Lymphokine research最新文献

Full length cDNAs encoding the human and murine p70 genes were isolated using polymerase chain reaction (PCR) and conventional cDNA library screening techniques, respectively. To validate their functional potential, expression vectors containing human, murine and chimeric human/murine p70 cDNAs were transfected into the murine IL-3-dependent cell line FDC-P1. Transfected cells expressed a combination of high and low-affinity IL-2 binding sites while parental cells displayed only the low-affinity sites associated with expression of endogenous p55 IL-2 receptor chains. The role of the transfected p70 chains in formation of the high-affinity receptor sites was confirmed by the finding that the species-specific inhibitory antibody TU27 blocked high-affinity binding to human p70 and chimeric human/murine p70-expressing cells while having no effect on cells transfected with the murine p70 cDNA construct. As consequences of the expression of the transfected p70 chains, the cells responded to IL-2 with increased levels of endogenous p55 receptor subunit and both short and long-term proliferation. These studies substantiate the role of the p70 receptor chain in functional responses to IL-2 and provide a model system for future dissection of the structure/function relationships of the IL-2 receptor.

The production of interleukin 1 beta (IL1 beta) in lipopolysaccharide (LPS) stimulated monocytes was inhibited by 98% using antisense phosphorothioate oligonucleotides complementary to the 5' untranslated and exon 6 regions of IL1 beta mRNA. A sense phosphorothioate oligonucleotide failed to inhibit IL1 beta production. The inhibition of IL1 beta synthesis was not due to reduced cell viability and [35S]methionine incorporation showed that it could not be accounted for by an overall inhibition in protein synthesis. Tumour necrosis factor (TNF) and IL1 alpha production was also inhibited but to a lesser extent than IL1 beta. The use of antisense phosphorothioate oligonucleotides to inhibit IL1 production should enable the complex pathways of IL1 regulation to be elucidated and provide information on the biological role of these cytokines.

The development and characterization of a lyophilized dosage form for recombinant Interleukin-1 beta is described. Included in the evaluation of the drug product are accelerated and long-term stability studies utilizing a number of biophysical techniques (reverse-phase HPLC, SDS-PAGE, isoelectric focusing and ELISA). Data collected with these methods were examined for correlations with biological activity assessments provided by an in-vitro cell culture system (mouse thymocyte proliferation). Results of these studies demonstrate that a lyophilized dosage form of Interleukin-1 beta can be prepared which retains its potency for at least 1 year when stored at ambient temperature. The analytical methodology used to assess the physicochemical integrity of the protein provided a sensitive and reproducible means of predicting changes in biological activity.

Tumor Necrosis Factor (TNF) has been implicated in the early metabolic events following acute tissue injury or sepsis; it increases blood levels of glucocorticoids and glucagon or the cellular responses to the hormones. To examine whether stress-related hormones have any effect on macrophage activation by TNF, human monocyte-derived macrophages were exposed to somatostatin (S), ACTH, angiotensin (An), insulin (I), epinephrine (E), and glucagon (G) at physiologic concentrations. 125I-TNF binding as well as the ability of TNF to activate macrophages to kill an intracellular pathogen (Mycobacterium avium) were measured. While treatment with recombinant interferon gamma increased the number of TNF receptors by 53 +/- 8%, E, I, G, S, ACTH and An decreased the number of receptors by 81 +/- 6%, 83 +/- 6%, 15 +/- 5%, 83 +/- 4%, 17 +/- 4% and 21 +/- 4%, respectively. Treatment with I, E, and S also decreased the ability of macrophages to kill M. avium by 30 +/- 1%, 20 +/- 6%, and 51 +/- 2%, respectively. These in vitro results suggest that stress hormones influence TNF-mediated activation of macrophages.

Bacterial lipopolysaccharide (LPS)-induced production of three known endogenous pyrogens, interferon alpha (IFN-alpha), interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) was studied during in vitro differentiation of human peripheral blood monocytes. In freshly seeded cells, secretion of IL-1 and TNF-alpha, but not IFN-alpha were readily induced by LPS. After 24 hr and for up to 14 days of culture, monocytes became irreversibly tolerant to LPS for the release of IL-1. IFN-alpha secretion was not induced by LPS in cells cultured for up to 8 days. Monocytes also became tolerant to LPS for TNF-alpha production 48 hr after an initial stimulation with LPS. This tolerant state, however, was transient, lasting from 6 to 8 days, after which competence for TNF secretion resumed. These observations demonstrate that regulation of production of IL-1, TNF-alpha and IFN-alpha by human mononuclear phagocytes is mutually independent, related to the stage of cell differentiation and modulated by cell stimulation. Since in vitro tolerance to LPS mimics the in vivo tolerance to LPS with respect to fever, we speculate that they are closely related.

Lymphocytes from mouse and men can be stimulated by a variety of cellular signals including concanavalin A and the interferons to generate cells capable of non-specific down regulation of the generation of immune responses. The studies reported here extend this finding to show that such regulatory cells are also generated during the course of a mixed lymphocyte response and following stimulation of the CD3 component of the T cell receptor with anti-CD3 antibody. Regulatory activity was assessed by the addition of putative regulatory cells to mixed lymphocyte cultures and measuring the generation of cytolytic T cell activity in these cultures. The action of such non-specific regulatory cells was blocked by antiserum to the N-terminal sequence of soluble immune response suppressor (SIRS). In addition, generation of the regulatory cell population was inhibited by antiserum to the 55 kd subunit of the IL-2 receptor. A survey of cytokines showed that while IL-2 was able to stimulate the generation of non-specific regulatory cell activity, IL-1,3,6,7, and 10 as well as TNF alpha and TGF beta were unable to generate such activity. IL-2 was unable to generate non-specific suppressive activity in the presence of anti-IL-2 receptor antibody. The regulatory activity of cells generated by IL-2 was inhibited by the anti-SIRS antiserum.

Recent evidence suggests that a Ca++, phospholipid, diacylglycerol-dependent protein kinase, protein kinase C, plays a role in the activation of cytotoxic T lymphocytes by target cells. In this investigation we have examined the role of protein kinase C in human NK cell-mediated cytolysis of K-562 cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited human NK cell-mediated cytolysis in a dose dependent manner. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a specific inhibitor of cyclic nucleotide dependent protein kinases had no effect on human NK cell-mediated cytolysis of K562 cells. There is little or no effect on protein synthesis or N-glycosylation activity in human NK cells by H-7. The relative inhibitory ability of the two inhibitors suggest that protein kinase C, acting synergistically with Ca++ mobilization, plays a role in the early stages of human NK cell-mediated cytolysis of K562 target cells.

Pertussis toxin, and also cholera toxin are capable of inhibiting the effects of LPS in the elicitation of the generalized Schwartzman reaction. This is a potentially lethal generalized thrombo-haemorrhagic hypersensitivity and inflammatory-type response that occurs after two consecutive injections of LPS. The two exotoxins furnish significant protection against the lethal outcome of this reaction. It is known that the acute haematological and haemodynamic changes are accompanied by alterations in the levels of various endogenous mediators: glucocorticoid hormones, prostaglandins, arachidonic acid metabolites, cytokines and proteases. In vitro effects of LPS on murine leukocyte cell lines can be antagonized by pertussis toxin, implicating a Gi-like regulatory protein in the mediation of these effects. Experiments designed to study the involvement of particular second messenger systems (cAMP and phosphatidylinositol) used by LPS in vivo, revealed that the protective effects conferred by these exotoxins are associated with the antagonization of alterations caused by LPS. No correlation was found between the levels of IL-6 and the mortality rate in this experimental mouse model. The results indicate that G proteins play a role in the generation of the Schwartzman reaction and open a new approach for pharmacological intervention in endotoxemia and in clinical settings with Gram-negative sepsis.

The possible role of Interleukin 6 (IL-6) and tumour necrosis factor (TNF) in the regulation of the acute phase response to injury was studied in thirteen subjects undergoing elective surgery or suffering from multiple accidental injuries. The temporal changes in the circulating concentrations of these cytokines were related to the circulating acute phase protein concentrations (C-reactive protein and alpha 1 antichymotrypsin), white cell count, blood loss and duration of surgery. Circulating acute phase protein concentrations rose in all subjects during a thirty hour period following injury but none of the subjects showed a detectable rise in circulating concentrations of TNF. Peak circulating concentrations of IL-6 were detected between 4-6 hours after surgery/trauma but these showed a poor correlation with blood loss, fever, white cell count or duration of surgery. The peak concentrations of IL-6 typically occurred before the rise in circulating acute phase protein concentration. The data do not suggest a role for circulating TNF in the regulation of the acute phase response to injury. In contrast, IL-6 appears to be variably involved in the acute phase response but its precise role in mediating fever, leucocytosis and synthesis of acute phase proteins is as yet uncertain.

Recombinant bovine interleukin-1 beta (rBoIL-1 beta) was administered to calves in conjunction with a bovine herpesvirus-1 (BHV-1) vaccine. All calves were immunized against BHV-1 and three groups received rBoIL-1 beta at 33, 100, or 330 ng/kg on days 1 and 15; control animals received physiological saline. All calves were challenged with BHV-1 on day 22. Total leukocytes were increased by rBoIL-1 beta, primarily by causing neutrophilia and monocytosis; CD4/CD8 ratios tended to be increased in rBoIL-1 beta-treated animals. Serum neutralizing antibody titers and cytotoxic responses against BHV-1-infected bovine kidney fibroblasts were increased and virus excretion was decreased in rBoIL-1 beta-treated calves. On days 58 and 59, control and 100 ng/kg rBoIL-1 beta-treated calves were injected with dexamethasone (.04 mg/kg). Virus excretion was less and clinical signs of BHV-1 infection were lower in rBoIL-1 beta-treated calves after dexamethasone injection. These data suggest that rBoIL-1 beta may be an effective adjuvant to BHV-1 immunization.