0:05, **p < 0:01,***p < 0:001, followed by Bonferroni correction multiple comparison test.
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Effect of ABEE on CD3þ, CD4þ, CD8þ T Cell Percentages and CD19þ
B Cell Percentages
Flow cytomertic analysis was used to monitor the effect of the test material on T-cellresponse. In unsensitized conditions, no significant results were observed on CD3þ,CD4þ, CD8þ, and CD19 lymphocyte percentages as compared to the control. In contrast,in sensitized conditions, ABEE at all doses resulted in a significant dose dependant increasein CD3þ, CD4þ, and CD8þ T-cell subsets and also on CD19 B-cell lymphocyte per-centages. Cyclophosphomide, as expected, resulted in the significant reduction of CD3þ,CD4þ, CD8þ, and CD 19 percentages as compared to the control. Maximal immunosti-mulatory effects for ABEE were observed at a dose of 50mg/kg with up regulation ofCD3þ, CD4þ, CD8þ, and CD19 cell counts (see Fig. 3). The effects were observed todecrease at the higher dose of 100mg/kg on T-cell subsets as well as B-Cell lymphocytepercentages.
Effect on Th1 and Th2 Cytokines
To determine the effect of ABEE on Th1/Th2 immunity selected cytokine levels weremeasured in vivo using flow cytometry.
Effect on Th1 Cytokines
Under unsensitized conditions, no significant increase in cytokine levels was observed.Levamisole resulted in a 59.23% and 64.9% increase in IL-2 and IFN-� levels, whereas
Dose (mg/kg)
OD
- 5
70 n
m (
Mea
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EM
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Control (
SRBC se
nsize
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Leva
misole
Cyclophosp
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ABEE-6.25
ABEE-12.5
ABEE-25
ABEE-50
ABEE-100
0.0
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1.0
1.5
2.0 CON A LPS
** **
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***
***
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*
Figure 2. Effect of ABEE on lymphocyte proliferation. Splenocytes (5� 106 viable cells/(mLwell)) from thedifferent experimental groups were cultured in the presence of mitogens (LPS or Con A) stimulus. Histogramsrepresent mean � S.E.M. absorbance units. Statistical significance was evaluated using One Way ANNOVA
followed by the Bonferroni test for multiple comparisons was used to compute p-values. *p < 0:05, **p < 0:01,***p < 0:001 vs. control.
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(A)
(B)
Figure 3. (A) Flow cytometry scatter plots for T-cell populations (CD4þ T-helper cells subset and CD8þ
cytolytic/cytotoxic T-cells). The plots represent events for one representative mouse from each group of the
control (sensitized), levamisole and ABEE (6.25–100mg/kg). The gating and the quadrants are set according tothe standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry scatter plots for T-cell populations(CD3þ T-helper cells subset and CD19þ B-cells). The plots represent events for one representative mouse fromeach group of the control (sensitized), levamisole and ABEE (6.25–100mg/kg). The gating and the quadrants are
set according to the standard procedures of the BD-LSR flow cytometer.
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cyclophosphomide caused a noted suppression for both cytokines. Treatment with extractof A. bracteosa also resulted in dose dependent increase of IL-2 and IFN-� cytokines(Figs. 4 and 5). IL-2 and IFN-� levels are stimulated maximally at 50mg/kg (56.7%stimulation in case of IL-2 (p < 0:001) and 67.37% (p < 0:001) for IFN compared to thecontrol), but with an increase in dosage their levels are reduced. ABEE shows a biphasicactivity of ABEE where up regulation and down regulation of these cytokine levels wasobserved at lower and higher doses, respectively.
Effect on Th2 Cytokines
As reflected in Fig. 6, IL-4 is stimulated maximally in groups receiving 50mg/kg ABEEwhere 60.19% stimulation is observed. However, the stimulation decreased with a furtherincrease in dosage.
Discussion
In the last decade there has been a huge upsurge in the use of plant extracts which targetT-helper lymphocytes for the modulation of immune responses in arthritic disorders.
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Figure 4. The histograms represent the intracellular concentration of the two Th1 cytokines — IL-2 (A) andIFN-� (B) after treatment with ABEE in different groups. Data is expressed as mean � S.E.M. N ¼ 6; *Denotes
comparison between control (sensitized) group I and other experimental groups. The p values were calculatedby One Way ANNOVA. *p < 0:05, **p < 0:01, ***p < 0:001, followed by Bonferroni correction multiplecomparison test.
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Immune homeostasis is maintained by a fine equilibrium between Th1 and Th2 lymphocytesubsets. Hence, the modulation of Th1/Th2 has emerged as a vital target for immuno-modulation. Immunomodulators are categorized as Th1, Th2 or mixed Th1/Th2 agentsdepending on their effeminacy towards T-helper lymphocyte subsets. AB has been pre-viously known to exhibit different pharmacological properties; however, so far no sys-tematic study has been conducted to assess the regulatory effects on Th1–Th2 immuneresponses. Herein, we reported significant biphasic immunomodulatory effects of AB onsystemic Th1–Th2 homeostasis.
Th1 and Th2 cells are regarded as “system supervisors” of the immune framework. Th1cells mediate cellular immune responses (Type 1 pathway), and Th2 cells mediate thehumoral immune responses (Type 2 pathway). Functionally, lymphocytes are associatedwith inflammation and cell mediated responses and also provide immunity againstmycobacterial infections such as tuberculosis, leishmania etc. and viruses such as influ-enza. Th2 cells provide support to B-cells for antibody production and protection againsthelminthes, allergy and other viral infections (for example measles). Th1 cells are alsoassociated with autoimmune diseases and graft rejection. Type 1 and Type 2 pathwaystogether provide an extraordinarily effective defense system (Romagnani, 2000). Leva-misole is a potent immunostimulant, which restores the suppressed immune functions of B-cells, T-cells, monocytes and macrophages (Bozic et al., 2003; Argani and Akhtarishojaie,
Figure 5. Flow cytometry histogram representation for signature Th-1 cytokine, IL-2 and IFN-�. Each histogram
represents the counts for one representative mouse from each group of the control (sensitized), levamisole andABEE-50mg/kg. The histograms area is acquired according to the standard procedures of the BD-LSR flowcytometer.
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2006). Hence, a comparative study of levamisole and the test extract (ABEE) was designedto evaluate Th1–Th2 modulating properties in vivo in SRBC immunized Balb/c mice.Interestingly, ABEE depicted dual biphasic dose dependant potentiation of both Th1 aswell as Th2 immunity. At higher doses the stimulatory effects showed a downward trend.This indicated a unique immunnomodulatory profile for AB.
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Figure 6. (A) The histograms represent the intracellular concentration of the IL-4 on treatment with ABEE and
Levamisole. Data is expressed as mean � S.E.M. N ¼ 6; *Denotes comparison between the control (sensitized)group I and other experimental groups. The p values were calculated by One Way ANNOVA. *p < 0:05,**p < 0:01, ***p < 0:001, followed by Bonferroni correction multiple comparison test. (B) Flow cytometry
histogram representation for Th-2 cytokine IL-4, the signature cytokine of the Th2 response. The histogramrepresents the counts for one representative mouse from each group of the control (sensitized), levamisole andABEE-50mg/kg. The histograms are acquired according to the standard procedures of the BD-LSR flowcytometer.
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The humoral immunity involves the interaction of B cells with the antigen and theirsubsequent proliferation and differentiation into antibody-secreting plasma cells. Antibodyfunctions as the effector of the humoral response by binding to antigen and neutralizing itor facilitating its elimination by cross-linking to form clusters that are more readily ingestedby phagocytic cells. The T-helper, Type 2 pathway (humoral immunity) was analyzed interms of the increase in circulating antibody titers, post immunization with SRBC. Asexpected, levamisole (2.5mg/kg) resulted in generalized immunostimulation, as evidentfrom the raised HA titers, while cyclphosphomide (100mg/kg) showed drastic inhibition ofthe tires. ABEE, at different graded doses of 6.25–100mg/kg, was found to cause anincrease in antibody titers, in relation to the control and levamisole with maximal effectseen at 50mg/kg. At higher doses, the response was found to decrease.
Delayed type hypersensitivity (DTH) is an index of cell mediated immunity (Th-1pathway). Treatment with ABEE, was seen to enhance the DTH reaction in response toSRBC immunization as evident from the increase in food pad thickness. ABEE demon-strated maximum stimulation of DTH reaction at a dose of 50mg/kg. These findingsdepicted optimum stimulatory effects for ABEE at higher doses on T-lymphocytes andother adjunct cell types needful for the formulation of DTH response (Benacerraf, 1978).DTH is marked by huge influx of macrophages and other inflammatory cells (Luster et al.,1982). It requires the specific recognition of a given antigen by activated T lymphocytes,which subsequently proliferate and release cytokines. These in turn increase vascularpermeability, induce vasodilatation and macrophage activation, promoting increased pha-gocytic activity. The overall effect is mediated via the cytokines released by Th1 cells torecruit and activate the macrophages, thereby promoting increased phagocytic activity(Cher and Mosmann, 1987).
The ability to invoke efficacious T and B lymphocyte immunity can be depicted by thepotentiation of lymphocyte proliferation reactions. In the paradigm of T-lymphocytes,helper T-cellspromote the antibody secretion from the B-lymphocytes and cause thecytotoxic lymphocytes to aid phagocytes for ingestion and elimination of intracellularpathogens. B-lymphocytes secrete antibodies, which aid in removing extracellular patho-gens and also neutralize toxins (Abbas et al., 1996). The proliferation assay with ABEEindicated a significant promotion both with CON A and LPS stimulated proliferation at anoptimum dose of 50mg/kg, which however got reduced at 100mg/kg dose, indicating areduction in stimulation of Th1 and Th2 pathway at higher doses.
In addition, evidence that lends support to our hypothesis of unique T-helper lym-phocyte activation and stimulation patterns by ABEE was the significant potentiating effecton CD3þ, CD4þ, and CD8þ lymphocyte subsets. Our findings using flow cytometryrevealed a notable increase in CD3þ, CD4þ, and CD8þ percentages in peripheral blood forABEE both at the same dosages as seen above with CON A stimulation studies. Similarupregulation was noted on CD 19þ B-cell percentages, which suggested the dual immuneactivation of both Th1 (T-cell) as well as Th2 (B-cell) pathways. It is already known that B-cells mediate antibody production and other immune functions through CD4þ and CD8þ
cells (Constant et al., 1997). Various studies have reported positive correlations betweencellular and humoral immunity and CD4þ/CD8þ percentages in immune altered disease
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states such as cancer, arthritis, etc. Also, T cells that express CD4þ are found to beincreased whenever there is a general expansion due to the active immunological activity ofT-lymphocyte subsets, for example as seen in RA (Lucy et al., 2004).
The Th1/Th2 framework of immune system largely rests on the dichotomy of cytokineprofiles corresponding to the two subsets of helper T lymphocytes (Oberholzer et al.,2000). In order to validate the role of T-helper cell derived cytokines for the mixedimmunomodulatory effects of the test extracts on Th1/Th2 immunity and elucidate furtherthe dose dependent dual Th1/Th2 modulating activity, we proceeded with the estimation ofTh1 (IL-2 and IFN-�) and Th2 (IL-4) cytokines in SRBC immunized mice using flowcytometry. Our findings were consistent with the earlier results on antibody titers andT-cell/B-cell activation and proliferation. Levamisole showed a generalized increasedconcentration in Th1/Th2 intracellular cytokine concentration. The pattern of cytokines,IL-2, IFN-� and IL-4 production in the groups treated with different doses of the extract ofAB correlated with the earlier results displayed with antibody production, DTH responseand T cell/B cell proliferation studies. Noteworthy to mention, the maximal stimulation ofall the cytokines production (Th1/Th2) was seen at 50mg/kg dose.
Upon antigenic stimulation, the Th-1 immune response is marked by release of IFN-�,IL-2 and TNF-� and Th-2 immune reactions by IL-4 secretion. Cytokines are multi-functional and so remain vital at the different stages of immune response (Oberholzer et al.,2000). IL-4 (Type 2 pathway) mediates the antibody production via B-cells, eosinophilsand mast cells. IL-2 stimulates Th-1 and cytotoxic cells to enhance the additional pro-liferation and differentiation of CD4þ cells, B-cells and activate macrophages. IFN-�secreted by the Th1 cells drives the inflammatory pathway (McInnes and Schett, 2007) andis required for selective immunity to intracellular bacteria, viruses, and protozoa, whereasTh2 produces IL-4 required for optimal antibody production to T-cell dependant antigens.During the initial stages, IL-2 and IFN-� augment the differentiation and proliferation ofTh1 cells, but in the later stages, these bring about “apoptosis” of the repeatedly stimulatedeffector cells. (Mosmann and Coffman, 1989). Hence, Th1 and Th2 proliferation rates areonly dividends of the concentrations of their cytokine profiles.
In safety studies, ABEE was found to be safe up to 2500mg/kg. Neither a sign of anymortality, nor any toxicological consequences were observed in mice, signifying that AB issafe to use. Thus, our study clearly demonstrated that AB could stimulate T-cell mediatedeffects in a biphasic manner. The biphasic response dose dependent upregulation of Th1/Th2 immunity by AB could be attributed to the synergistic effects of the different bio-chemical constituents present in the extract. There are numerous examples where plantderived extracts have shown such selectivity in immunotherapy. For instance, in Ginseng(Lee and Han, 2006), Withania somnifera (Davis and Kuttan, 2000, 2002) and Tinosporacordifolia (Thatte et al., 1994; Kapil and Sharma, 1997; Nair et al., 2004) the differentbiochemical constituents have mediated the immnomodulatory effects through differenttargets. Such immunoactive mixtures have been reported to deliver synergistic moietieswhich simultaneously or concurrently modulate the different limbs of immune matrix e.g.,in inflammation, infection, cancer, etc. and restore the immune homeostasis (Patwardhan,
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2005). In conclusion, AB exhibited selectivity in immune therapy which could be useful ineffective drug discovery from these botanicals.
Conclusion
The present study therefore concludes AB to possess mixed immunostimulatory effects onboth Th1 and Th2 immunity. Such plant derived extracts could deliver such moieties thatmay have applications in such conditions where broader stimulation of Th1/Th2 paradigmrequired. Its apparent safety over long-term administration is encouraging enough towarrant further studies to explore its possible therapeutic use. Standardized extracts of ABcould provide newer adjuvant moites for the safer modulaton of host immunity. It is furtherrecommended that for an insight into the molecular mechanism of such effects, bioassayguided fractionation and in depth studies on transcripiton factors for cytokines is required.The work, with respect to the mechanistic understanding of immunomodulating effects andother biochemical properties of AB, is still continuing in our lab.
Acknowledgments
This research was supported by a research grant from the University of Kashmir, J&K,India.
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