Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Figures & data

Table 1.  Toxicity profile of ethanolic extract of A. lanata.

	Control^a	Vehicle^a	5 mg/kg BW^a	10 mg/kg BW^a	50 mg/kg BW^a	100 mg/kg BW^a
Mortality^b (D/T)	0/8	0/8	0/8	0/8	0/8	3/8*
Behavior changes^b	None	None	None	None	None	Hypo-activity
Body weight change (g)	+1.72 ± 0.38	+1.76 ± 0.52	+1.81 ± 0.47	+1.67 ± 0.50	+1.26 ± 0.39	−1.16 ± 0.84*
Organ weight^b
Liver	5.64 ± 0.43	5.62 ± 0.59	5.67 ± 0.39	5.37 ± 0.69	5.21 ± 0.46	4.76 ± 0.36*
Spleen	0.39 ± 0.01	0.40 ± 0.03	0.41 ± 0.04	0.45 ± 0.05	0.42 ± 0.03	0.38 ± 0.05
Thymus	0.10 ± 0.01	0.10 ± 0.02	0.11 ± 0.02	0.14 ± 0.03	0.12 ± 0.03	0.08 ± 0.04
Kidney	1.35 ± 0.21	1.37 ± 0.12	1.35 ± 0.26	1.36 ± 0.29	1.30 ± 0.21	1.27 ± 0.24
Lungs	0.63 ± 0.05	0.64 ± 0.03	0.66 ± 0.04	0.61 ± 0.07	0.62 ± 0.04	0.62 ± 0.05
Serum ALP (U/ml)	13.62 ± 0.94	13.68 ± 0.76	13.51 ± 0.64	13.76 ± 0.86	15.38 ± 1.24	28.62 ± 1.76*
Serum GPT (U/ml)	63.81 ± 4.92	63.17 ± 3.96	63.92 ± 5.28	63.57 ± 4.82	67.13 ± 5.72	93.21 ± 7.39*
Blood urea (mg/dl)	40.65 ± 3.51	40.85 ± 2.38	40.05 ± 3.74	40.72 ± 4.53	49.13 ± 5.04	68.15 ± 5.68*
Serum creatinine (mg/dl)	0.86 ± 0.05	0.88 ± 0.07	0.87 ± 0.04	0.88 ± 0.05	0.94 ± 0.06	1.94 ± 0.13*

^aVehicle control received 1% gum acacia; experimental groups received A. lanata ethanolic extract (at indicated doses) IP daily for 14  days. All mice were examined each day for signs of toxicity (behavioral changes, diarrhea, and mortality). All mice were euthanized by  cervical dislocation 1 day (Day 15) after the final treatment.

^bValues are mean ± SD. Organ weight as g/100 g body weight; D/T, dead/treated mice; None, no toxic symptoms noted.

*p < 0.05, significantly different from vehicle control.

Figure 1.  Effect of A. lanata on natural killer cell activity. BALB/c mice were divided into three groups (n = 36/group). Group I animals were induced to form ascites by IP injection of Ehrlich ascites carcinoma cells (EAC, 1 × 10⁶ cells/mouse) and kept as untreated tumor-bearing controls. Group II animals were treated with A. lanata extract (10 mg/kg BW/day, IP) for 5 consecutive days. Group III mice received five daily doses of A. lanata and then EAC cells (1 × 10⁶ cells/animal, IP) 24 h after the final extract treatment. Animals were then sacrificed at different timepoints (n = 3/group/timepoint) thereafter. At necropsy, the spleen was removed and processed to generate single-cell suspensions; these materials were then used as effector cells to determine NK cell activity in a [⁵¹Cr]-release assay. Results are expressed as mean (± SD) percentage cell lysis.

Figure 2.  Effect of A. lanata on antibody-dependent cellular cytotoxicity. BALB/c mice were divided into three groups (n = 36 per group) and treated as outlined in the legend to Figure 1. Mice were then sacrificed at different timepoints (n = 3 per group/timepoint) after tumor induction. At necropsy, the spleen was removed and processed to single-cell suspensions; these materials were then used as effector cells to determine ADCC in a [⁵¹Cr]-release assay. Results are expressed as mean (± SD) percentage cell lysis.

Figure 3.  Effect of A. lanata on antibody-dependent complement-mediated cytotoxicity. BALB/c mice were divided into three groups (n = 36 per group) and treated as outlined in the legend to Figure 1. Mice were then sacrificed at different timepoints (n = 3 per group/timepoint) after tumor induction. At necropsy, blood was collected and serum then separated and incubated at 37°C for 3 h with EAC cells (1 × 10⁶ cells/100 µl) and complement (50 µl, neat). The extent of cytotoxicity was then assessed by a trypan blue exclusion method. Results are expressed as mean (± SD) percentage cell lysis.

Table 2.  Effect of A. lanata on serum cytokine profile.

Treatment	IL-2 (pg/ml)	IFNg (pg/ml)
Normal	10.44 ± 0.58	262.71 ± 12.08
Tumor control	7.77 ± 0.32*	1993.22 ± 117.36*
A. lanata	18.29 ± 0.48*	3183.90 ± 14.03*
Tumor + A. lanata	12.80 ± 0.30*	2888.14 ± 26.79*

Blood was collected from normal, tumor control, and A. lanata- treated tumor-bearing mice on Day 6 after tumor inoculation.  Serum was separated and the cytokine level estimated by ELISA.

Results are expressed as mean ± SD (n = 6/group). * p < 0.05 vs  value for normal mice.

Table 3.  Effect of A. lanata on in vivo CTL generation and survival of animals (System A).

Treatment	Number of days survived	% ILS
EL4 alone	31.4 ± 2.6
EL4 + A. lanata	43.4 ± 3.6*	38.22
EL4 + normal alloimmunized effector cells	37.8 ± 1.2	20.38
EL4 + normal alloimmunized effector cells + 5 doses of A. lanata	45.3 ± 2.7*	44.27
EL4 + A. lanata-treated alloimmunized spleen cells	46.7 ± 2.2*	48.73
EL4 + A. lanata-treated alloimmunized spleen cells + 5 doses of A. lanata	54.6 ± 3.1*	73.89

All mice received EL4 cells (5 × 10⁴ cells) with effector cells  produced in vivo by different types of treatments with  A. lanata. CTL activity was determined by the Winn’s  neutralization assay and animals were observed for survival.

Results are expressed as mean ± SD (n = 6/group). * p < 0.05 vs  value for EL4-only (alone) mice.

Table 4.  Effect of A. lanata on in vitro CTL generation and survival of animals (System B).

Treatment	Number of days survived	% ILS
EL4 alone	31.4 ± 2.6
EL4 + normal co-cultured spleen cells	39.7 ± 1.6	26.43
EL4 + normal co-cultured spleen cells + 5 doses of A. lanata	44.9 ± 2.1*	42.99
EL4 + A. lanata -treated co-cultured spleen cells	47.2 ± 2.4*	50.32
EL4 + A. lanata treated co-cultured spleen cells + 5 dose of A. lanata	53.7 ± 2.8*	71.02

All mice received EL4 cells (5 × 10⁴ cells) with effector cells  produced in in vitro conditions. CTL activity was determined  by the Winn’s neutralization assay. The animals were treated  with A. lanata, and observed for survival.

Results are expressed as mean ± SD (n = 6/group). * p < 0.05 vs  value for EL4-only (alone) mice.

Table 5.  Effect of A. lanata on in vitro CTL production and survival of animals (System C).

Treatment	Number of days survived	% ILS
EL4 alone	31.4 ± 2.6
EL4 + normal co-cultured spleen cells	39.7 ± 1.6	17.0
EL4 + A. lanata-treated co-cultured spleen cells	52.6 ± 2.3*	79.8

All mice received EL4 cells (5 × 10⁴ cells) and effector cells  produced in vitro culture conditions in the presence or  absence of A. lanata for 5 days at 37°C. CTL activity was  determined by the Winn’s neutralization assay. Animals were  observed for survival.

Results are expressed as mean ± SD (n = 6/group). * p < 0.05 vs  value for EL4-only (alone) mice.

Table 6.  Effect of A. lanata on cytokine production during in vivo CTL generation (System A).

Treatment	IL-2 (pg/ml)	IFNg (pg/ml)
Normal	10.26 ± 0.64	282.94 ± 69.18
EL4 alone	7.58 ± 0.41	1958.43 ± 46.85
EL4 + A. lanata	2.58 ± 0.39*	2683.82 ± 37.62*
EL4 + normal alloimmunized effector cell	8.20 ± 0.19	2294.28 ± 49.16
EL4 + normal alloimmunized effector cell + 5 doses of A. lanata	14.73 ± 1.04*	3183.24 ± 34.32*
EL4 + A. lanata treated alloimmunized spleen cells	16.16 ± 0.76*	3304.73 ± 58.25*
EL4 + A. lanata treated alloimmunized spleen cell + 5 doses of A. lanata	19.38 ± 0.93*	3461.03 ± 62.84*

All mice received EL4 cells (5 × 10⁴ cells) with effector cells  produced in vivo by different types of treatment with A. lanata.  CTL activity was determined by the Winn’s neutralization  assay and animals were observed for survival. Blood was  collected from each animal by tail vein bleeding 2 days after  the final dose of A. lanata. Serum IL-2 and IFNg levels were  determined using ELISA kit.

Results are expressed as mean ± SD (n = 6/group). * p < 0.05 vs  value for normal mice.