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 Medicine DOI: 10.1142/S0192415X14500256

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Effect of Ajuga bracteosa on Systemic T-Cell Immunity 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 countries against gout, hepatitis, pneumonia, rheumatism, and various neuro inflammatory disorders. The aim of this study was to investigate the possible immunoregulatory effects of the ethanolic extract of Ajuga bracteosa (ABEE) on systemic Th1/ Th2 immunity in SRBC immunized Balb/C mice. Animals were orally administered with graded doses of ABEE from 6.25 mg/kg to 100 mg/kg. Post sub-cutaneous immunization with SRBCs and circulating antibody titers, DTH responses and splenocyte proliferation was monitored as markers of Th2 and Th1 responses. Cyclophosphamide and levamisole were used as controls. Lymphocyte immunophenotying (CD4/CD8 cell counts) and intracellular Th1/Th2 cytokine concentrations were determined using flow cytometry. Treatment with ABEE demonstrated significant biphasic immunostimulation of effector T-helper immunity. ABEE at 50 mg/kg dose resulted in maximal increase in antibody titers, DTH responses and CD4þ/CD8þ T-cell percentages indicating maximal activation and proliferation of T and B lymphocytes at this dose. ABEE, at the same dose, also showed maximal up regulation of LPS and CON A stimulated 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 (100 mg/kg), significant down regulation of all these effector T-helper (Th) immune responses was observed. The study therefore suggests mixed biphasic immunostimulatory Th1/Th2 activity of ABEE that could support its immunoadjuvant potential. 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, India190008. Tel: (+91) 099-0620-6662, Fax: (+91) 019-4246-3980, E-mail: [email protected]

375

376

A. NISAR et al.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Introduction Currently there is a huge upsurge in the use of plant derived extracts for the modulation of immune response with respect to T-helper (Th) immunity. Th lymphocyte homeostasis is vital in engineering all the diverse parameters of the immune system with the cytokine response, B-cell activation at one end and stimulation of macrophages, evasion of extracellular toxins, intracellular pathogens at the other end. During the last decade, T-helper lymphocyte regulation has emerged as a potential target for immunomodulation (Roger, 2004). Based on their cytokine profiles, T-helper lymphocytes are derived into two distinct phenotype subsets of Th1 and Th2 effector cells. Th1 cells drive the cellular immunity while Th2 cells drive the humoral immunity (Mosmann and Coffman, 1989). There are several examples in experimental models where in modulation of the Th1/Th2 balance has regulated the outcome in several disease states, for example cancer, AIDS, etc. The trends indicate that such agents, which restore a well balanced Th1/Th2 response, are best suited for optimal immunotherapy suited and these agents could exhibit stimulatory, suppressive or regulatory activities on the system (Patwardhan and Gautam, 2005). Herein we report, mixed immunostimulatory effects of Ajuga barcteosa (AB) on murine immune response using SRBC as an antigenic stimulus. AB Wall ex Benth. is a perennial herb, belonging to the family of Labitacea of the genus Ajuga. It comprises about 50 species distributed in Pakistan, Arabia, East Africa, and North America. It is distributed in subtropical and temperate regions from Kashmir to Nepal in Western Himalayas and also in Pakistan, Afghanistan, China, and Malaysia (Ali and Nasir, 1990; Kirtikar and Basu, 1918). In Pakistan, it is found in northern hilly areas, where in the local Punjabi language it is called koribooti due to its bitter taste. A. bracteosa is found on rock crevices as an erect or ascending hairy herb. The flowers are white or purplish-violet tinged from lower surface in distant, axillary whorls in spikes. Ayurveda describes A. bracteosa as “Neelkanthi” and is a rich medicinal herb used for ages in traditional medicine for the treatment of gout, rheumatism, and various neuro inflammatory disorders (Wealth of India, 1985). It also has been used traditionally in a variety of inflammatory disorders (Indian Herbal Pharmacopoeia, 1998). Also, the leaves of A. bracteosa have been used as a stimulant, diuretic, astringent, and febrifuge (Ali and Nasir, 1990). The juice of the plant is applied as hot fermentations against carcinomas and also plastered on burns and insect bites. Even the seeds of the plant are used to relieve stomach ache and diarrhea (Perry and Metzger, 1980). In India it has also been used for a long time as a traditional remedy for malaria (Chandel and Bagai, 2010). Also in Taiwan, A. bracteosa has been used traditionally for the treatment of various inflammatory 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 dissolve blood 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 of joint pain and has been reported to upregulate the synthesis of collagen in aged model rats (Ono et al., 2008).

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

377

Ajuga bracteosa contains sphingolides, bractic acid, diterpenoids, and withanoloides, which have been found to have various enzyme inhibiting activities such as that of Lipoxygenase and cholinesterase (Riaz et al., 2007). Previously, AB has been credited with cancer 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 to evaluate the modulating effects of AB on T-helper lymphocyte homeostasis in SRBC immunized Balb/c mice using flow cytometry. Our results suggested that A. barcteosa has profound immunostimulatory effects with dual Th1/Th2 responses which could indicate its use as immunoadjuvant in several disease conditions. Materials and Methods Chemicals Fluorescein isothiocyanate (FITC)-labeled anti-CD4 mouse monoclonal antibody; Phycoerytherin (PE)-labeled anti-CD8, IL-2, IFN-, and IL-4 mouse monoclonal antibodies; FACS lysing solution (BD Biosciences, USA); Cyclophosphamide, levamisole, E. coli polysaccharide (LPS), Concanavalin A (Con A) (Sigma-Aldrich, India); Gum acacia (Hi Media, India). Unless otherwise specified, all other chemicals were purchased from Hi Media 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 in Kashmir. The plants were correctly identified by the Centre of Biodiversity and Taxonomy, University of Kashmir, Srinagar, India and authenticated by a botanist, Dr. Irshad Wanchoo. A voucher sample is retained and deposited at Central Herbarium, Department of Botany, University of Kashmir, India as 1760 KASH (26/06/2010) [Ajuga bracteosa]. The material was ensued to be free from pathogens, aflatoxins, pesticidal residues, and heavy metals according to guidelines of WHO, 1998. Preparation of Extracts The authentically identified whole parts of plant material were shade dried and then powdered. About 1.2 kg of A. bracteosa was subjected to Soxhlet extraction separately with absolute ethanol (EtOH). The solvent was removed under reduced pressure on a vacuum rotary evaporator to get a dark brownish extract of A. bracteosa referred as ABEE extract (yield: 39.2%). The crude extract was stored at 4  C for experimental use. The extract was studied at doses ranging from 6.25 mg/kg to 100 mg/kg and the test material for experimentation was prepared as fresh suspension each time using 1% sterile gum acacia.

378

A. NISAR et al.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Animals All experimental procedures used in the present study were in accordance with institutional guidelines for animal research (CPCSEA, 2003). The study was conducted on male Balb/c mice obtained from healthy animal colony at the Department of Pharmacology, Indian Institute 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. Standard laboratory chow (Amrut Mills, Nashik, India) and water were given ad libitum. Institutional 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, under ether 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 the up 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 2500 mg/kg orally by gavage. The animals were observed for toxic symptoms continuously for the first 4 h after dosing. Finally, the number of survivors was noted after 24 h and these animals were then maintained for a further 13 days with observations made daily. The animals were observed for any changes in general behavior, weight, mortality, or other physiological activities. Drugs Levamisole (2.5 mg/kg) was used as a positive control and cyclophosphamide (100 mg/kg) as an immunosuppressive agent. They were administered orally during the study and prepared fresh in 1% gum acacia. Antigenic Stimulus Fresh sheep red blood cells (SRBC) were collected aseptically in cold Alsevers solution from the jugular vein of animals housed at IIIM, Jammu. The cells were washed thrice with sterile pyrogen free normal saline (0.9% NaCl, w/v). For immunization and challenge each mouse received 5  10 9 cells/ml i.p. at required time schedule, Day 1 and Day 7. Under our assay conditions, 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% gum acacia (Sensitized control); Group II received Levamisole (2.5 mg/kg) (Positive control);

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

379

Group III; Cyclophosphomide (50 mg/kg) (Negative control); Group IV: ABEE (6.25 mg/kg); Group V: ABEE (12.5 mg/kg); Group VI: ABEE (25 mg/kg); Group VII: ABEE (50 mg/kg); Group VIII: ABEE (100 mg/kg). All the test extracts and standard drugs dissolved in 1% guma acacia were administered orally for 14 days in a dose volume of 0.2 ml. All the groups were given antigenic treatment using SRBC on Day 1 and Day 7.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

SRBC Specific Humoral Immune Responses The estimation of circulating antibody titres was done using a standard haemaglutination test (Nelson and Mildenhall, 1967). Blood samples were collected in micro centrifuge tubes from individual animals by retro-orbital plexus on Day 7 for primary antibody titer and for secondary antibody titer on Day 15. Serum was separated and briefly equal volumes of individual serum samples of each group were pooled. Two-fold dilutions of pooled serum samples were made in 25 l volumes of normal saline in a micro titration plate to which were added 25 l of 1% suspension of SRBC in saline. After mixing, the plates were incubated at room temperature for 1 h and examined for haemagglutination titer under the microscope. The reciprocal of the highest dilution of the test serum giving agglutination was taken as the antibody titer. The mean titer values of the drug and test extracts treated groups were compared to those of the control. SRBC Specific Cellular Immune Responses To assess SRBC induced delayed type hypersensitivity (DTH) responses in mice, the test extracts ABEE and other agents, as per the protocol, were administered 2 h after SRBC injection and once daily on consecutive days. On Day 7 SRBC primed mice were challenged and immunized with 20 l of SRBC antigen (5  10 9 cells) in the right hind footpad and 50 l of normal saline was given in the left hind footpad. The difference between the left and right paw thickness/swelling of foot was measured using a speromicrometer (0.01 mm 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 experimental groups, under aseptic conditions and then homogenized in complete medium, RPMI 1640 medium supplemented with 12 mM HEPES-buffered. Splenocytes were sedimented by centrifugation at 300  3 g for 7 min at 4  C, washed and re-suspended in RPMI 1640 medium containing 10% heat inactivated FCS, penicillin G (100 U/ml), streptomycin (100 g/ml), amphotericin B (0.25 g/ml), 2-mercaptoethanol (50 M), 2 mM L-glutamine (Sigma) and 1 mM sodium pyurvate. The cell count was determined with a haemocytometer by the Trypan blue dye exclusion technique. Cell suspensions (1  10 7 viable cells/ ml/well) were pipetted into 96 well flat bottom plates (Costar, Cambridge, MA) and cultured 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 containing humid 5% CO2 followed by the addition of 20 l MTT solution (5 mg/ml) to each

380

A. NISAR et al.

well and incubated for 4 h. The plates were then centrifuged (1400 g) (5 min). Untransformed 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 after 15 min to assay the proliferation responses.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Lymphocyte Immunophenotyping The analysis of T-helper subsets, namely CD4 þ (T-helper cells), CD8 þ (cytotoxic cells) and B-cell count (CD19 þ ), was performed using multiparametric flow cytometric analysis on peripheral blood. Mouse monoclonal antibodies conjugated to a fluorochrome and directed 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 of whole blood was added to each tube. Monoclonal abs conjugated to a flurochrome and directed against CD3, CD4, CD8, and CD19 were added directly to the tubes. After mixing and incubating at room temperature for 30 min in the dark, FACS lysing solution was added. The samples were then incubated for 10 min at 37  C and spun at 300–400 g. Supernatant was aspirated and washed thrice with phosphate buffer saline (7.4). After washing, enumeration of lymphocytes subsets was done using a flow cytometer (BD, Biosciences) using Cell Quest Pro Software (BD, Biosciences). 10,000 events were collected to analyze CD3 þ T-cells, namely CD4 þ , CD8 þ T cells and also CD19 þ B-cells. Intracellular Cytokine Estimation The detection of cytokines was performed in peripheral blood based on BD Biosciences protocol end reported method. Briefly, to 100 l of peripheral blood collected from the animals, CD4 þ and CD8 þ monoclonal antibody (mabs) was added. The tubes were mixed and incubated at 37  C in the dark followed by the addition of FACS lysing solution. Subsequently the samples were incubated and centrifuged for 10 min at 10,000 rpm. Cells were washed, permeabilized and stained with phycoerythrin (PE) coupled IL-2, IL-4, and IFN- mabs. The cells stained were then acquired using a flow cytometer (BD, Biosciences). 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 subpopulations. The resulting numbers are percentages of cytokine expression of those subpopulations. Data Analysis and Statistical Considerations Data is expressed as mean  S.E.M. Statistical significance of differences was assessed by one way ANOVA followed by the Bonferroni test for multiple comparisons. The level of significance was set at p < 0:05. Percent immunomodulatory activity in different experimental groups was derived using following method: % Modulatory activity ¼

ðTest group  Sensitized control groupÞ  100: Sensitized control group

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

381

Results Effect on Normal Behaviour and Acute Safety

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Groups of animals treated with ABEE at all graded doses up to 2500 mg/kg showed no signs of any abnormal behavior and no mortality was observed, which indicates that the test extracts were well tolerated up to 2500 mg/kg and hence are safe to use. A broad range of oral dosages of the test extracts (6.25–100 mg/kg) was taken up to get dose response and to 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 the humoral and cell mediated responses was established. Humoral Responses ABEE, when administered orally at 6.25–100 mg/kg doses, also resulted in a dose dependent increase in antibody titers. Maximal immunnomodulatory action was observed at 50 mg/kg dose with 56.7% increase in primary and 64.2% in secondary antibody synthesis. However, when the dose was raised to 100 mg/kg primary and secondary antibody titers decreased to 26.6% and 25%, respectively indicating biphasic modulatory activity for ABEE (Table 1). Under similar conditions, levamisole (positive control) and cyclophophomide (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 Control (sensitized) Levamisole Cyclophosphomide ABEE ABEE ABEE ABEE ABEE

Doses (mg/kg) SRBC 2.5 100 6.25 12.5 25 50 100

Day 7 6.0 8.2 4.8 6.4 6.8 8.0 9.4 7.6

       

0.18 0.16*** (36.7") 0.20*** (20.0#) 0.18 (6.7") 0.12* (13.4") 0.14*** (33.4") 0.10*** (56.7") 0.14** (26.6 ")

Day 15 5.6 8.0 3.8 6.2 6.8 8.2 9.2 7.0

       

0.22 0.24*** (42.8") 0.14*** (32.1#) 0.18 (10.71") 0.16** (21.4") 0.26*** (46.4") 0.18*** (64.2") 0.28*** (25")

Note: a HATitres were determined on day 7 for primary antibody synthesis and day 15 for secondary antibody synthesis, (") stimulaton, (#) suppression. b Number of mice are six for each group. c Data is expressed as mean  S.E.M. N ¼ 6; Percent modulatory activity was calculated using formulae given Sec. 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.

382

A. NISAR et al.

Cellular Responses: SRBC Specific DTH Responses Maximal increase in foot pad thickness was observed in treatment with ABEE at 50 mg/kg dose with 67.85% and 59.18% stimulation after 24 h and 48 h, respectively. However, when the dose was raised to 100 mg/kg, the DTH responses after 24 h comparatively was reduced as seen in Fig. 1. Levamisole, under similar conditions, resulted in 53.57% up regulation ( p < 0:001) of DTH response after 24 h compared to the control.

We investigated the effects of ABEE on the proliferation of spleen cells in the presence of CON A (T-cell mitogen) and LPS (B-cell mitogen) (Fig. 2). In the case of group II, the levamisole group, proliferative responses to CON A and LPS increased by 56.7% and 61.9%, respectively. In group III, receiving cyclophosphomide 46.67% and 36.4% immunosuppression was, respectively, observed for CON A and LPS induced proliferation studies. ABEE (6.25–100 mg/kg) resulted in a significant increase in the proliferation index. In the case of CON A treated cells, maximal increase of 68.05% proliferation as compared to the control (sensitized) was seen in cells obtained from a group treated with ABEE 50 mg/kg. The percentage increase, however, decreased in cells from group VIII treated with 100 mg/kg ABEE (38.86%). In the presence of LPS, ABEE enhanced the cellular proliferation to 18.25%, 36.86%, 48.8%, 61.09%, and 36.17% at 6.25, 12.5, 25, 50, and 100 mg/kg, respectively (Fig. 2).

2.5 Paw oedema (Mean ± SEM)

2.0

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

*** ***

***

***

***

***

1.5

***

***

***

**

1.0 ***

0.5

hr s 48

hr s

0.0 24

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Effect of ABEE on Lymphocyte Proliferation

Figure 1. Immunomodulatory effect of ABEE on SRBC induced cellular immune response (DTH) in vivo. 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 calculated by One Way ANNOVA. *p < 0:05, **p < 0:01, ***p < 0:001, followed by Bonferroni correction multiple comparison test.

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

OD - 570 nm (Mean ± SEM )

2.0

383

LPS

CON A

*** ***

1.5

***

***

* ***

1.0

*** **

***

**

***

0.5

0 AB EE -1 0

AB EE -5 0

AB EE -2 5

.5 AB EE -1 2

AB EE -6 .2 5

) Le va m Cy iso clo le ph os ph om id e

ze d iƟ ns se RB C l( S nt ro Co

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

0.0

Dose (mg/kg)

Figure 2. Effect of ABEE on lymphocyte proliferation. Splenocytes (5  10 6 viable cells/(mLwell)) from the different experimental groups were cultured in the presence of mitogens (LPS or Con A) stimulus. Histograms represent 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.

Effect of ABEE on CD3 þ , CD4 þ , CD8 þ T Cell Percentages and CD19 þ B Cell Percentages Flow cytomertic analysis was used to monitor the effect of the test material on T-cell response. In unsensitized conditions, no significant results were observed on CD3 þ , CD4 þ , CD8 þ , and CD19 lymphocyte percentages as compared to the control. In contrast, in sensitized conditions, ABEE at all doses resulted in a significant dose dependant increase in CD3 þ , CD4 þ , and CD8 þ T-cell subsets and also on CD19 B-cell lymphocyte percentages. Cyclophosphomide, as expected, resulted in the significant reduction of CD3 þ , CD4 þ , CD8 þ , and CD 19 percentages as compared to the control. Maximal immunostimulatory effects for ABEE were observed at a dose of 50 mg/kg with up regulation of CD3 þ , CD4 þ , CD8 þ , and CD19 cell counts (see Fig. 3). The effects were observed to decrease at the higher dose of 100 mg/kg on T-cell subsets as well as B-Cell lymphocyte percentages. Effect on Th1 and Th2 Cytokines To determine the effect of ABEE on Th1/Th2 immunity selected cytokine levels were measured 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

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

384

A. NISAR et al.

(A)

(B) Figure 3. (A) Flow cytometry scatter plots for T-cell populations (CD4 þ T-helper cells subset and CD8 þ cytolytic/cytotoxic T-cells). The plots represent events for one representative mouse from each group of the control (sensitized), levamisole and ABEE (6.25–100 mg/kg). The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer. (B) Flow cytometry scatter plots for T-cell populations (CD3 þ T-helper cells subset and CD19 þ B-cells). The plots represent events for one representative mouse from each group of the control (sensitized), levamisole and ABEE (6.25–100 mg/kg). The gating and the quadrants are set according to the standard procedures of the BD-LSR flow cytometer.

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA 15

*** *** *

**

10

***

***

5

***

se l( tro on C

C

(A)

ze yc le d) lo va ph m os iso ph le om id A e B EE 6. A 2 B EE 5 -1 2 .5 A B EE 2 A 5 B E A E-5 B EE 0 -1 00

iti ns

ns iti ze yc le d) lo va ) ph m i os so ph le om id A e B EE A -6.2 B EE 5 -1 2. A 5 B EE A -25 B EE -5 A B EE 0 -1 00

0

se l( tro on

***

10

C

5

*** ***

(Mean ± SEM )

***

% Cytokine secretion in whole blood

(Mean ± SEM )

% Cytokine secretion in whole blood

15

IFN-γ

IL-2

0

C

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

20

385

(B)

Figure 4. The histograms represent the intracellular concentration of the two Th1 cytokines — IL-2 (A) and IFN- (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 calculated by One Way ANNOVA. *p < 0:05, **p < 0:01, ***p < 0:001, followed by Bonferroni correction multiple comparison test.

cyclophosphomide caused a noted suppression for both cytokines. Treatment with extract of 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 50 mg/kg (56.7% stimulation in case of IL-2 (p < 0:001) and 67.37% (p < 0:001) for IFN compared to the control), but with an increase in dosage their levels are reduced. ABEE shows a biphasic activity of ABEE where up regulation and down regulation of these cytokine levels was observed at lower and higher doses, respectively. Effect on Th2 Cytokines As reflected in Fig. 6, IL-4 is stimulated maximally in groups receiving 50 mg/kg ABEE where 60.19% stimulation is observed. However, the stimulation decreased with a further increase in dosage. Discussion In the last decade there has been a huge upsurge in the use of plant extracts which target T-helper lymphocytes for the modulation of immune responses in arthritic disorders.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

386

A. NISAR et al.

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 and ABEE-50 mg/kg. The histograms area is acquired according to the standard procedures of the BD-LSR flow cytometer.

Immune homeostasis is maintained by a fine equilibrium between Th1 and Th2 lymphocyte subsets. Hence, the modulation of Th1/Th2 has emerged as a vital target for immunomodulation. Immunomodulators are categorized as Th1, Th2 or mixed Th1/Th2 agents depending on their effeminacy towards T-helper lymphocyte subsets. AB has been previously known to exhibit different pharmacological properties; however, so far no systematic study has been conducted to assess the regulatory effects on Th1–Th2 immune responses. Herein, we reported significant biphasic immunomodulatory effects of AB on systemic Th1–Th2 homeostasis. Th1 and Th2 cells are regarded as “system supervisors” of the immune framework. Th1 cells mediate cellular immune responses (Type 1 pathway), and Th2 cells mediate the humoral immune responses (Type 2 pathway). Functionally, lymphocytes are associated with inflammation and cell mediated responses and also provide immunity against mycobacterial infections such as tuberculosis, leishmania etc. and viruses such as influenza. Th2 cells provide support to B-cells for antibody production and protection against helminthes, allergy and other viral infections (for example measles). Th1 cells are also associated with autoimmune diseases and graft rejection. Type 1 and Type 2 pathways together provide an extraordinarily effective defense system (Romagnani, 2000). Levamisole is a potent immunostimulant, which restores the suppressed immune functions of Bcells, T-cells, monocytes and macrophages (Bozic et al., 2003; Argani and Akhtarishojaie,

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

*** 8

*** ***

***

* (Mean ± SEM )

% Cytokine secretion in whole blood

IL-4

6

4

*** 2

C

on

tro

l(

se

ns

iti ze yc le d) lo va ph m os iso ph le om id A e B EE 6 A .2 B EE 5 -1 2. A 5 B EE A -25 B EE -5 A B EE 0 -1 00

0

C

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

10

387

(A)

(B) 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 histogram represents the counts for one representative mouse from each group of the control (sensitized), levamisole and ABEE-50 mg/kg. The histograms are acquired according to the standard procedures of the BD-LSR flow cytometer.

2006). Hence, a comparative study of levamisole and the test extract (ABEE) was designed to evaluate Th1–Th2 modulating properties in vivo in SRBC immunized Balb/c mice. Interestingly, ABEE depicted dual biphasic dose dependant potentiation of both Th1 as well as Th2 immunity. At higher doses the stimulatory effects showed a downward trend. This indicated a unique immunnomodulatory profile for AB.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

388

A. NISAR et al.

The humoral immunity involves the interaction of B cells with the antigen and their subsequent proliferation and differentiation into antibody-secreting plasma cells. Antibody functions as the effector of the humoral response by binding to antigen and neutralizing it or facilitating its elimination by cross-linking to form clusters that are more readily ingested by phagocytic cells. The T-helper, Type 2 pathway (humoral immunity) was analyzed in terms of the increase in circulating antibody titers, post immunization with SRBC. As expected, levamisole (2.5 mg/kg) resulted in generalized immunostimulation, as evident from the raised HA titers, while cyclphosphomide (100 mg/kg) showed drastic inhibition of the tires. ABEE, at different graded doses of 6.25–100 mg/kg, was found to cause an increase in antibody titers, in relation to the control and levamisole with maximal effect seen at 50 mg/kg. At higher doses, the response was found to decrease. Delayed type hypersensitivity (DTH) is an index of cell mediated immunity (Th-1 pathway). Treatment with ABEE, was seen to enhance the DTH reaction in response to SRBC immunization as evident from the increase in food pad thickness. ABEE demonstrated maximum stimulation of DTH reaction at a dose of 50 mg/kg. These findings depicted optimum stimulatory effects for ABEE at higher doses on T-lymphocytes and other 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 vascular permeability, induce vasodilatation and macrophage activation, promoting increased phagocytic activity. The overall effect is mediated via the cytokines released by Th1 cells to recruit 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 the potentiation of lymphocyte proliferation reactions. In the paradigm of T-lymphocytes, helper T-cellspromote the antibody secretion from the B-lymphocytes and cause the cytotoxic lymphocytes to aid phagocytes for ingestion and elimination of intracellular pathogens. B-lymphocytes secrete antibodies, which aid in removing extracellular pathogens and also neutralize toxins (Abbas et al., 1996). The proliferation assay with ABEE indicated a significant promotion both with CON A and LPS stimulated proliferation at an optimum dose of 50 mg/kg, which however got reduced at 100 mg/kg dose, indicating a reduction in stimulation of Th1 and Th2 pathway at higher doses. In addition, evidence that lends support to our hypothesis of unique T-helper lymphocyte activation and stimulation patterns by ABEE was the significant potentiating effect on CD3 þ , CD4 þ , and CD8 þ lymphocyte subsets. Our findings using flow cytometry revealed a notable increase in CD3 þ , CD4 þ , and CD8 þ percentages in peripheral blood for ABEE both at the same dosages as seen above with CON A stimulation studies. Similar upregulation was noted on CD 19 þ B-cell percentages, which suggested the dual immune activation of both Th1 (T-cell) as well as Th2 (B-cell) pathways. It is already known that Bcells mediate antibody production and other immune functions through CD4 þ and CD8 þ cells (Constant et al., 1997). Various studies have reported positive correlations between cellular and humoral immunity and CD4 þ /CD8 þ percentages in immune altered disease

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

389

states such as cancer, arthritis, etc. Also, T cells that express CD4 þ are found to be increased whenever there is a general expansion due to the active immunological activity of T-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 cytokine profiles 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 mixed immunomodulatory effects of the test extracts on Th1/Th2 immunity and elucidate further the dose dependent dual Th1/Th2 modulating activity, we proceeded with the estimation of Th1 (IL-2 and IFN-) and Th2 (IL-4) cytokines in SRBC immunized mice using flow cytometry. Our findings were consistent with the earlier results on antibody titers and T-cell/B-cell activation and proliferation. Levamisole showed a generalized increased concentration 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 of AB correlated with the earlier results displayed with antibody production, DTH response and T cell/B cell proliferation studies. Noteworthy to mention, the maximal stimulation of all the cytokines production (Th1/Th2) was seen at 50 mg/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 multifunctional 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, eosinophils and mast cells. IL-2 stimulates Th-1 and cytotoxic cells to enhance the additional proliferation and differentiation of CD4 þ cells, B-cells and activate macrophages. IFN- secreted by the Th1 cells drives the inflammatory pathway (McInnes and Schett, 2007) and is required for selective immunity to intracellular bacteria, viruses, and protozoa, whereas Th2 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 of Th1 cells, but in the later stages, these bring about “apoptosis” of the repeatedly stimulated effector cells. (Mosmann and Coffman, 1989). Hence, Th1 and Th2 proliferation rates are only dividends of the concentrations of their cytokine profiles. In safety studies, ABEE was found to be safe up to 2500 mg/kg. Neither a sign of any mortality, nor any toxicological consequences were observed in mice, signifying that AB is safe to use. Thus, our study clearly demonstrated that AB could stimulate T-cell mediated effects 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 biochemical constituents present in the extract. There are numerous examples where plant derived extracts have shown such selectivity in immunotherapy. For instance, in Ginseng (Lee and Han, 2006), Withania somnifera (Davis and Kuttan, 2000, 2002) and Tinospora cordifolia (Thatte et al., 1994; Kapil and Sharma, 1997; Nair et al., 2004) the different biochemical constituents have mediated the immnomodulatory effects through different targets. Such immunoactive mixtures have been reported to deliver synergistic moieties which simultaneously or concurrently modulate the different limbs of immune matrix e.g., in inflammation, infection, cancer, etc. and restore the immune homeostasis (Patwardhan,

390

A. NISAR et al.

2005). In conclusion, AB exhibited selectivity in immune therapy which could be useful in effective drug discovery from these botanicals.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Conclusion The present study therefore concludes AB to possess mixed immunostimulatory effects on both Th1 and Th2 immunity. Such plant derived extracts could deliver such moieties that may have applications in such conditions where broader stimulation of Th1/Th2 paradigm required. Its apparent safety over long-term administration is encouraging enough to warrant further studies to explore its possible therapeutic use. Standardized extracts of AB could provide newer adjuvant moites for the safer modulaton of host immunity. It is further recommended that for an insight into the molecular mechanism of such effects, bioassay guided fractionation and in depth studies on transcripiton factors for cytokines is required. The work, with respect to the mechanistic understanding of immunomodulating effects and other 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. References Abbas, A.K., K.M. Murphy and A. Sher. Functional diversity of helper T lymphocytes. Nature 383: 787–793, 1996. Ali, S.I. and Y.J. Nasir. Flora of Pakistan. BCC & T Press, University of Karachi, 1990, Vol. 192, p. 14. Argani, H. and E. Akhtarishojaie. Levamizole enhances immune responsiveness to intra-dermal and intra-muscular hepatitis B vaccination in chronic hemodialysis patients. J. Immune Based Ther. Vaccines 4: 3, 2006. Bafna, A. and S. Mishra. Antioxidant and immunomodulatory activity of the alkaloidal fraction of Cissampelos pareira linn. Sci. Pharm. 78: 21–31, 2010. Benacerraf, B. A hypothesis to relate the specificity of T lymphocytes and the activity of I regionspecific Ir genes in macrophages and B lymphocytes. J. Immunol. 120: 1809–1812, 1978. Bozic, F., V. Bilic and I. Valpotic. Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs. J. Vet. Pharmacol. Ther. 26: 225–231, 2003. Chandel, S. and U. Bagai. Antiplasmodial activity of Ajuga bracteosa against Plasmodium berghei infected BALB/c mice. Indian J. Med. Res. 131: 440–444, 2010. Cher, D.J. and T.R. Mosmann. Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones. J. Immunol. 138: 3688–3694, 1987. Chiu, N.Y. and K.H. Chang. The Illustrated Medicinal Plants of Taiwan. SMC, 1992, Vol. 3. Constant, S.L. and K. Bottomly. Induction of Th1 and Th2 CD4þ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15: 297–322, 1997. CPCSEA, Government of India. Committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines for laboratory animal facility. Indian J. Pharm. 35: 257–274, 2003.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

IMUUNOSTIMULATORY EFFECTS OF AJUGA BRACTEOSA

391

Davis, L. and G. Kuttan. Immunomodulatory activity of Withania somnifera. J. Ethnopharmacol. 71: 193–200, 2000. Davis, L. and G. Kuttan. Effect of Withania somnifera on cell mediated immune responses in mice. J. Exp. Clin. Cancer Res. 21: 585–590, 2002. Ghufran, M.A., R.A. Qureshi, A. Batool, T.P. Kondratyuk, J.M. Guilford and L.E. Marler. Evaluation of selected indigenous medicinal plants from the western Himalayas for cytotoxicity and as potential cancer chemopreventive agents. Pharm. Biol. 47: 533–538, 2009. Hsieh, W.T., Y.T. Liu and W.C. Lin. Anti-inflammatory properties of Ajuga bracteosa in vivo and in vitro study and their effects on mouse model of liver fibrosis. J. Ethnopharmacol. 135: 116– 125, 2011. Indian Herbal Pharmacopoeia. Joint Publication of Indian Drugs Manufacturer’s Association and Regional Research Laboratory, Jammu–Tawi, India, 1998, Vol. 1, pp. 165–173. Kapil, A. and S. Sharma. Immunopotentiating compounds from Tinospora cordifolia. J. Ethnopharmacol. 58: 89–95, 1997. Kirtikar, K.R. and B.D. Basu. In: L.M. Basu (eds.) Indian Medicinal Plants, 2nd Ed. The Indian Press, Allahabad, pp. 1158–1159. Lee, J.H. and Y. Han. Ginsenoside Rg1 helps mice resist to disseminated candidiasis by Th1 type differentiation of CD4þ T cell. Int. Immunopharmacol. 6: 1424–1430, 2006. Lucy, B. HIV: getting to the bottom of CD4 T cell loss. Nat. Rev. Microbiol. 2: 853, 2004. Luster, M.I., J.H. Dean and G.A. Boorman. Cell-mediated immunity and its application in toxicology. Environ. Health Perspect. 43: 31–36, 1982. McInnes, I.B. and G. Schett. Cytokines in the pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7: 429–442, 2007. Mosmann, T.R. and R.L. Coffman. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7: 145–173, 1989. Nair, P.K., S. Rodriguez, R. Ramachandran, A. Alamo, S.J. Melnick, E. Escalon, P.I. Garcia, Jr., S.F. Wnuk and C. Ramachandran. Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia. Int. Immunopharmacol. 4: 1645–1659, 2004. Nelson, D.S. and P. Mildenhall. Studies on cytophillic antibodies. The production by mice of macrophage cytophillic antibodies to sheep erythrocytes, relationship to the production of other antibodies and development of delayed type hypersensitivity. Aust. J. Exp. Biol. Med. Sci. 45: 113–130, 1967. Oberholzer, A., C. Oberholzer and L.L. Moldawer. Cytokine signaling–regulation of the immune response in normal and critically ill states. Crit. Care Med. 28: N3–N12, 2000. OECD. Organization for economic cooperation and development (OECD), Guidelines for Testing of Chemicals. Guideline 423, Acute Oral Toxicity–Acute Toxic Class Method, Adopted, March 22, 1996. Ono, Y., Y. Fukaya, S. Imai and T. Yamakuni. Beneficial effects of Ajuga decumbens on osteoporosis and arthritis. Biol. Pharm. Bull. 31: 1199–1204, 2008. Patel, D.G., O.D. Gulati and S.D. Gokhale. Positive inotropic action of an alkaloidal fraction from Ajuga bracteosa Well ex Benth. Indian J. Physiol. Pharmacol. 6: 224–230, 1962. Patwardhan, B. Ethnopharmacology and drug discovery. J. Ethnopharmacol. 100: 50–52, 2005. Patwardhan, B. and M. Gautam. Botanical immunodrugs: scope and opportunities. Drug Discov. Today 10: 495–502, 2005. Perry, L.M. and J. Metzger. Medicinal Plants of East and Southeast Asia. The MIT Press Cambridge, London, 1980, p. 184. Riaz, N., S.A. Nawaz, N. Mukhtar, A. Malik, N. Afza, S. Ali, S. Ullah, P. Muhammad and M.I. Choudhary. Isolation and enzyme-inhibition studies of the chemical constituents from Ajuga bracteosa. Chem. Biodivers. 4: 72–83, 2007.

392

A. NISAR et al.

Am. J. Chin. Med. 2014.42:375-392. Downloaded from www.worldscientific.com by UNIVERSIT OF SOUTHERN CALIFORNIA on 04/09/14. For personal use only.

Roger, P.M. Revival of the regulatory T cell: new targets for drug development. Drug Discov. Today 9: 310–316, 2004. Romagnani, G. Cytokines and the Th1/Th2 Paradigm. In: F. Balkwill (ed.) The Cytokine Network. Oxford University Press, Oxford, 2000. Thatte, U.M., S.G. Rao and S.A. Dahanukar. Tinospora cordifolia induces colony stimulating activity in serum. J. Postgrad. Med. 40: 202–203, 1994. Wealth of India. The Dictionary of Indian Raw Materials and Industrial Products. Council of Scientific and Industrial Research, New Delhi, India, 1985, Vol. I, p. 121. World Health organization (WHO). Quality Control Guideline for Medicinal Plant Materials. 1998, p. 111.

Th2 immunostimulatory effects.

Ajuga bracteosa (AB) has been widely used in folk medicine in Asian countries against gout, hepatitis, pneumonia, rheumatism, and various neuro inflam...
2MB Sizes 5 Downloads 3 Views