concanavalin-a and Carcinoma--Lewis-Lung

Polymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models.. Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01).. In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.

Topics: Administration, Oral; Animals; Carcinoma, Lewis Lung; Concanavalin A; Cytokines; Dendritic Cells; Disease Models, Animal; Escherichia coli; Female; Injections, Intravenous; Lipopolysaccharides; Lung; Lung Neoplasms; Lymphocytes; Macrophages, Peritoneal; Melanoma, Experimental; Mice, Inbred C57BL; Phenotype; Polymyxin B; Spleen

Our previous study isolated an anti-fatigue polysaccharide (HRWP) from the Hippophae rhamnoides berry. In this study, using ion-exchange chromatography and gel filtration chromatography in turn, a water-soluble homogenous polysaccharide HRWP-A was isolated from HRWP. Structural analysis determined that HRWP-A was a polysaccharide with repeating units of (1→4)-β-d-galactopyranosyluronic residues, of which 85.16% were esterified with methyl groups. An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma (LLC) growth in tumor-bearing mice. Further experiments suggested that the antitumor effect of HRWP-A might be mediated through immunostimulating activity, as it enhances the lymphocyte proliferation, augments the macrophage activities, as well as promoting NK cell activity and CTL cytotoxicity in tumor-bearing mice. To our knowledge, this is the first report on a natural antitumor high-methoxyl homogalacturonan pectin from the H. rhamnoides berry-a compound that acts as a potential immunostimulant and anticancer adjuvant.

Topics: Animals; Antineoplastic Agents; Body Weight; Carbon-13 Magnetic Resonance Spectroscopy; Carcinoma, Lewis Lung; Concanavalin A; Cytotoxicity, Immunologic; Fruit; Hippophae; Immunologic Factors; Killer Cells, Natural; Lipopolysaccharides; Macrophages; Male; Mice, Inbred C57BL; Nitric Oxide; Pectins; Phagocytosis; Proton Magnetic Resonance Spectroscopy; Spectroscopy, Fourier Transform Infrared; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

Tumor-bearing (TB) patients and TB animal models show a wide array of immunologic deficits. Heparan sulfate (HS) has been shown to both improve immune cell proliferative responses and to induce Th1 cytokine responses in normal animals. These HS effects, if harnessed, would be of great benefit to TB patients. The present study focused on replicating previous HS-induced Th1 and proliferative response results as well as extrapolating the beneficial immunomodulatory effects to an experimental model derived from TB animals of Lewis lung cell carcinoma. Lewis Lung Carcinoma (LLC)-TB and control mouse splenocytes were assessed for proliferation and cytokine response to concanavalin A (Con A) with 1 and 3 days' exposure to HS. Our results found HS treatment stimulated splenocyte proliferation to Con A in control mice splenocytes after 1 and 3 days of treatment, although HS proliferative effects were not seen in unfractionated TB cultures. Furthermore, cytokine studies revealed normal splenocytes treated with HS had increased levels of both Th1 and Th2 cytokines. Surprisingly, HS treated TB-splenocytes showed suppressed cytokine levels. Of particular interest was the decreased levels of the Th2 cytokine IL-4 in TB-derived samples. In conclusion, we found that HS did show immune-modulator properties in both normal and TB environments. Our studies reinforced the possibility that HS could one day be used as an immune-modulating therapeutic agent.

Topics: Animals; Antigen Presentation; Carcinoma, Lewis Lung; Concanavalin A; Cytokines; Dendritic Cells; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Female; Heparitin Sulfate; Immunologic Factors; Interleukin-4; Macrophages; Mice; Mice, Inbred C57BL; Spleen; T-Lymphocytes; Th1 Cells; Th2 Cells

The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-gamma-dependent fashion. Intraperitoneal administration of IFN-gamma stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-gamma treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-gamma in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.

Topics: Adoptive Transfer; Animals; Bone Marrow; Carcinoma, Lewis Lung; Cell Communication; Chemical and Drug Induced Liver Injury; Chemokine CXCL10; Chemokine CXCL9; Chemokines, CXC; Concanavalin A; In Vitro Techniques; Interferon-gamma; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Chemokine; Receptors, CXCR3; Recombinant Proteins; Spleen; T-Lymphocytes

Deficits in immune cell responses have been reported in cancer patients. We used the murine Lewis lung carcinoma (LLC) model to better understand these deficits. The goal of this study was to determine if the immune responses of LLC tumor-bearing (TB) mice differ from control mice and whether the difference could be attributed to either antigen-presenting cells (FPC) or to T cells. Tumors were first allowed to grow in vivo for approximately 2 weeks. Splenocytes were then isolated for in vitro proliferation and cytokine release studies. The results showed a decrease in mitogen-stimulated proliferation by unfractionated splenocyte cultures from TB mice when compared to control mice in response to concanavalin A (Con A), a T-cell mitogen. Decreased responses were also observed when the APC spleen cell fraction from TB mice was cultured with normal T cells, although proliferation was more prominently reduced in cultures of TB T cells plus normal APC. Also, splenocytes from TB mice secreted significantly increased levels of IFN-gamma, IL-4, and IL-10. Admixing APC from control mice with TB T cells significantly decreased levels of IL-4 and IL-10 secretion as compared to the levels secreted by cocultures of TB T cells and TB APC. The decreased cytokine profile in the presence of normal APC despite the presence of TB T cells suggests that APC contributes to the immune dysfunction, including Th skewing of tumor bearers, possibly through their influence on T-cell expansion and cytokine production. Finally, our assessment of the APC population contributing to the observed immune dysfunction--i.e., dendritic cells or macrophages--showed that the proliferation of TB T cells was decreased regardless of the APC population with which they were cocultured. However, normal T-cell proliferation was only reduced by the addition of TB macrophages and not by the addition of TB dendritic cells. In conclusion, our results demonstrate that LLC TB mice have a skewed immune response characterized by a decreased proliferative response with both T cells and APC affected by the presence of tumor.

Topics: Animals; Antigen-Presenting Cells; Carcinoma, Lewis Lung; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Concanavalin A; Cytokines; Dendritic Cells; Female; Flow Cytometry; Interferon-gamma; Interleukin-10; Interleukin-4; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Spleen

Immune aberration in cancer patients can be at least partly ascribed to an accumulation of immature myeloid cells and monocytes/macrophages with immunosuppressive functions. Mice implanted with Lewis lung carcinoma 2 (LL/2) cells show marked splenomegaly as the tumors progress, and this condition is accompanied by impaired T cell activities. We characterized the cells that accumulated in the spleens of LL/2 tumor-bearing mice and attempted to restore the normal cell population by employing interleukin-2 (IL-2). Flow cytometric analysis revealed that the cells expressing Mac1, B7, NK-K1, Gra-1, and MHC class II antigens on their surfaces drastically decreased in number when LL/2 had been engineered to produce IL-2. IL-2 also restored the concanavalin A (ConA)-mediated proliferative response and IL-2 production of the spleen cells. The in vivo growth of IL-2-producing tumors was significantly slower than that of parental LL/2 cells. Therefore, local IL-2 production may reverse systemic immune abnormality by stopping myeloid cell accumulation.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Lewis Lung; Cell Line, Tumor; Concanavalin A; Female; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Myeloid Cells; Neoplasm Transplantation; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Transfection; Vascular Endothelial Growth Factor A