Lymphokine research最新文献

An immunoenzymatic detection method of in-situ hybridization reactions for interleukin 1 (IL-1) beta was established, using sulphonated probes. As model system we used unstimulated and lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMNC). After hybridization, sulphone groups were targeted with a monoclonal antibody, and bound antibody was visualized by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. In unstimulated PBMNC both the control and the IL-1 beta specific c-DNA probes were negative, whilst a large proportion of LPS treated PBMNC was positive with the sulphonated IL-1 beta plasmid only. This method may be a powerful alternative to radio-isotopic labeling. Since the entire procedure can be performed within one day, it may be applied for routine diagnostic purposes.

Tuberculosis is characterised by necrosis in the lesions and in skin-test sites, and by fever and weight loss. In contrast, other diseases with chronic T cell mediated responses, such as uncomplicated leprosy and sarcoidosis, have non-necrotising lesions with little systemic upset. Crude sonicates of M. tuberculosis and M. leprae prepare skin sites for TNF-mediated damage via a pathway which unexpectedly appears to involve CD8+ T cells, and both mycobacteria contain potent triggers of TNF release (lipoarabinomannan and peptidoglycan derivatives). These observations can partially explain the pathology of tuberculosis, but fail to explain why similar events do not normally occur in leprosy. It now seems likely that the answer lies in the existence of novel regulatory pathways. A recently recognised correlate (or consequence) of diseases characterised by T cell-dependent tissue-damaging pathology and cytokine release, is an increase in the level of agalactosyl IgG. This behaves like a T cell-dependent acute phase reactant, and is raised in tuberculosis, rheumatoid arthritis, and Crohn's disease, but not in sarcoidosis or uncomplicated leprosy. Thus it may act as a marker for a type of pathology of very broad significance, though its functional role remains obscure.

Since granulocyte-macrophage colony-stimulating factor (GM-CSF) has previously been shown to activate macrophages, it was of particular interest to study its effect on synthesis and release of tumor necrosis factor-alpha (TNF-alpha). GM-CSF alone was incapable of activating murine peritoneal macrophages to TNF-alpha release. However, in response to lipopolysaccharide (LPS), GM-CSF was found to prime macrophages for enhanced TNF-alpha production. This priming effect was short-lived and was superseded by the contrary, an unresponsiveness to LPS. The suppressed response was due to a delayed production of prostaglandin E2 (PGE2) which did not affect GM-CSF-enhanced TNF-alpha gene transcription but blocked TNF-alpha production. When PGE2 synthesis was inhibited by indomethacin, the priming effect of GM-CSF was entirely reconstituted. Thus, GM-CSF initially primes for TNF-alpha and subsequently for PGE2 release which, taken together, may represent an autoregulatory feed-back system that could restrict macrophage activation.

TNF alpha may play a critical role in many physiologic and pathophysiologic responses. It shows a bifunctional regulatory nature of both inhibiting tumor cell proliferation and enhancing growth and differentiation of other cells. TNF alpha acts as a mediator, binding to specific high affinity receptors to elicit biological responses. TNF alpha may be an essential local and systemic mediator in the pathogenesis of cachexia. By inhibiting the activity of lipogenic enzymes, it affects lipid mobilization from adipose tissue and adipocyte differentiation. The ability of TNF alpha to inhibit anabolic processes in adipocytes and preadipocytes is not unique and has been displayed, though to a lesser degree, by other cytokines such as LT, IFN-gamma, and IL-1. In infected animals, the effect of TNF alpha on LPL of inhibiting anabolic processes and enhancing secretion of FFA by adipocytes, may be important in providing additional energy needed by the immune system to combat invasion. However, the relationship between the suppression of the anabolic processes and the cytotoxicity is uncertain and prolonged action may lead to wasting. The evidence suggests that in the adipocyte, TNF alpha is the unique molecule that suppresses LPL through the selective inhibition of mRNA production. As a consequence of the decrease in the enzyme amounts, there is a reduction in TG-uptake and lipid deposition contributing to cachexia. Neutralizing the action of TNF alpha may reverse the cachectic processes and potentially improve the condition. Further research into the induction, synthesis and regulation of TNF alpha production is necessary. Modern molecular biology techniques should serve as useful models in understanding the regulation of TNF alpha functions in response to stimuli. Development of faster and more sensitive detection methods would improve the measurement of the low concentrations of cytokines.

Human recombinant IL-1 alpha stimulates in a dose-dependent fashion the production of 5-lipoxygenase-derived LTC4 and, to a lesser extent, of cyclooxygenase-derived eicosanoids (PGE2, 6-keto PGF1 alpha, TXB2) by murine resident peritoneal macrophages. Enhancement of eicosanoid production was evident on unstimulated macrophages, but only marginal on macrophages phagocytosing zymosan. The effect of IL-1 was similarly achieved at physiological (37 degrees C) as well as at higher (39 degrees C) temperatures. Human recombinant IL-1 beta, human natural IL-1, and partially purified murine IL-1-rich supernatant also stimulated eicosanoid production by macrophages, although IL-1 alpha appeared to be the most effective.

Specific immunoassays were used to measure IL-1 peptides in the serum and synovial fluid of patients with rheumatoid arthritis (RA) and in the serum of age-matched healthy controls. Patients with RA had raised levels of both IL-1 beta and IL-1 alpha in their sera compared to controls. Synovial fluid levels of IL-1 beta significantly correlated with immunoreactive IL-2 and soluble IL-2 receptor (sIL-2R). In addition, incubation of synovial fluid MNC with human recombinant (hr) IL-1 caused a dose-dependent increase in the level of sIL-2R in the cell supernatant. Finally, production of IL-1 beta and IL-6 from RA peripheral blood (PB) and synovial fluid (SF) MNC was examined. PBMNC spontaneously produced low levels of IL-1 beta and IL-6 that were augmented by the addition of hr IL-1 alpha. In contrast, SFMNC spontaneously produced high levels of IL-1 beta but only low levels of IL-6, again this production was augmented by the addition of hr IL-1 alpha. Taken together, the data suggests that IL-1 potentiates immune responses within the joint.

IL2 and IL3 are polypeptide growth factors that support the survival and proliferation of, respectively, activated T lymphocytes and a range of myeloid cell types. We have examined the involvement of tyrosine phosphorylation in IL2 and IL3 mediated signal transduction. Phosphotyrosyl proteins were immunoaffinity purified and analyzed by single and two dimensional gel electrophoresis. The majority of phosphotyrosyl proteins purified from human T lymphocytes and murine myeloid cells had identical 2 D electrophoretic mobilities, suggesting a high degree of evolutionary conservation. Several proteins in both cell types increased in tyrosine phosphorylation after factor stimulation, including pp200, pp180, pp92, and pp42. The 92 kD protein was the most highly modulated phosphoprotein identified, with increases in phosphorylation greater than 18 fold after 20 min of stimulation. These results suggest that signal transduction pathways for IL2 and IL3 involve tyrosine phosphorylation of protein substrates common to both lymphoid and myeloid linages.

We have observed a marked species specificity in the in vivo antitumor, shock-inducing and Interleukin-6 (IL-6)-inducing activities of human recombinant TNF (hrTNF) versus murine rTNF (mrTNF) in mouse models. We also have observed that this species specificity can be abolished when synergistic factors are co-administered. We now present data showing that the profiles of induced IL-6 and TNF after lethal and non-lethal doses of endotoxin are different. These results are discussed in the context of a model of dual signalling for the induction of the major post-endotoxin phenomena that takes into consideration the involvement of Interleukin-1 (IL-1) in the in vivo actions of TNF.

The purpose of these studies was to determine whether human peripheral blood polymorphonuclear cells (PMNC) can be directly activated by lymphokines or bacterial products to lyse tumorigenic cells under in vitro conditions. Preparations of PMNC with different levels of purity were isolated by countercurrent elutriation. PMNC were incubated with recombinant interferon gamma and muramyldipeptide, with lipopolysaccharide, or with the synthetic lipopeptide CGP 31362. Only PMNC preparations containing 3-5% mononuclear cells lysed allogeneic melanoma cells. Homogeneous populations of PMNC did not. The addition of 5% monocytes to homogeneous populations of PMNC triggered PMNC-mediated tumor lysis. Cell-to-cell contact was not required in this process since culture supernatant of blood monocytes incubated with lipopolysaccharide (but not with medium) could trigger tumor cell lysis by PMNC. PMNC lysed both allogeneic tumorigenic (melanoma) and allogeneic nontumorigenic (fibroblast) cells, whereas activated monocytes lysed only tumorigenic cells. In conclusion, PMNC are triggered to lyse target cells by blood monocytes activated by bacterial products or by lymphokines.