Lymphokine research最新文献

Liposomes containing membrane-IL-1, Iak, and the antigen conalbumin were evaluated as "synthetic macrophages." The role of these three molecules in macrophage-T cell interaction was studied by testing the ability of such synthetic macrophages to induce the proliferation of a T cell clone specific to conalbumin (the D10 cell line) or immune spleen cells sensitized three times in vivo with conalbumin. In the latter case, splenic macrophages were eliminated by adherence and a lysomotropic agent. The antigen conalbumin was presented on the surface of the liposomes in two forms: as native undigested protein or as a complex of Iak and naturally processed peptides isolated from peritoneal macrophages incubated with conalbumin. When the liposomes presented the Ia antigen associated with conalbumin-peptides and membrane-IL-1, the proliferation of both the D10 and the immune spleen cells was high and maximal. The proliferation response of both cell types was completely dependent on the presence of membrane-IL-1, since no proliferation occurred when liposomes were prepared with only conalbumin-peptides bound to Iak. Therefore, it could be concluded that processed antigen associated with class II MHC antigen and membrane-IL-1 were required for T cell activation. When the liposomes presented native conalbumin, Iak, and membrane-IL-1, significant proliferation occurred, but if the liposomes lacked membrane-IL-1, the proliferation of the T cell clone and the spleen cells decreased to only 40-50% of the previous signal. In this case native conalbumin and class II antigen alone were required for T cell activation, while membrane-IL-1 alone amplified the response. When the liposomes were made with only Iak and membrane-IL-1, lacking either form of conalbumin, there was no proliferation of antigen-specific target cells. These results indicated that in this synthetic system, membrane-IL-1 is essential for the proliferative response of antigen-specific T cells when conalbumin is presented as processed peptides in association with Iak.

The pharmacokinetic parameters and distribution of the adjuvant synthetic nonapeptide VQGEESNDK, corresponding to the fragment in position 163-171 in human IL-1, were analyzed after administration to rabbit through different routes. The radiolabeled peptide did not bind to plasma proteins and, when inoculated i.v., it disappeared very rapidly from the circulation, with a t1/2 alpha of 1 min and a t 1/2 beta of 166 min. Upon administration through i.m., s.c. and oral route, the Cmax was reached between 30 and 90 min after inoculum and ranged between 7 and 4% of the administered dose. Organ distribution showed that most of the radioactivity was concentrated in kidneys and excreted in urine. From Sephadex G-10 chromatography, about 60% of the peptide recovered in the urine 4h after i.v. inoculum was intact, whereas it was more than 85% degraded when administered by other routes. The amount of intact peptide recovered in the urine correlated with the biological effectiveness through different routes, suggesting that the adjuvant effect in vivo is exerted by the intact peptide, rather than by its metabolites.

Alpha-mannosidase was tested for its ability to inhibit lytic activity of nonadherent, mononuclear peripheral blood leukocytes (PBL) against K-562 target cells. Pretreatment of effector cells (60 min., 37 degrees C, pH 7.3) with this enzyme, prior to and after exhaustive dialysis, was examined. Nondialyzed enzyme preparations completely inhibited NK lytic function at all concentrations tested (1.0, 0.5, and 0.25 units/ml). On the other hand, dialyzed enzyme preparations had no inhibitory effect on NK lytic function over the same range of concentrations. The inhibitory effects of the nondialyzed enzyme were due to the presence of (NH4)2SO4, which could be removed by dialysis. Studies were also performed to determine whether enzyme treatment of effector cells resulted in hexose release from cell surface structures. Treatment of effector cells with alpha-mannosidase (dialyzed preparation) resulted in a dose dependent release of mannose. These data demonstrate that NK cell lytic function is not inhibited by pretreatment of effector cells with alpha-mannosidase even though mannose is quantitatively released from cell surface oligosaccharide structures. These results suggest that NK lytic function does not involve an effector cell surface structure bearing terminal alpha-linked mannose residues as previously reported.

Infection with the human immunodeficiency virus (HIV) is characterized by a long and variable asymptomatic phase followed by the development of the acquired immunodeficiency syndrome (AIDS) in a substantial proportion of individuals within 10 years. An understanding of the mechanism by which the infection progresses in vivo to an ultimate destruction of the immune system is essential to the design of rational approaches to anti-viral therapy, immune reconstitution, and vaccine development. To delineate these complex processes, we have established an in vitro model of latent or chronic HIV infection and have used this model to examine ways in which HIV replication can be upregulated. We have shown that cytokines are important mediators of HIV replication in chronically HIV-infected cells. Thus, the virus has incorporated itself into the human immune system and utilizes for its own propagation many of the cellular and molecular mechanisms which the immune system uses to maintain its homeostatic regulation. Studies in this area will not only shed important light on the immunopathogenesis of HIV, but will certainly lead to greater depth in our understanding of the normal function of the human immune system.

The synthetic precursor Ia of lipid A (compound 406), lacking dodecanoic and tetradecanoic fatty acids, is inactive in inducing IL-1, but is able to modulate the production and the release of LPS-induced IL-1.

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-associated tumor necrosis factor (TNF) production in patients as well as a TNF-inducing membrane constituent of tumor cells have been reported. In a murine fibrosarcoma model we analyzed TNF production during growth of a tumor transplant. In situ hybridization showed that a gradually increasing number of cells within the tumor tissue became positive for TNFmRNA. Also, in spleen cells of tumor-bearing mice TNFmRNA became more abundant in later stages of tumor growth compared to early stages. In plasma of these animals, however, TNF activity was not detected at any time even after stimulation with bacterial endotoxin. Neutralization with monoclonal antibodies of endogenous TNF during tumor growth did not affect the growth rate of the tumor, indicating that either the antibodies did not reach the relevant TNF production and action sites or that endogenously produced TNF did not play a significant role in this tumor model.

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.

We describe five murine monoclonal antibodies that are specific for human IL-4. At least three spatially distinct epitopes on the hIL-4 molecule are recognized by this panel of antibodies which allowed development of a sensitive sandwich EIA specific for hIL-4. The EIA is capable of detecting 200 pg/ml of hIL-4 and exhibits no detectable crossreactivity to seven other human cytokines examined. In addition to the EIA these antibodies are also useful in a number of other techniques for investigating hIL-4. Recombinant hIL-4 can be easily and efficiently purified by affinity chromatography using these mAbs. Western blotting with two of the antibodies detects as little as 1.8 ng/ml of hIL-4 in a 50 microliters sample. Finally, we present a method for cytoplasmic localization of hIL-4 by a fluorescence staining method utilizing a hIL-4 specific mAb.

Peripheral blood mononuclear cells (PBMC) that had been stimulated with a polyclonal B cell activator, pokeweed mitogen (PWM), were examined for mitogenic responsiveness to interleukins (IL) and colony stimulating factors (CSF) or other cytokines using recombinant preparations from E. coli or Cos cells expressing their cloned cDNAs. PWM-stimulated PBMC (PWM-blasts) were found to be responsive to IL-3 depending upon the concentrations of the agent. Mitogenic response of PWM-blasts in the reaction to IL-3 was suppressed by the addition of a specific monoclonal antibody against IL-3 but not by anti-IL-2 serum, excluding the possibility that the DNA replication induced by IL-3 is a result of the proliferative response of IL-2 responder cells to IL-2, which might be produced during the culture of PWM-blasts in the presence of IL-3. The PWM-blasts were also responsive to IL-2 and IL-4 but not to other interleukins, namely, IL-1, IL-5 and IL-6, nor to granulocyte/macrophage (GM)-, granulocyte (G)- nor macrophage (M)-CSFs. They were also not responsive to interferon gamma. Similar responsiveness to IL-3 and IL-4 was found in the PBMC stimulated with a B-cell mitogen, Staphylococcus aureus Cowan 1 (Sac-1), but not in the PBMC stimulated with T cell mitogens, PHA or ConA. These results suggest that the human PBMC stimulated with B cell mitogens such as PWM or Sac-1 contain activated IL-3- and IL-4- responsive cells in sufficient number to detect these lymphokine activities.(ABSTRACT TRUNCATED AT 250 WORDS)