Activation of bovine neutrophils by recombinant interferon-gamma

The effect of recombinant bovine interferon-gamma (r-IFN-gamma) on neutrophil functions was investigated and compared to the effects of an unpurified lymphokine preparation. Incubation of purified bovine neutrophils with r-IFN-gamma or antigen-induced lymphokine for 2.5 hr at 37 degrees C resulted in impairment of the ability of neutrophils to migrate under agarose, and an enhancement of their ability to mediate antibody-dependent and antibody-independent cell-mediated cytotoxicity against chicken erythrocytes. Neither the lymphokine preparation nor the r-IFN-gamma had any influence on Staphylococcus aureus ingestion, or iodination by neutrophils. The lymphokine preparation enhanced cytochrome c reduction by neutrophils and was weakly chemotactic, whereas the r-IFN-gamma had neither of these effects. Only 5 min of r-IFN-gamma preincubation with neutrophils were needed to trigger protein synthesis by the neutrophils resulting in inhibition of random migration. Therefore, recombinant interferon-gamma acts as a neutrophil migration inhibition factor and a neutrophil activation factor resulting in enhanced neutrophil-mediated antibody-dependent and -independent cell-mediated cytotoxicity. Many, but not all, of the in vitro effects of an unpurified lymphokine preparation on neutrophil function can be attributed to the interferon-gamma contained in the lymphokine.     

Activation of neutrophils by recombinant interleukin 6

The cytokine interleukin 6 (IL-6) has been shown to have multiple biological activities against many cellular targets. The present studies were designed to determine whether these activities extended to the neutrophil (PMN). Initially, we investigated the ability of IL-6 to modulate PMN-mediated antibody-dependent cellular cytotoxicity. The presence of IL-6 stimulated 51Cr release from labeled, opsonized targets by 67.1% (from 21.6 +/- 1.4% to 36.1 +/- 1.3% at 10 U of IL-6 (P less than 0.01)). IL-6 was not directly toxic to the target cells and stimulation of ADCC was shown to occur across a range of effector-to-target ratios. To investigate the basis of the capacity of IL-6 to stimulate PMN, we studied the effects of IL-6 on PMN chemotaxis, degranulation, and the respiratory burst. IL-6 was not chemotactic or chemokinetic for PMN. However, IL-6 stimulated lysozyme secretion from 14.1 +/- 2.5 to 23.7 +/- 3.6% at 100 U (P less than 0.01). IL-6 was a complete secretagogue, being able to induce the secretion of both the secretory granule marker lactoferrin (11.2 +/- 2.0 to 23.5 +/- 2.2%) and the primary granule marker beta-glucuronidase (5.0 +/- 1.0 to 18.2 +/- 4.0%). IL-6 was not able to directly stimulate the PMN respiratory burst. However, IL-6 did "prime" PMN, enhancing superoxide secretion by fMLP (10(-7) M)-treated PMN by 50.8% (5.9 +/- 1.0 to 8.9 +/- 1.5 nmol superoxide at 100 U of IL-6; P less than 0.01) and PMA (5.0 nM) by 54.3% (8.1 +/- 2.6 to 12.5 +/- 3.6 nmol; P less than 0.05). In conclusion, IL-6 is a PMN stimulant, enhancing the toxicity of PMN in an antibody-dependent cellular cytotoxicity assay. Enhanced cytotoxicity may have been mediated, at least in part, by the stimulation of secretion of toxic components from PMN targets and by the priming of stimulating respiratory burst activity.     

Activation of recombinant human SK4 channels by metal cations

The effects of metal cations on the activation of recombinant human SK4 (also known as hIK1 or hKCa4) channels, expressed in HEK 293 cells, were tested using patch clamp recording. Of the nine metals tested, cobalt, iron, magnesium, and zinc did not activate the SK4 channels when applied, at concentrations up to 100 microM, to the inside of SK4 channel-expressing membrane patches. Barium, cadmium, calcium, lead, and strontium activated SK4 channels in a concentration-dependent manner. The rank order of potency was at Ca2+ > Pb2+ > Cd2+ > Sr2+ > Ba2+.     

Interaction of duodenase with human blood serpins

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, alpha 1-protease inhibitor (alpha 1-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for alpha 1-PI and ACT, respectively. The presence of a stable enzyme-inhibitory complex duodenase-alpha 1-PI was confirmed by SDS-PAGE. No formation of the duodenase-ACT complex was demonstrated; instead, the band of the cleaved inhibitor was indicated upon the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (Ka, M-1 s-1) were 2.4 +/- 0.3 x 10(5) for alpha 1-PI and 3.0 +/- 0.4 x 10(5) for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.     

Activation and enzymatic characterization of recombinant human kallikrein 8

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM.     

Activation of the interferon system enzymes by recombinant alpha2-interferon in patients with chronic hepatitis B

Treatment of patients suffering from chronic hepatitis B with recombinant leukocytic interferon (reaferon) increased the levels of circulating interferon and activated interferon-dependent enzymes such as 2-5A-synthetase and histone kinase. Activation of the enzymes was observed for 1 to 2 weeks. After that period it was maintained at the required levels with intramuscular administration of 1-3 million units of reaferon 2 or 3 times a week. In parallel with increasing of the levels of the interferon system enzymes there was observed a decrease in the level of aminotransferase. The reaction of the viral antigens to the treatment with reaferon was not the same: HBe antigen and antibodies to HBe antigen disappeared, the content of HBs antigen and antibodies to delta-interferon did not change.     

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.     

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k _a) were 11, 6.8, and 17 mM^?1·sec^?1 for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k _a). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3′, rACT P3-P4′, rACT P4-P3′, and rACT P6-P4′) was studied. The inhibition type ([E]₀ = 1·10^?7 M, 25°C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K _i) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.     

Activation, proteolytic processing, and peptide specificity of recombinant cardosin A

Cardosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Here we describe the activation and proteolytic processing of recombinant procardosin A. The cleavage sites involved in this multi-step autocatalytic process were determined, some of them using a novel method for C-terminal sequence analysis. Even though the two-chain recombinant enzyme displayed similar properties as natural cardosin A, a single-chain mutant form was engineered based on the processing results and produced in Escherichia coli. Determination of its primary specificity using two combinatorial peptide libraries revealed that this mutant form behaved like the natural enzyme. The primary specificity of the enzyme closely resembles those of cathepsin D and plasmepsins, suggesting that cardosin A shares the same peptide scissile bond preferences of its vacuolar/lysosomal mammalian and protozoan homologues.     

An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI

Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).     

Activation of protein kinase C results in down-modulation of different recombinant GABAA-channels.

Different combinations of cloned rat brain subunits of the GABAA receptor were expressed in Xenopus oocytes. The effect of the phorbol ester PMA, an activator of protein kinase C, on the expressed GABA-gated ion current was determined. Ion currents were diminished by beta-PMA, but not by the control substance alpha-PMA, irrespective of the subunit combination studied. The mechanism of current decrease was investigated in more detail for the subunit combination alpha 5 beta 2 gamma 2. The reversal potential of the current remained unaffected, while the maximal current amplitude was decreased and the apparent Ka for GABA-dependent channel gating was shifted to higher concentrations.     

Study on interaction between duodenase protease with dual specificity and inhibitors of bowman-birk family

The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 (n)M respectively.     

Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species

Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.     

Activation of macrophages by peroxidases

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.     

Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts.

Duodenase is a 29-kDa serine endopeptidase that displays selective trypsin- and chymotrypsin-like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm-infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFalpha, known to up-regulate PAR2 expression in other systems, and IL-1beta, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPgammaS binding to fibroblast membranes indicating that a G-protein-coupled receptor may mediate the effects of duodenase. Duodenase-induced DNA synthesis and GTPgammaS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 microm) inhibited this response completely, indicating a key role for PtdIns 3-kinase. Furthermore, duodenase induced a 2.3 plus minus 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteolytic mechanism.