Lymphokine research最新文献

The colony-stimulating factor 1 receptor (CSF-1R) is a cell surface glycoprotein consisting of an extracellular ligand-binding domain, a single membrane-spanning segment, and an intracellular tyrosine kinase domain. Binding of CSF-1 activates the receptor kinase, leading to "autophosphorylation" of receptor subunits and the concomitant phosphorylation of a series of cellular proteins on tyrosine residues. The diverse effects of CSF-1 on mononuclear phagocyte proliferation, differentiation, survival, and macrophage effector function appear to reflect the ability of CSF-1R to simultaneously modulate the activities of a series of intracellular proteins that function in relaying biochemical signals. Sequences surrounding sites of ligand-induced tyrosine phosphorylation within CSF-1R may serve as targets for interactions with cellular effector proteins whose activities are modified by receptor binding, tyrosine phosphorylation, or both. The specificity of the cellular response to CSF-1 may depend, at least in part, on the differential coupling of the receptor to these "downstream" effectors in different cell types.

Activated macrophages mediate cytotoxicity against tumor targets and thus may modulate development and growth of metastatic tumor cells. Macrophage colony stimulating factor (M-CSF) has a potential role in activating mature macrophages to a cytotoxic state. We employed a murine Kupffer cell (KC) model of cytotoxicity against a tumor necrosis factor (TNF) - sensitive murine colon adenocarcinoma cell line (MCA26) to evaluate the ability of recombinant human M-CSF (rhM-CSF) 1) to act alone as a KC-activating agent and 2) to enhance KC cytotoxicity against MCA26 cells in association with known macrophage activating compounds. rhM-CSF produced a dose-dependent increase in TNF release by KC in vitro with a parallel increase in MCA26 killing. KC activated by rhM-CSF produced less TNF and concomitantly demonstrated a lower cytotoxicity against MCA26 cells when compared with KC activated by gamma interferon (gamma IFN) with or without lipopolysaccharide (LPS). M-CSF did not act in a synergistic fashion with gamma IFN and LPS to increase TNF secretion or cytotoxicity against MCA26 cells. rhM-CSF thus acts as a single agent capable of activating murine KC to a cytotoxic state but does not cooperate with classical priming/triggering signals to achieve KC activation.

T cell fibronectin (FN) is a product of antigen and mitogen activated human, murine and guinea pig T lymphocytes. Operationally and functionally, T cell FN is a lymphokine associated with delayed hypersensitivity. T cell FN acts at femtomolar concentrations to agglutinate mononuclear phagocytes and translocate monocytes and neutrophils through model extracellular matrices, and is 1.1 x 10(4) to 2.3 x 10(6) times more potent than other FN for these activities. It does not act on peripheral blood lymphocytes. Macrophage agglutination mediated by T cell FN requires cellular metabolism and depends on interactions between multiple classes of cell surface protein receptors and FN gelatin- and cell-binding domains. In contrast, translocation of cells through artificial matrices mediated by T cell FN is a biophysical process dependent on interactions between surface heparan sulfates on responding cells and FN amino-terminal heparin-binding and gelatin-binding domains. The correlation between the ability of cloned murine T cell lines to produce FN and their ability to transfer delayed hypersensitivity reactions suggests that secretion of T cell FN may be an important element in the initiation of these responses. The double activity of T cell FN could allow it to enhance influx of phagocytic effector cells and retain monocytes at tissue sites of T cell activation.

The human keratinocyte cell line COLO-16 expresses mRNA homologous to human IL-1 alpha and IL-beta (transcript sizes 2.3 and 1.6 kb, respectively). A 1.2 kbp cDNA was selected with a human IL-1 beta probe from a lambda gt11 library constructed using poly A+ RNA from COLO-16 cells. Sequence analysis revealed that this cDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1 beta. When this cDNA was expressed in COS cells using a mammalian expression vector, IL-1 activity was detected in the cell conditioned supernatants using assays for D10-T-cell, thymocyte and fibroblast proliferation. Western analysis of lysates from COS cells transfected with this clone revealed the presence of a -17 kDa protein which reacted with antisera to human IL-1 beta. This protein was the same size as the processed form of IL-1 beta present in COLO-16 cells suggesting that this cDNA encodes the mature form of IL-1 beta. COLO-16 cells contain proteins of -30 kDa and 17 kDa which are immunoreactive with specific antisera for human IL-1 alpha and human IL-1 beta. Despite the presence of four-fold greater amounts of immunoreactive IL-1 beta protein than IL-1 alpha in cell lysates, all the IL-1 activity in the lysate could be neutralized by antisera to IL-1 alpha. IL-1 beta comprised only 25% of the IL-1 activity in the cell-conditioned media, all remaining activity was neutralized by antisera to IL-1 alpha. Whereas IL-1 alpha protein in both cell lysates and conditioned supernatants was predominantly in the processed -17 kDa form, IL-1 beta proteins were primarily of the processed and inactive 30kDa species. This apparent inability of keratinocytes to process IL-1 beta may explain our observations that the IL-1 activity secreted by COLO-16 cells is principally due to IL-1 alpha.

The protein-bound polysaccharide extracted from a fungus, PSK, has been used as a biological response modifier in the treatment of cancer patients in Japan for over ten years. Although the antitumor mechanism of PSK is not fully understood, host-mediated antitumor activity has been claimed to play a significant role. The administration of PSK to tumor-bearing rodents inhibited tumor growth and modulated immune responses. To clarify the potential immunomodulating activities of PSK, we examined the direct effect of PSK on cytokine gene expression and production in human peripheral blood mononuclear cells (PBMC) in vitro. As determined by Northern blotting, PSK was a potent inducer of gene expression for IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor (TNF-alpha) and monocyte chemotactic and activating factor (MCAF), but not for IL-2 and lymphotoxin (LT). Expression of mRNA occurred at 1-3 hr in a dose dependent manner using from 5-400 micrograms/ml of PSK. Furthermore, these cytokines were also produced in response to PSK as detected by ELISA, RIA or bioassays. We speculate that these cytokines may mediate immunoenhancing actions of PSK in vivo.

Tumor necrosis factor-alpha (TNF) is a pluripotent protein produced by cells of the monocyte-macrophage lineage. It has important pro-inflammatory functions and is thought to be involved in the pathogenesis of viral-bacterial lung infections of cattle. Binding and internalization of gold-labeled TNF (TNF-G10) by bovine alveolar macrophages as well as by peripheral blood mononuclear leukocytes (PBML) and polymorphonuclear granulocytes (PMN) have therefore been investigated in conjunction with studies of the effect of TNF on some leukocyte functions. TNF-G10 bound to all leukocyte types to varying extents, which however, did not correlate with the influence of TNF on cell functions. Thus, PMN appeared to possess the lowest number of TNF-receptors, but were nevertheless the most sensitive to functional modulation by TNF. The expression of TNF-receptors appeared to be regulated by other cytokines such as IFN-gamma and IL-2, with IFN-gamma down-regulating receptor expression on all cell types, and IL-2 up-regulating receptor expression on lymphocytes. In macrophages internalization of TNF-G10 occurred via clathrin-coated structures, whereas in lymphocytes and PMN the endosomes appeared to lack distinct coating.

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.

We analyzed the proliferative response of the growth factor-dependent murine cell lines FDCp1, DA1-a, 32DC1, Ea3.15, 7TD1, BCL1 and of femural bone marrow cells for their sensitivity to various cytokines, viz. rhIL-1 beta, rhTNF, rhIL-2, mIL-3, rmIL-4, rmIL-5, rhIL-6, rhG-CSF and rmGM-CSF. We also tested for IL-1 and TNF-mediated cytokine secretion by several T cell lines and thymocytes. In all T cell systems, IL-1 alpha and IL-1 beta were equally active in the induction of cytokine production, except for the rat/mouse T cell hybridoma PC60. This cell line exhibited a 10-fold difference in specific activity for the induction of cytokine secretion between rhIL-1 alpha and the other human or murine IL-1 species. Furthermore, IL-1 and IL-2 synergistically induced PC60 cells to produce a factor, which was preferentially active on FDCp1-cells, provisionally called FDCp1-growth factor. SDS-PAGE analysis of partially purified FDCp1-GF showed 19 kDa and 24 kDa-associated biological activities. Amino-terminal and internal amino acid sequences of both bands were determined and on this basis, we identified FDCp1-GF as rat GM-CSF.

Tumor necrosis factor (TNF) alpha and TNF beta both inhibited proliferation of cultured human osteoblastic SaOS-2 cells. TNF alpha also inhibited alkaline phosphatase (ALP) activity in the cells. The TNF alpha-induced inhibition of proliferation and ALP activity was further potentiated by interferon (IFN) gamma. These findings indicate that human SaOS-2 cells, fulfilling several criteria for osteoblasts, respond to TNF alpha and IFN gamma, resulting in decceleration of their maturation.