白细胞移动抑制因子(LMIF)释放的测定及条件性抑制LMIF的作用

目的选用与迟发过敏（ＤＴＨ）反应密切相关的白细胞移动抑制因子（ＬＭＩＦ）的体外测定方法，分析在条件性免疫抑制反应中ＬＭＩＦ活性的变化。方法ＬＭＩＦ测定是以间接毛细管方法结合分光光度计，测定游出细胞的吸光度（Ａ值）。在条件性反应训练和建立ＤＴＨ反应模型的第７天，取耳称重，同时取小鼠肠系膜淋巴结细胞，制备ＬＭＩＦ。结果ＬＭＩＦ在条件反应组的作用明显减弱（Ａ值升高），与不经过条件性训练的对照组相比，差异有非常显著意义（Ｐ＜０．０１，方差分析，ｎ＝７），但与非条件反应组相比没有差异，说明已形成了条件性抑制ＬＭＩＦ的反应。结论本工作建立了稳定的ＬＭＩＦ吸光度检测法，建立了环磷酰胺条件性抑制ＬＭＩＦ的模型。为深入分析条件性免疫抑制反应的血清中的介导物质提供了一个方便的体外检测手段     

Leukocyte migration inhibitory factor (LMIF) profile in primary and secondary immunodeficiency disease.

Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease.     

Cell-mediated immunity in ferrets delayed dermal hypersensitivity,lymphocyte transformation,and macrophage migration inhibitory factor production

Cell-mediated immune reactions — delayed dermal hypersensitivity, lymphocyte transformation, and macrophage migration inhibitory factor production — were investigated in the ferret. Ferrets immunized with streptokinase in complete Freund's adjuvant had skin test responses characterized by induration with little erythema; skin test biopsies showed mononuclear cell infiltration. In vitro transformation of peripheral blood or splenic lymphocytes was elicited by phytohemagglutinin, concanavalin A, pokeweed mitogen, and streptokinase; macrophage migration inhibitory factor was produced by spleen cells from immunized ferrets. These studies confirm the usefulness of certain tests of cell-mediated immunity in ferrets.     

Lack of correspondence between virus-induced human leukocyte interferon and concanavalin A-induced human migration inhibitory factor (MIF)

Summary Virus-induced human leukocyte interferon did not contain demonstrable MIF activity and MIF-rich fractions produced by concanavalin A stimulation of human lymphocytes lacked interferon activity.     

Macrophage migration inhibitory factor (MIF) enhances palmitic acid- and glucose-induced murine beta cell dysfunction and destruction in vitro

Although several reports suggest a potentially deleterious role of macrophage migration inhibitory factor (MIF) in type 2 diabetes (T2D) pathology, it is still unclear how this pro-inflammatory cytokine acts on pancreatic beta cells. The aim of the present study was to evaluate MIF effects on murine beta cells in the in vitro settings mimicking T2D-associated conditions. Results indicate that recombinant MIF further increased apoptosis of pancreatic islets or MIN6 cells upon exposure to palmitic acid or glucose. This was accompanied by upregulation of several pro-apoptotic molecules. Furthermore, MIF potentiated nutrient-induced islet cell dysfunction, as revealed by lower glucose oxidation rate, ATP content, and depolarized mitochondrial membrane. The final outcome was potentiation of mitochondrial apoptotic pathway. The observed upregulation of nutrient-induced islet cell dysfunction and apoptosis by MIF implicates that silencing MIF may be beneficial for maintaining integrity of endocrine pancreas in obesity-associated T2D.     

Beta-lactam antibiotics and human lymphocyte function: The in vitro effect on blastogenesis,lymphokine production and suppressor cell functions

The effects of penicillin, cephalothin and cefoxitin on lymphokine production and mitogen stimulation of human peripheral blood lymphocytes and suppressor cell functions in man were studied with concentrations achievable in serum. Penicillin (50 and 100 μg/ml) and cephalosporins (50 and 100 μg/ml) directly stimulated production of the lymphokine leukocyte aggregating factor (LAgF) by unstimulated lymphocytes and enhanced mitogen-stimulated production of this lymphokine. Neither β-lactam derivative interfered directly with neutrophil aggregation. Using the same concentration, penicillin did not suppress but had either no effect on or slightly increased the unstimulated and mitogen-stimulated lymphocyte transformation responses, whereas cephalosporins significantly suppressed both responses. Suppression of blastogenesis by the latter could be mediated through suppressor cell enhancement as indicated by the significant suppressed lymphocyte transformation observed while using the supernatant of cephalosporin-stimulated and cultured lymphocytes. This suppressive activity was more pronounced with cephalothin than with cefoxitin while it was not observed with penicillin which rather stimulated thymidine uptake. These findings suggest that enhancement of lymphokine production and suppression of lymphocyte transformation by cephalosporins are not contradictory and reflect a stimulating effect on two different lymphocyte subsets. The enhancement of suppressor function might account for the lower incidence of late hypersensitivity reactions observed in patients treated with the cephalosporins compared with those treated with penicillins.     

In vitro effect of interferon alpha-2b on T lymphocyte transformation and leukocyte migration inhibition in patients with chronic brucellosis

The in vitro effect of IFN-a on lymphocyte transformation and specific immune response against Brucella antigens was studied in 33 patients with chronic brucellosis and 10 normal controls. The following immunologic in vitro tests were applied: PHA activated lymphocyte transformation test using Bromodeoxyuridine and a monoclonal antibody in the presence and absence of 50 and 100 IU IFN Alpha-2b and leukocyte migration inhibition test against Brucella antigens in the presence and absence of 100 and 500 IU of IFN Alpha-2b. Patients were further divided to 2 subgroups according to a positive or negative migration inhibition test. Our results showed that T lymphocyte transformation was similar in patients and controls and that the addition of 50 IU IFN resulted in a significant increase of transforming cells whereas in the concentration of 100 IU IFN only anergic patients and controls responded positively. IFN also resulted in a significant leukocyte migration inhibition only in anergic patients and controls. These findings suggest that the chronic infection is not due to a generalized cellular immunodeficiency state and that IFN Alpha-2b might be a promising therapeutic approach in anergic patients.     

Correlation of PPD and BCG-induced leukocyte migration inhibition,delayed cutaneous hypersensitivity,lymphocyte transformation in vitro and humoral antibodies to PPD in man

In a study of healthy human individuals a complete lack of correlation between the results of the agarose leukocyte migration inhibition (LMI) test, using purified protein derivative (of tuberculin) (PPD) and Bacillus Calmette Guérin (BCG) as antigens, and delayed cutaneous hypersensitivity and lymphocyte transformation in vitro to PPD was found. There was a reasonable correlation between PPD- and BCG-induced LMI. Antibodies to PPD proved to have no influence on PPD-induced LMI. Purified polymorphonuclear leukocytes, whether derived from donors sensitive to PPD in the agarose LMI test or from nonsensitive donors, did not show migration inhibition to PPD. It was concluded that polymorphonuclear leukocytes and lymphocytes need to be present simultaneously for migration inhibition of peripheral blood leukocytes by PPD. Furthermore, because a consistent relation with conventional parameters of cell-mediated immunity was lacking, it is doubtful whether the agarose LM1 test can be considered as an alternative parameter of this kind of immunity.     

Transforming growth factor beta (TGF-beta) potentiates the inhibitory effect of retinoic acid on human breast carcinoma (MCF-7) cell proliferation

Anchorage-dependent and -independent MCF-7 cell growth was dose-dependently inhibited by retinoic acid (RA) but was insensitive to TGF-beta (from 1 to 100 pM). Growth of MCF-7 monolayer cultures was inhibited (50%) when exposed to 10(-6) M RA. RA was unable to completely inhibit MCF-7 cell proliferation, as concentrations above 10(-6) M were rapidly cytotoxic. However, the combination of TGF-beta and RA resulted an increase in RA inhibitory effect on MCF-7 monolayer growth and a 80% reduction in colony formation in soft agar. These results demonstrate that although TGF-beta does not inhibit the growth of MCF-7 cells, it potentiates the antiproliferative effect of RA, suggesting that it may play a part, albeit indirect, in the regulation of MCF-7 cell growth.     

The effects of cyclic AMP on leucocyte inhibitory factor (LIF) production and on the inhibition of leucocyte migration.

The effect of drugs known to increase intracellular levels of cyllic AMP were studied in the leucocyte migration ihibition system. It was found that cyclic AMP, dibutyryl cyclic AMP, theophyline, and prostaglandins E1 and E2 inhibited the production of leucocyte inhibiting factor by HA pulsed lymphocytes Inhibition only occured when the drugs were present during or after the PHA pulse. In addition it was found that these drugs enhanced the migration of polymorphonuclear leucocytes (PMN), in this system. Electrophoretic mobility of PMN cells was not altered by these drug indicating that the effect is not due to changes in membrane charge. However, granulocyte adhesion was reduced in the presence of these drug suggesting that adhesion is of primary importance in the migration of polymorphonuclear leucocytes out of capillary tubes. The findings show that cyclic AMP is important in modulating both cell-mediated and inflammatory responses.     

Phytohaemagglutinin (PHA) induced lymphocyte transformation and toxoplasma gondii antibody studies in primary biliary cirrhosis. Evidence of impaired cell-mediated immunity

Phytohaemagglutinin (PHA) induced dose-response lymphocyte transformation has been studied in a group of patients with primary biliary cirrhosis. There is a marked reduction in responsiveness, particularly with low doses of PHA. These observations, together with the finding of a raised incidence (7%) of high titre Toxoplasma gondii antibody in primary biliary cirrhosis patients, are adduced as evidence of impaired cell-mediated immunity in this disease.     

B8, DR3 antigens and production of human leucocyte migration inhibitory factor (LIF) by mononuclear cells stimulated with concanavalin A (Con A)

LIF release by Con A stimulated mononuclear cells was evaluated in 67 randomly selected healthy Sicilians typed for HLA antigens. The results show that B8 and/or DR3 positive subjects release less LIF than negative ones, suggesting that this immunological response might be controlled by HLA-linked immune response (Ir) gene(s).     

Further purification of neutrophil migration inhibition factor from T lymphocytes (NIF-T): evidence that NIF-T and leukocyte inhibitory factor (LIF) are immunologically distinct

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) purified by antibody affinity chromatography and gel filtration chromatography was radioiodinated and identified as a 26,000-MW protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). NIF-T was identified by elution of biological activity from gel fractions and selective adsorption of a radioiodinated mediator by HL60 cells differentiated in the presence of dimethyl sulfoxide (DMSO) to develop receptors for NIF-T. A goat neutralizing antibody for NIF-T neutralized and immunoprecipitated migration inhibition activity in the conditioned medium from Mo T-lymphoblast cells and human peripheral blood lymphocytes (PBL) cultured with concanavalin A (Con A), but not from RPMI 1788 B-lymphoblast cells with leukocyte migration inhibitory factor (LIF) activity. These studies distinguish NIF-T both chemically and immunologically from LIF.     

Effects of dialyzable leukocyte extracts (DLEs) with transfer factor activity on leukocyte migration in vitro. III. Characterization of the antigen-independent migration inhibition factor in DLEs as a neutrophil immobilizing factor.

In earlier evaluations of the agarose LMI assay as an in vitro test for studying the nature and mechanism of action of TF, we reported the existence of a component in human DLEs which caused noncytotoxic inhibition of the random migration of human PMNs. The LMI was not dependent on the stimulation of viable mononuclear leukocytes by antigen or mitogen to effect the release of mediators of cellular immunity such as LIF; rather, the LMI was promoted by the direct action of a preexisting component in DLEs on PMNs. We now present evidence that this "antigen-independent" LMI activity in DLE'S is similar to a NIF shown previously by Goetzl and co-workers to be present in acid extracts of leukocytes and to be released by phagocytosing PMNs. The comparison is drawn from several parameters: (1) cellular origin, (2) molecular weight, (3) target cell, (4) susceptibility to inactivation by heating or by incubation with pronase, trypsin, or chymotrypsin, and (5) ability to cause noncytotoxic inhibition of random migration or chemotaxis of PMNs.     

Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate induction of migration and invasion of glioma cells in vitro and in vivo: Differential inhibitory effects of flavonoids

In an earlier study, we reported that nitric oxide is involved in lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate-induced malignant transformation via increases in metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression in rat glioma C6 cells, however the mechanism has remained undefined. Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate alone, induced transformation in glioma C6 cells (but not in human glioblastoma cells GBM-8401 cells) without affecting their viability. An increase in inducible nitric oxide synthase protein expression, nitric oxide production, and metalloproteinase 9 enzyme activity is identified lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-treated C6 cells, however lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate and 12-o-tetradecanoylphorbol 13-acetate (but not lipopolysaccharide) addition shows the similar inductive pattern on metalloproteinase 9 enzyme activity without affecting inducible nitric oxide synthase protein expression and nitric oxide production in GBM-8401 cells. Treatment of C6 cells with lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate increases the expression of phosphorylated extracellular regulated protein kinases and Jun N-terminal kinases, but not p38, proteins, and an addition of the extracellular regulated protein kinases inhibitor PD98059 or Jun N-terminal kinases inhibitors SP600125, but not the p38 inhibitor SB203580, significantly blocked lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced inducible nitric oxide synthase protein expression and metalloproteinase 9 enzyme activity accompanied by blocking morphological transformation in C6 cells. Among 19 structurally related flavonoids, kaempferol and wogonin exhibit significant inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, and attenuation of inducible nitric oxide synthase, phosphorylated extracellular regulated protein kinases protein expression, and metalloproteinase 9 enzyme activity was observed. 2'-OH flavone at a dose of 100 microM inhibition of lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events via apoptosis induction is identified. Furthermore, lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate, induces tumoral invasion and migration in vitro and in vivo, and those are blocked by kaempferol and wogonin addition. These data suggest that combination of lipopolysaccharide and 12-o-tetradecanoylphorbol 13-acetate promotes tumoral progression via activating metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression, which is located downstream of mitogen-activated protein kinases activation, in rat glioma cells C6. Kaempferol and wogonin exhibit effective inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events, and thus possess the potential for further development.     

Differential effects of glial cell line-derived neurotrophic factor on A9 and A10 dopamine neuron survival in vitro

Glial cell-line derived neurotrophic factor (GDNF) enhances dopamine (DA) cell survival and fiber outgrowth, and may be beneficial in enhancing cell restorative strategies for Parkinson's disease (PD). However, GDNF may have different roles for transplanted DA cell sub-types. The present in vitro study investigated the effect of GDNF on the survival of rat DA cells displaying a phenotype consistent with either the substantia nigra [A9 cells immunopositive for tyrosine hydroxylase (TH) and G-protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2)] or with the ventral tegmental area [A10 cells immunopositive for TH and calbindin]. It was found that a single exposure of GDNF enhanced the number of DA cells of an A9 phenotype, without affecting DA cells of an A10 phenotype. Conversely, repeated GDNF exposure did not alter the survival of A9 phenotypic cells, but doubled the percentage of A10 cells. It was concluded that GDNF administration may affect dopaminergic cells differently depending on time and degree of GDNF exposure. For cell transplantation in PD, long-term GDNF administration may result in detrimental effects for transplanted A9 TH+ cells as this may introduce competition with A10 TH+ cells for survival and fiber outgrowth into the host striatum. These results may have important implications for clinical neural transplantation in PD.     

Differential effects of agarose and poly(lactic-co-glycolic acid) on dendritic cell maturation

Application of biomaterials in combination products in which the biomaterial is presented to the host with a biological component prompts the need for understanding the biomaterial-associated adjuvant effect in the immune response against antigens associated with such a product. We have previously demonstrated that a polymer commonly used in tissue engineering and vaccine delivery, poly(lactic-co-glycolic acid) (PLGA), exerts an adjuvant effect in vivo, which was supported by PLGA-induced dendritic cell (DC) maturation in vitro. In this study, the effects of agarose and PLGA on DC maturation were compared in vitro to establish differential biomaterial effects. Human monocyte-derived DCs were treated with agarose or PLGA microparticles or films, and their maturation effect was measured as expression of costimulatory and MHC class II molecules, allostimulatory capacity, and proinflammatory cytokine secretion. Direct comparison of DC maturation phenotype indicated that PLGA was a stronger stimulus of DC maturation than agarose, and this maturation was not affected by microparticle phagocytosis. However, agarose-treated DCs showed higher activation of nuclear factor kappaB (NFkappaB) 24 h after the initial stimulation of DCs. Taken together, these results demonstrate differential biomaterial effects on DC maturation, substantiating the maturation effect of PLGA, and provide screening methods for biomaterial adjuvant effect for applications in combination products.     

维甲酸调变的巨噬细胞在体外对A549癌细胞的作用

The tumoricidal activity of mouse macrophage (M phi) activated with retinoic acid (RA) and the effect of RA in combination with CP on mouse M phi were studied with phase contrast microscopy, light microscopy, and 3HTdR labelling technique. The results of our studies suggest that: (1) RA can activate mouse peritoneal M phi both in vivo and in vitro, and the RA-activated M phi are cytostatic and cytolytic to A549 cells. The degree of activation is related to the concentration or dosage of RA given, and (2) RA can enhance the function of CP-activated M phi. A summation effect was obtained by giving RA in combination with CP. This effect was related to the dosage of RA. When optimal dosage was given in combination with CP, a synergistic action in activating M phi could be obtained. Morphological changes could be seen under phase contrast microscope and light microscope in killed tumor cells.     

Macrophage migration inhibitory factor (MIF): its essential role in the immune system and cell growth.

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in inflammatory and immune responses. MIF was originally discovered as a lymphokine involved in delayed hypersensitivity and various macrophage functions, including phagocytosis, spreading, and tumoricidal activity. Recently, MIF was reevaluated as a proinflammatory cytokine and pituitary-derived hormone potentiating endotoxemia. This protein is ubiquitously expressed in various organs, such as the brain and kidney. Among cytokines, MIF is unique in terms of its abundant expression and storage within the cytoplasm and, further, for its counteraction against glucocorticoids. MIF has unexpectedly been found to convert D-dopachrome, an enantiomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole. However, its physiologic significance remains to be elucidated. It was demonstrated that anti-MIF antibodies effectively suppress tumor growth and tumor-associated angiogenesis, suggesting that MIF is involved not only in inflammatory and immune responses but also in tumor cell growth. At present, MIF cannot be clearly categorized as either a cytokine, hormone, or enzyme. This review presents the latest findings on the role of MIF in the immune system and in cell growth, with regard to tumorigenesis and wound repair, and discusses its potential functions in various pathophysiologic states.