Normal Splenocytes Research Articles

In vivo Heparan Sulfate Treatment Alters the Immune Response of Normal and LLC-Bearing Mice

Despite the large amount of research dedicated to the understanding and treatment of tumor growth, the majority of cancers continue to lack effective therapeutic options. As in the case of most solid tumors, growth requires evasion of the host immune system. Our previous work using the Lewis Lung Carcinoma (LLC) model of tumor bearing (TB)-mice has shown several tumor-induced immune suppressing effects to be present. These effects include a decreased T-cell proliferative response to Con A and altered cytokine secretion patterns that favor neither a Th1 nor a Th2 response. To address these immune alterations, immune modulating approaches have been a central area of study. Of the many potential immune modulating compounds, we believe promising therapeutic potential lies in the heparin family. Heparan sulfate (HS), in particular, has been shown to increase T-cell proliferative response in non TB-mouse splenocytes as well as promotion of a beneficial Th1 response. In this paper, we studied the potential of HS to decrease tumor burden via in vivo treatment of TB-mice. Results showed both normal and TB-mice splenocytes had a dose response change in proliferation as a result of HS treatment. Furthermore, splenocytes from HS treated TB-mice showed a potentially beneficial decrease in basal level proliferation. On gross examination, HS treatment produced a decrease in tumor surface necrosis with a visible (2 ± 1.8%) surface necrotic area in treated mice as opposed to a (43 ± 16%) surface necrotic area in untreated mice. HS treatment decreased TB-mice splenomegaly when comparing mice spleen weights in treated (0.3 ± 0.05 g) vs. untreated (0.14 ± 0.02 g) groups. These results show a potential role of HS as an immune modulating agent with antitumor properties.

Adenovirus-mediated CTLA4Ig and OX40Ig gene transfer induces long-term survival of cardiac allografts in rats

To investigate the potential role of CTLA4Ig gene and OX40Ig protein in inducing transplantation tolerance and the mechanisms thereof. Thirty Lewis rats underwent transplantation of the hearts of DA rats and then randomly divided into five equal groups: control group, blank virus AdEGFP treated group (adenovirus containing EGFP at the dose of 1-5 x 10(9) pfu/ml was infused via portal vein immediately after the operation), AdCTLA4Ig treated group, AdOX40Ig treated group, and AdCTLA4Ig-IRES-OX40Ig treated group. The cardiac allograft survival was monitored by daily palpation. The total cessation of beating was defined as rejection and was confirmed by histology. Peripheral venous blood samples were collected 0, 3, 7, 10, 14, 21 and 28 days after the administration of adenovirus. ELISA was used to detect the expression of CTLA4Ig and OX40Ig. Twenty days after the heart transplantation single splenocyte suspension was prepared from surviving Lewis rats to be used as responder. The spleens of the normal donor-DA rats and the third strain DA rats to prepare single cell suspension of the same density to perform mixed lymphocyte reaction (MLR). Then recombinant IL-2 was added into the mixed MLR system to observe t\if the MLR could be reversed. Twenty days after the heart transplantation the splenocytes of the tolerating Lewis rats were injected into the lingual vein of the normal Lewis rats to observe the delayed type hypersensitivity (DTH) of the transferred Lewis rat to normal rat splenocytes. RT-PCR was used to detect the mRNA expression of IL-2, interferon-gamma, IL-4, and IL-10. The survival time of the AdCTLA4Ig-IRES-OX40Ig treated group was 151.5 d +/- 42.6 d, significantly longer than those of the AdOX40Ig treated group (60.2 d +/- 11.4 d (P = 0.003), AdCTLA4Ig (43.2 d +/- 11.1 d, P = 0.0026), control group (5.7 d +/- 0.5 d, P = 0.000 43), and AdEGFP treated group (5.2 d +/- 0.4 d, P = 0.000 43). CTLA4Ig and/or OX40Ig proteins were expressed at a high level in the adenoviral treated rats. Compared with the control group the splenocytes of the AdCTLA4Ig-IRES-OX40Ig, AdCTLA4Ig, and AdOX40Ig treated groups displayed donor-specific hyporesponsiveness (P = 0.0016, 0.0026 and 0.001), which could be partly reversed by the addition of exogenous IL-2. Moreover, the hyporesponsiveness could be transferred to the same strain rats through adoptive transfer. In comparison with the normal controls, the expression of Th1 type cytokines, such as IL-2 and IFN-gamma, was significantly decreased in the tolerating rats and significantly increased in the rats with rejection; however the expression of the Th2 type cytokines, such as IL03 and IL-10, was significantly increased in the tolerating rats and significantly decreased in the rats with rejection, showing a deviation of Th1/Th2 type cytokines. AdCTLA4Ig-IRES-OX40Ig-mediated genes transfer renders prolonged expression of CTLA4Ig and OX40Ig in Lewis recipient rats, leading to a long-term survival of cardiac allografts. The induced tolerance is donor-specific, and the mechanisms may be associated with T cell anergy, deviation of Th1/Th2, and the regulatory T cells.

B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors

The B7/CD28 co-stimulatory pathway plays a critical role in T cell activation and differentiation. Our previous study demonstrated that administration of B7.2-Ig fusion proteins to tumor-bearing mice elicits IL-4-dependent, CD8+ T cell-mediated tumor regression. Here, we investigated whether B7.2-Ig stimulation of tumor-sensitized CD8+ CTL precursors during in vitro antigen re-sensitization actually results in their differentiation into mature CTLs and if so, whether such a process depends on IL-4 signals. Splenocytes from tumor-sensitized (tumor-bearing or tumor-immunized) mice exhibited low levels of anti-tumor CTL responses upon culturing alone, but induced strikingly enhanced CTL responses when stimulated in vitro with B7.2-Ig fusion proteins. Because CTLs were not generated from normal splenocytes even by B7.2-Ig stimulation, the expression of the B7.2-Ig effect required the in vivo tumor sensitization of CD8+ CTL precursors. Administration of anti-CD4 or anti-CD40 ligand (CD40L) to mice before tumor sensitization resulted in almost complete inhibition of CTL responses generated in the subsequent culture containing B7.2-Ig. In contrast, anti-IL-4 did not influence in vivo tumor sensitization required for CTL induction. However, B7.2-Ig stimulation of tumor-sensitized splenocytes enhanced IL-4 production and neutralization of this IL-4 with anti-IL-4 potently down-regulated CTL responses. These results indicate that B7.2-Ig enhances IL-4-dependent differentiation of anti-tumor CD8+ CTL precursors that can be sensitized in vivo depending on collaboration with CD4+ T cells involving CD40L function.

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells

AbstractThe stromal compartments of hematopoietic organs (eg, spleen) are known to influence the viability and growth of diseased hematopoietic progenitors. Here we have used Friend murine leukemia virus (F-MuLV)–induced erythroleukemia to investigate factors of the splenic microenvironment that may make it fertile for the expansion and survival of malignant erythroblasts. We found that splenectomized, erythroleukemic mice exhibited extended survival compared with age-matched sham controls. In vitro, the proliferation of primary erythroleukemic cells cocultured with leukemic-derived splenic adherent cells or their conditioned media was found to be significantly higher than that observed in cocultures with healthy-derived adherent splenic cells. Cytokine protein arrays revealed that F-MuLV–infected splenocytes secreted elevated levels of interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), macrophage chemoattractant protein-5 (MCP-5), soluble tumor necrosis factor receptor-1 (sTNFR1), IL-12p70, tumor necrosis factor-α (TNF-α), and IL-2 over normal splenocytes. Medium supplemented with both VEGF-A and MCP-5 could sustain proliferation of primary erythroleukemic cells in vitro, and significant proliferative suppression was observed upon addition of neutralizing antibodies to either of these factors. Furthermore, in vivo administration of a neutralizing antibody to VEGF-A extended survival times of erythroleukemic mice in comparison with controls. These findings suggest that VEGF-A and MCP-5 are potentially pivotal paracrine mediators occurring within the diseased splenic microenvironment capable of promoting disease acceleration and expansion of erythroleukemic blasts.

Ginsan improved Th1 immune response inhibited by gamma radiation

Gamma radiation causes suppression of the immune function, and immune properties are related to cytokine production. In the present study, the polysaccharide, Ginsan, purified from an ethanol-insoluble fraction of Ginseng (Panax ginseng C.A. Meyer, Araliaceae) water extract was studied to assess its effects on the immunosuppressive activities of gamma radiation. Ginsan was found to stimulate murine normal splenocytes by inducing the mRNA expressions of Th1 and Th2 type cytokines, and also restore the mRNA expression of IFN-gamma, Th1 cytokine, after its inhibition by whole-body gamma irradiation. Therefore, Ginsan was found to restore the T lymphocytes function that had been suppressed by gamma irradiation in allogenic MLR (mixed lymphocyte reactions). However, Ginsan exhibited no excessive stimulatory effects on the control group. The above results indicated that Ginsan may constitute a new noble agent for the improvement of gamma radiation-induced immunosuppression.

Two Related Cinnamic Acid Derivatives from Brazilian Honey Bee Propolis, Baccharin and Drupanin, Induce Growth Inhibition in Allografted Sarcoma S-180 in Mice

Honey bee propolis is rich in cinnamic acid derivatives. Baccharin and drupanin from Brazilian honey bee propolis are cinnamic acid derivatives that contain prenyl moieties. We previously isolated these two compounds and demonstrated that they induce an apoptotic event in several tumor cell lines. In this study, we examined the tumoricidal activity of baccharin and drupanin in mice allografted with sarcoma S-180 and also studied the genotoxic effects on normal splenocytes using the alkaline single cell gel (comet) assay. We found that both baccharin and drupanin effectively suppressed growth of the tumor. Furthermore, these compounds induced a significant genotoxic effect on the tumor cells in comparison with normal splenocytes. Thus, baccharin and drupanin are potent tumor suppressive components of honeybee propolis.

The anti-cancer effects of poi(Colocasia esculenta) on colonic adenocarcinoma cellsIn vitro

Hawaiians tend to have lower incidence rates of colorectal cancer and it was hypothesized that this may be due to ethnic differences in diet, specifically, their consumption of poi, a starchy paste made from the taro (Colocasia esulenta L.) plant corm. Soluble extracts of poi were incubated at 100 mg/mL in vitro for antiproliferative activity against the rat YYT colon cancer cell line. (3)H-thymidine incorporation studies were conducted to demonstrate that the poi inhibited the proliferation of these cancer cells in a dose-dependent manner. The greatest suppression of YYT colon cancer growth occurred when 25% concentration was used. When poi was incubated with the YYT cells after 2 days, the YYT cells underwent apoptotic changes as evidenced by a positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stain. Poi enhanced the proliferation of normal mouse splenocyte control cells, suggesting that poi is not simply toxic to all cells but even has a positive immunostimulatory role. By flow cytometry, T cells (CD4+ and CD8+) were predominantly activated by the poi. Although numerous factors can contribute to the risk of colon cancer, perhaps poi consumption may contribute to the lower colon cancer rates among Hawaiians by two distinct mechanisms. First, by inducing apoptosis within colon cancer cells; second, by non-specifically activating lymphocytes, which in turn can lyse cancerous cells. Our results suggest for the first time that poi may have novel tumor specific anti-cancer activities and future research is suggested with animal studies and human clinical trials.

Involvement of the immune response in the cure of metastatic murine CT-26 colon carcinoma by low electric field-enhanced chemotherapy

Low electric field cancer treatment-enhanced chemotherapy (LEFCT-EC) is a new treatment modality that combines chemotherapeutic agents and low electric field stimulation. LEFCT-EC was found to destroy malignant mouse tumors and cause massive death of tumor cells. This may enable the immune system cells to efficiently recognize and eliminate tumor cells at the primary tumor site and at metastatic foci. Mice with 15 mm diameter intracutaneous colon carcinomas (CT-26) were injected with BCNU (35 mg/kg), and 2 min later the tumors were exposed to low electric fields (intensity 40 V/cm, pulse duration 180 micros, frequency 500 Hz) for 12 min (LEFCT-EC). We found that treatment with LEFCT-EC achieved complete cure of 93% of the animals. In comparison, electric fields alone (13% cure), chemotherapy alone (0%), surgery (15%) or a combination of surgery and bis-chloroethyl-nitrosurea, carmustine (BCNU; 84%) treatments resulted in lower cure rates. After treatment and cure with LEFCT-EC, 50% of the cured mice developed resistance to a tumor challenge (surgery + BCNU only 15%). Furthermore, splenocytes from cured animals protected naive animals from a tumorigenic dose of tumor cells. Separation of spleen cells into lymphocyte subpopulations indicated a major role for CD4 and CD8 T cells in this protection. FACS analysis revealed restoration of normal splenocyte subpopulation proportions impaired by cytotoxic chemotherapy. Our results suggest that LEFCT-EC can directly destroy primary tumors and facilitate the destruction of metastatic disease by enforcement of antitumor immune responses.

A New Multi-Parameter Flow Cytometric Assay for Monitoring Lymphoma Growth and Spread in a Pre-Clinical Murine Model for Human Lymphoma.

Murine lymphoma models are commonly used to test the efficacy of idiotype-based vaccines for B-cell lymphoma prior to human trials. Mouse survival has been used as an indirect measure of the tumor response to the vaccine. In this study, we describe a multiparameter flow cytometric (MPFC) assay that enables direct assessment of the tumor at the inoculation site and detection of lymphoma metastases. In initial experiments, we show that 38C13 mouse lymphoma cells expressed an aberrant phenotype compared to normal splenocytes. The combination of CD45, CD19, B220 and an anti-idiotypic antibody (S1C5) along with a sequential gating technique defined the 38C13 cells with high sensitivity and specificity. The specificity of the MPFC assay for 38C13 lymphoma cells was greater than a gating strategy using light scatter and idiotype antibody binding. The MPFC assay was used to monitor 38C13 tumor kinetics in the C3H/HeN mouse and demonstrated tumor growth at the inoculation site and metastases to the lymph nodes and bone marrow. The presence of 38C13 metastases in lymphoid organs and bone marrow predicted by the MPFC assay were confirmed by histology and immunohistochemistry (IHC). The MPFC assay will enable investigators to monitor 38C13 lymphoma tumor response to individual vaccines or other lymphoma therapy prior to clinical trials. Furthermore, the MPFC concept could be readily applied to other models of human B cell lymphoma.

Modification of mitogen-driven lymphoproliferation by ceftriaxone in normal and immunocompromised mice

Cyclophosphamide-treated mice are proposed as a model to assay immunomodulation by antimicrobial agents in immunocompromised animals. Cyclophosphamide-treated BALB/c mice developed temporary leukopenia, myelopenia and spleen atrophy that was followed by splenomegaly. Cells from both atrophic and hypertrophic spleens exhibited impaired responses to mitogens and suppressed the mitogen-driven proliferation of normal splenocytes. This experimental model was applied to the study of immunomodulation by ceftriaxone. Ceftriaxone did not worsen the cyclophosphamide-damaged immunity mechanisms. On the contrary, cyclophosphamide-induced suppression of lymphoproliferation in response to concanavalin A was attenuated by ceftriaxone.

Lymphocyte regulation of neuropeptide gene expression after neuronal injury.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are induced strongly in neurons after several types of injury, and exhibit neuroprotective actions in vitro and in vivo. It is thought that changes in expression of neuropeptides and other molecules in injured neurons are mediated by new factors produced in Schwann and immune cells at the injury site, a loss of target-derived factors, or a combination of mediators. To begin to determine the role of the inflammatory mediators, we investigated axotomy-induced changes in VIP and PACAP gene expression in the facial motor nucleus in severe combined immunodeficient (SCID) mice, and in mice with targeted mutations in specific cytokine genes. In normal mice, VIP and PACAP mRNA was induced strongly in facial motor neurons 4 days after axotomy. The increase in PACAP mRNA was blocked selectively in SCID mice, indicating that mechanisms responsible for VIP and PACAP gene induction are not identical. The loss of PACAP gene expression in SCID mice after axotomy was fully reversed by an infusion of normal splenocytes, suggesting that PACAP mRNA induction requires inflammatory mediators. PACAP and VIP mRNA inductions, however, were maintained in mice lacking leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), and in mice lacking both receptors for tumor necrosis factor alpha (TNFalpha). The data suggest that an inflammatory response, most likely involving T lymphocytes, is necessary for the axotomy-induced increase in PACAP but not in VIP. LIF, IL-6, and TNFalpha, however, are not required for this response to injury.

N-Acetylcysteine enhances UV-mediated caspase-3 activation, fragmentation of E2F-4, and apoptosis in human C8161 melanoma: inhibition by ectopic Bcl-2 expression

Redox imbalance due to oxidative stress or excessive antioxidant levels can alter apoptotic responses. Recently, antioxidants like N-acetylcysteine (NAC) were reported to inhibit H 2O 2-mediated necrotic cell death, although they were inactive against apoptosis induced by other agents like etoposide. NAC was also found to kill preferentially tumor cells compared to normal fibroblasts at 20–50 mM, but these concentrations are lethal to normal splenocytes. We now demonstrate that 10 mM NAC, a non-toxic concentration, can enhance the UV radiation-mediated apoptosis of human C8161 melanoma cells. Compared to treatment with UV radiation alone, combination treatment with NAC doubled the ratio of activated caspase-3 to pro-caspase-3 and produced greater fragmentation of the retinoblastoma protein and the E2F-4 transcription factor without affecting the E2F-1 protein. These effects of joint NAC–UV radiation treatment were counteracted by the overexpression of the bcl-2 gene. To our knowledge, this report is the first to: (i) demonstrate a synergy between DNA-damaging agents, like UV radiation, and antioxidants, like NAC, and (ii) show that a Bcl-2-inhibitable E2F-4 fragmentation occurs concurrently with caspase-3 activation and apoptosis.

A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin–agarose or a trypsin–agarose column. In both cases, SDS–PAGE showed two bands of M r 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K is for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6×10 −7 and 1.3×10 −5 M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

Immunobiological features of the galactoside lectin L-Lc isolated from the Argentine mistletoe Ligaria cuneifolia

Ligaria cuneifolia has been used in Argentine folk medicine and is currently employed as substitute for the European mistletoe ( Viscum album) as hypotensor agent. Extracts from V. album are widely used in cancer therapy and the antineoplasic effect is attributed to their cytostatic/cytotoxic and immunomodulatory actions. When studying immunomodulatory effects of L. cuneifolia extracts ( Lc extracts), they inhibited proliferation of murine mitogen-activated lymphocytes, leukaemic lymphocytes (LB) and breast tumour cells (MMT). The aim of this work was to isolate and identify lectins from Lc extracts and investigate their immunobiological actions. A galactoside lectin (L-Lc) of 57 kDa was isolated. A polyclonal antiserum obtained against Lc extract recognised both L-Lc and MLI ( V. album lectin), suggesting the possibility of shared epitopes. Treatment of LB tumour cells with L-Lc (0.01 and 0.1 μg/ml) produced up to 40.0±6.9% inhibition of cell growth, which seems partly mediated by apoptosis (apoptosis of L-Lc treated cells 58.4±10.3% versus non-treated cells 38.1±8.8%; P<0.05), analysed by acridine orange and ethidium bromide staining. Inhibitory effect on ConA stimulated splenocyte growth was non-significant, while a mitogenic effect was observed on normal murine splenocytes and MMT cells. L-Lc in non-cytotoxic concentrations (250 ng/ml) modified mRNA expression of IL-10 but neither that of TGF-β nor of IL-2 produced by LB cells. In addition, 43.9±0.5% reduction in NO production by LPS-stimulated murine macrophages was found. Finally, survival rates of LB tumour-bearing mice treated or not with Lc extract or L-Lc failed to show significant differences.

Conservation of Relative Chromosome Positioning in Normal and Cancer Cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM−/− mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.

FR901228, an inhibitor of histone deacetylases, increases the cellular responsiveness to IL-6 type cytokines by enhancing the expression of receptor proteins.

The related members of the interleukin-6 (IL-6) family of cytokines, leukemia inhibitory factor (LIF), oncostatin M (OSM) and IL-6 are inflammatory mediators that control differentiated cell functions as well as proliferation. The cellular responsiveness to these cytokines is largely determined by the expression of the appropriate receptor proteins. The receptor expression profile for each cell type is established during differentiation and is often altered during oncogenic transformation. Since inhibition of histone deacetylases (HDAC) has the potential to re-activate epigenetically silenced genes, we asked whether inhibition of HDAC enhances the expression of IL-6 cytokine receptors and, thus, increase desirable cytokine responses. We demonstrate that treatment with FR901228 (FR), an HDAC inhibitor, increases the responsiveness to LIF in different cell types, including normal fibroblasts, epithelial cells, macrophages and splenocytes, as well as various tumor cell lines. Depending on the cell type, FR treatment also enhances the responsiveness to OSM and IL-6. These effects involve a transcriptional induction of the cytokine receptor subunits LIFRalpha, OSMRbeta, gp130, or the transcription factor STAT3. FR-specific induction of LIFRalpha occurs independently of de novo protein synthesis and cell proliferation and is mediated in part by the CBP/p300 coactivator. Chromatin immunoprecipitation experiments indicate that the expression of LIFRalpha and gp130 genes correlates with the level of acetylated histone 3 associated with the receptor promoter regions. The FR-stimulated expression of IL-6-type cytokine receptors in certain tumor cells also provided improved conditions for suppression of cell growth by taking advantage of the growth inhibitory effect of these cytokines.

Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol

AbstractCb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to protein-coding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion inCb2, exon-1B/exon-2 Cb2messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2complementary DNA and a myeloid cell line carrying a virus insertion in Cb2(ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoatractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG–induced migration of B220-, CD19-, immunoglobulin M–, and immunoglobulin D–expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration.

Phosphatidylinositol 3-Kinase p85 Adaptor Function in T-cells: CO-STIMULATION AND REGULATION OF CYTOKINE TRANSCRIPTION INDEPENDENT OF ASSOCIATED p110

Phosphatidylinositol 3-kinase (PI3K) is a key regulator of a variety of cellular functions from cytoskeletal organization, vesicular trafficking, and cell proliferation to apoptosis. The enzyme complex is comprised of an 85-kDa adaptor (p85) coupled to a 110-kDa catalytic subunit (p110). While the function of PI3K has been largely attributed to the generation of D-3 lipids, an unanswered question has been whether p85 with a number of motifs (SH2, SH3, BcR homology (BH) region) can generate independent intracellular signals. In this study, we demonstrate that p85 lacking p110 (Deltap85) can activate NFAT transcription in T-cell hybridomas and normal splenocytes. This up-regulatory effect was unaffected by inhibition of PI 3-kinase, and cooperated specifically with Rac1, but not related family members. Stimulation correlated with Rac1 binding and was lost with the deletion of the BH domain. Lastly, the CD28-Deltap85 chimera also cooperated with TcR/CD3 to provide co-signals that enhanced IL-2 transcription. Our findings identify for the first time p85 as an adaptor that operates independently of the classic PI 3-kinase catalytic pathway and further shows that this pathway can provide co-signals in the regulation of T-cell function.

Immunomodulating properties of Argentine plants with ethnomedicinal use.

Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties. Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatorium arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, IC50 ranged between 1-75 microg/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 microg/ml to produce the effect. On mitogenic activated splenocytes, IC50 ranged between < 1 to 85 microg/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation (IC50 0.5-1.5 microg/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active (IC50 5 microg/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus (IC50 11.2 and 17.3 microg/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.

Intestinal infection of BALB/c mice with Yersinia enterocolitica O9 causes major modifications in phenotype and functions of spleen cells.

Yersinia enterocolitica serotype O9 may cause a persistent intestinal infection with few or no symptoms in humans and in BALB/c mice. The present study demonstrated profound alterations in the immune status of BALB/c mice infected with Y. enterocolitica O9. Infected mice developed splenomegaly and phenotypic analysis of spleen cells revealed increases in CD3+ total T cells, CD4+ helper T cells, CD8+ cytotoxic T cells and CD11b+ phagocytic cells. Spleen cells from infected mice exhibited impaired responses to mitogens and suppressed the proliferation of normal splenocytes in response to mitogens. Suppression of responses to concanavalin A and heat-killed yersiniae was associated with increased production of gamma interferon and reactive nitrogen intermediates. Y. enterocolitica-infected mice resisted challenge with a lethal dose of the intracellular pathogen Listeria monocytogenes. These findings suggest that infection of mice with Y. enterocolitica O9 induces gamma-interferon-secreting cells that promote macrophage activation, mediating resistance to infection with L. monocytogenes, and macrophage production of reactive nitrogen intermediates, which results in in vitro inhibition of lymphocyte response to mitogens.