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 neutrophils by N-formyl chemotactic peptides

The response of neutrophils to N-formyl peptides is mediated via a specific 50,000- to 60,000-dalton (Mr) receptor. Real-time kinetic analysis indicated that most of the cellular responses elicited by this ligand began within 5-10 s of addition to the cells at 37 C. Of three possible biochemical changes measured that could serve as transducers or second messengers, two, i.e., increases in cyclic AMP (cAMP) and intracellular free Ca2+, occurred within 5 s of stimulus addition. In contrast, internalization of the ligand by cells showed a latency time of 20-30 s, which indicates that it probably plays no role in triggering later responses. Using pulse binding techniques that allow the level of a given response to be measured as a function of the measured level of surface receptor occupancy, we found that O2- production required up to 30% receptor occupancy to elicit 50% of maximal response. In contrast, secretion, cAMP changes, Ca2+ changes, and membrane potential changes required less than 5% occupancy. Within 5 s, occupied receptors were converted at the cell surface to a slowly dissociating form. The receptors, exhibiting apparent higher affinity, were transiently associated with the cell cytoskeleton as defined by their conversion to a Triton X-100-insoluble form. Internalized receptor-ligand complexes were transported, in large part, to the Golgi apparatus. Further analyses of the mechanism of stimulation of leukocytes have been performed with monoclonal antibodies directed against the neutrophil surface. Data with these antibodies, which are not directed to the N-formyl peptide receptor, reveal that some modulated the N-formyl peptide-mediated responses and other antibodies initiated responses of the cells.     

Modulation of bovine luteal cell synthetic capacity by interferon-gamma

Previous work from our laboratory has demonstrated that major histocompatibility complex (MHC) antigens are expressed on cultured bovine luteal cells following exposure to the T lymphocyte-derived cytokine, interferon-gamma (IFN-gamma). In light of these actions of IFN-gamma, it was of interest to investigate the effects of this cytokine on other aspects of luteal function. Therefore, bovine luteal cells were cultured for 7 days in the presence or absence of IFN-gamma, and luteal progesterone (P4), prostaglandin F2 alpha (PGF2 alpha), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) production were evaluated. After a 24-h exposure to IFN-gamma (100 U), both PGF2 alpha and 6-keto-PGF1 alpha production were decreased approximately 50% (p less than 0.05). However, as time in culture progressed, IFN-gamma markedly increased the synthesis of both prostaglandins approximately 400% above controls (p less than 0.05). Stimulation of prostaglandin production by IFN-gamma was abrogated by the addition of exogenous P4. During the period of IFN-gamma-stimulated prostaglandin synthesis, LH-stimulated P4 production was inhibited by IFN-gamma treatment. However, the suppression of P4 production by IFN-gamma was not mediated by the increase in prostaglandins since concomitant treatment with indomethacin did not reverse the inhibition of steroidogenesis. These results suggest that IFN-gamma, in addition to an indirect role in promoting immune response mechanisms, may also directly affect luteal function by enhancing luteal prostaglandin synthesis and by inhibiting luteal steroidogenesis.     

High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis

Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.     

Activation of recombinant proenteropeptidase by duodenase

Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single-chain bovine proenteropeptidase (kcat/Km = 2700 M(-1) s(-1)). The measured rate of proenteropeptidase cleavage by duodenase was about 70-fold lower compared with the rate of trypsin-mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.     

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.     

Stimulation of microtubule assembly by recombinant human interferon-alpha and interferon-gamma

Interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) have been demonstrated to stimulate microtubule assembly measured in the in vitro assembly system. The process is substoichiometric occurring when the interferon concentrations are below that of tubulin. IFN-gamma is a more potent effector than IFN-alpha. The critical tubulin concentration describing microtubule assembly decreases from 1.5 mg/ml measured in the absence of added effector to 1.05 mg/ml and 1.3 mg/ml when measured in the presence of 2.16.10(-6) M IFN-gamma and 3.06.10(-6) M IFN-alpha, respectively.     

Activation of bovine plasminogen by Streptococcus uberis

Abstract Thiosulfate and tetrathionate oxidation activity of Thiobacillus ferrooxidans were found to be absent in iron-growth cell as well as in the cells grown anaerobically on elemental sulfur. While the thiosulfate oxidase activity was absent in the cell-free extract of the above cells, the activity of rhodanese was present irrespective of the culture condition of T. ferrooxidans . It is thus conceivable that rhodanese is not involved in thiosulfate metabolism. During growth in presence of ferrous sulfate plus elemental sulfur, the thiosulfate/tetrathionate oxidation activity was absent till the oxidation of ferrous iron was complete and the cells harvested only in the latter period acquired the thiosulfate/tetrathionate oxidation activity. Thus it becomes evident that the inhibition of thiosulfate and tetrathionate oxidation is solely due to presence of ferrous iron.     

Reversibility of acid denaturation of recombinant interferon-gamma

It has been shown that interferon-gamma (IFN-gamma) loses activity after acid treatment and this property can be used to distinguish it from other types of interferons. Therefore, reversibility of acid denaturation of IFN-gamma was examined using the recombinant human protein. The fluorescence spectra showed that conformation of the protein is similar before and after acid treatment, suggesting reversibility of the acid denaturation. The antiviral activity of the protein was also identical in the same treatment. However, the antiviral activity was significantly reduced when it was determined by directly diluting the acidic samples into the assay medium containing high salts and serum proteins. Similar results were obtained with the recombinant murine IFN-gamma. This observation demonstrates that acid denaturation of the IFN-gamma is dependent on the way the protein is renatured, and hence that the difference in response to acid treatment between IFN-gamma and other interferons is quantitative rather than qualitative.     

牛γ 干扰素的表达及其抗病毒活性测定

通过RT-PCR从经ConA刺激诱导的奶牛脾脏淋巴细胞总RNA中扩增出牛γ干扰素 (BoIFN-γ) cDNA,克隆到真核载体pVAX1中,测序结果显示pVAX1中的插入序列BoIFN-γ基因与已报道序列一致。用重组质粒pVAX1-BoIFN-γ转染COS-7细胞并进行间接免疫荧光试验鉴定,结果显示BoIFN-γ在COS-7细胞中得到成功表达。将BoIFN-γ基因克隆到原核表达质粒pET-30a(+)、pGEX-6p-1后,分别转化重组表达菌BL21(DE3)、BL21后,通过对表达条件的优化,SDS-P     

Sites of phosphorylation in recombinant human interferon-gamma

Recombinant human interferon-gamma was phosphorylated with ATP and c-AMP-dependent protein kinase. After phosphorylation, interferon-gamma was separated from the adenosine phosphates and the kinase and analyzed by SDS-PAGE, reverse phase HPLC, and HPLC peptide mapping. Comparison of the S. aureus V8 protease maps of intact and phosphorylated interferon-gamma showed that the maps were identical except that one peptide fragment elutes earlier in the map of the phosphorylated sample. This peptide was identified as the C-terminal fragment containing two serinyl phosphorylation sites at positions 132 and 142. This phosphorylated interferon-gamma exhibited a slightly higher specific antiviral activity than the intact protein.     

Activation of neutrophils NADPH oxidase by PMA: Cytosol activity is translocated in phorbol-primed neutrophils

• 1.1. Translocation of cytosol activity in phorbol-primed neutrophils was studied.
• 2.2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA.
• 3.3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane.
• 4.4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane.
• 5.5. These observations suggested that cytosol activity was translocated in PMA-primed cells.

     

Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1.

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.     

Study of antigenic epitopes recognized by monoclonal antibodies to recombinant interferon-gamma

Seven hybridomas (BG 1-7) which secreted monoclonal antibodies against recombinant interferon-gamma were produced. The ascites fluids containing four of the seven monoclonal antibodies (BG 1-4) neutralized the antiviral activity of both natural and recombinant interferon-gamma. Competition between labeled and unlabeled monoclonal antibodies for interferon-gamma in a solid phase immunoassay showed that BG 1 was competed by both BG 3 and BG 4 but not by BG 2; BG 2 was competed by BG 3 but not by BG 1 nor by BG 4. These results suggest that human interferon-gamma has at least two antigenic epitopes; one of the epitopes reacted with BG 1 & BG 4 while the other reacted with BG 2; BG 3 either binds to a region overlapping with the other two epitopes or reacts with both epitopes. The antigenic epitopes recognized by these four neutralizing monoclonal antibodies are likely at or closely related to the active sites of interferon-gamma.     

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.     

Activation of plasminogen by the early bovine embryo

Activation of the plasma zymogen plasminogen to the enzyme plasmin by the early bovine embryo was evaluated. Sixteen-cell embryos to early morulae were collected at death from handmated synchronized and superovulated crossbred beef cows. Embryos were cultured in Ham's F-12 medium supplemented with 15 mg/ml bovine serum albumin containing 0, 15, 30, 60 or 120 micrograms/ml plasminogen in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were observed every day, and stage of development was recorded. Medium was collected at 24-h intervals, starting at initiation and continuing through 288 h of culture. Plasminogen activator and plasmin levels in the culture media were determined, using a caseinolytic assay. The percentages of embryos developing to the initiating hatching blastocyst, hatched blastocyst, attached blastocyst, and attached blastocyst with trophoblastic outgrowth stages were not significantly different between the five levels of plasminogen. Initiation and completion of hatching, however, accelerated as plasminogen concentration increased in the culture media. Plasminogen activator production, expressed as milliunits X ml-1 X h-1 X viable embryo-1, was low for the first 48 h of culture, increased between 48-120 h, and tended to plateau thereafter. Plasminogen activation, measured indirectly as the plasmin concentration in a microdrop of medium and expressed as microgram plasmin X ml-1 X h-1 X viable embryo-1, followed plasminogen activator production, and was consistently low for the first 48-72 h of culture. Embryonic activation of plasminogen increased sharply thereafter, and also plateaued after 120 h.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.