inflammatory drugs from plant sources that can be used for the treatment of inflammatory disorders. Ursolic acid, (UA; 3b-hydroxy-12-urs-12-en-28-oic acid), a natural pentacyclic triterpenoid carboxylic acid is present in a wide variety of plants, including apples, basil, bilberries, cranberries, peppermint, rosemary, oregano [22]. Several bio- chemical and pharmacological effects of UA such as anti- inflammatory, antioxidant, anti-proliferative, anti-cancer, anti- mutagenic, anti-atherosclerotic, anti-hypertensive, anti-leukemic and antiviral properties are reported in a number of experimental systems [23,24]. UA exhibited anti-inflammatory effects in RAW264.7 cells (Mouse monocyte macrophage cell line) by attenuating inducible nitric oxide synthase and cycloxygenase-2 expression [25,26]. The anti-proliferative, anti-tumor and anti- leukemic properties have been shown to be mediated via suppression of NF-kB activation and inhibiting the expression of NF-kB regulated genes like lipoxygenase, COX-2, MMP-9, and iNOS [27,28,29,30]. It is well known that activation of NF-kB, MAPKs, AP-1 and NF-AT following major histocompatible complex-T cell receptor (MHC-TCR) interaction is vital for the antigen induced lymphocyte proliferation, cytokine secretion and survival [31]. In resting T cells, NF-kB is sequestered into an inactive state by the cytoplasmic inhibitor of NF-kB (IkBa). T cell activation through TCR leads to the rapid activation of the IkB kinases (IKKs) via protein kinase C and results in phosphorylation and subsequent degradation of IkB proteins which allows nuclear translocation of NF-kB [32]. Since dysregulation of NF-kB function is associated with inflammation, any molecule that interferes with NF-kB activation is a potential candidate for therapeutic strategy in the treatment of inflammatory diseases. The present study was aimed to investigate anti-inflammatory properties of UA in murine lymphocytes. The molecular mechanism of action of UA for the observed anti-inflammatory activity was also studied. Results UA inhibited Con A and anti-CD3/CD28 mAb induced proliferation of lymphocytes in vitro The potential immunomodulatory effects of UA were studied by stimulating murine splenic lymphocytes with polyclonal T cell mitogen Con A or with plate bound anti-CD3 plus soluble anti- CD28 mAb in the presence or absence of UA. Lymphocyte proliferation induced by Con A or anti-CD3/CD28 mAb was assessed by CFSE dye dilution using a flowcytometer. As shown in figure 1A–D, UA inhibited Con A induced lymphocyte prolifer- ation in a dose dependent manner in vitro. UA at 5 mM completely inhibited both Con A and anti-CD3/CD28 mAb induced lymphocyte proliferation (Fig. 1B & D). This inhibition of proliferation may be due to inhibition of entry of cells into S phase of the cell cycle as evinced from cell cycle analysis (Fig. 1E). The fraction of cells in S+G2/M phase of cell cycle in UA treated lymphocytes stimulated with Con A was significantly lower than that in lymphocytes stimulated with Con A alone (Fig. 1E). There was a concomitant increase in the percentage of cells in G1 phase of cell cycle in UA treated lymphocytes stimulated with Con A than that in lymphocytes stimulated with Con A alone indicating that UA induced G1 phase arrest in activated T cells (Fig. 1E). This inhibition of mitogen and anti-CD3/CD28 mAb induced T cell proliferation was not due to induction of cell death as this inhibitory concentration of UA was found to be non-toxic to lymphocytes when assessed by propidium iodide (PI) staining (Fig. 1F&G). Anti-Inflammatory Effects of Ursolic Acid UA inhibited proliferation of CD4+ T cells, CD8+ T cells and B cells T cells (CD4+ and CD8+) and B cells are the two major cell types involved in the adaptive immune response following pathogenic invasion. We studied whether UA acts on both these cell types or is specific to a particular lineage of lymphocytes. As shown in Fig. 2A–F, UA inhibited anti-CD3/CD28 mAb induced proliferation of CD4+ T cells (Fig. 2A&B), CD8+ T cells (Fig. 2C&D) and LPS stimulated proliferation of B cells (Fig. 2E&F). UA inhibited Con A, anti-CD3/CD28 mAb and LPS induced cytokine secretion by lymphocytes, CD4+ T cells and macrophages in vitro Fig. 3 shows the secretion of IL-2, IL-4, IL-6 and IFN-c cytokines by lymphocytes stimulated with Con A or anti-CD3/ CD28 mAb in the presence or absence of UA. Lymphocytes stimulated with Con A or anti-CD3/CD28 mAb produced significantly higher levels of IL-2, IL-4, IL-6 and IFN-c cytokines (Fig. 3A & B). Treatment of cells with UA (5 mM) completely inhibited both Con A and anti-CD3/CD28 mAb induced secretion of IL-2, IL-4, IL-6 and IFN-c cytokines (Fig. 3A & B). These results were in agreement with the earlier results showing complete inhibition of Con A and anti-CD3/CD28 mAb induced proliferation of lymphocytes by UA at 5 mM (Fig. 1B&D). Similar anti-inflammatory effects of UA were observed on CD4+ T cells when they were stimulated with anti-CD3/CD28 mAb in the presence of UA. Treatment of purified CD4+ T cells with UA prior to stimulation with anti-CD3/CD28 mAb led to complete the inhibition of secretion of IL-2, IL-4, IL-6 and IFN-c (Fig. 3C). It was also observed that treatment of splenic adherent macrophages with UA prior to stimulation with LPS completely inhibited the secretion of IL-6, IL-1b and TNF-a cytokine (Fig. 3D). Inhibition of gene expression in activated CD4+ T cells by ursolic acid Quantitative real time RT-PCR for 6 genes that are known to be involved in T cell activation (IL-2), B cell activation (phospholipase C gamma 2 (Plcg2)), cell division/cycle (cell division cycle 25 homolog A (Cdc25a), E2F, growth arrest and DNA damage 45 gamma (Gadd45g) and minichromosome maintenance complex component 7 (Mcm7)) and functioning (IFN-g) was carried out. On activation with CD3/CD28 mAb the expression of all these genes was elevated in comparison to control (Fig. 3E). But treatment of cells with UA prior to activation with antiCD3/CD28 resulted in significant suppression of mRNA levels of these genes except for Plcg2 (Fig. 3E). UA inhibited up-regulation of activation markers and co- stimulatory molecules on both T and B cells Optimum T cell activation requires signaling through both T cell receptor as well as through co-stimulatory proteins. Activation of T cells only through TCR in the absence of a co-stimulatory signals leads to T cell anergy. Also, T cell upon activation up- regulate certain cell surface proteins which are necessary for complete T cell activation and effector functions. The effect of UA on T cell activation markers and co-stimulatory molecules was studied to determine whether UA inhibits T cell activation and induces T cell anergy. Fig. 4 shows the expression of early and late T cell activation markers CD69 (Fig. 4A), CD25 (IL-2R-a, Fig. 4B) and CD134 (Fig. 4C) and co-stimulatory molecule CD28 (Fig. 4D) in lymphocytes treated with UA (5 mM, 4 h) and stimulated with PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31318