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PubMed2003

Placental immunomodulator ferritin, a novel immunoregulator, suppresses experimental arthritis.

OBJECTIVE: To determine the effect of treatment with C48, the recombinant cytokine-like domain of the novel human placental immunomodulator ferritin (PLIF) immunoregulator, on zymosan-induced arthritis (ZIA) in mice and on adjuvant-induced arthritis (AIA) in rats. METHODS: The in vitro effect of PLIF/C48 was tested in mixed lymphocyte cultures (MLCs) of allogeneic mouse splenocytes. Arthritis was induced by intraarticular injection of zymosan into naive mice and by subcutaneous injection of Mycobacterium tuberculosis into rats. C48 was injected intraperitoneally daily from day 3 to day 9 or from day 7 to day 13 after induction of synovitis by zymosan, and every other day from day 2 to day 14 after induction of AIA. Swelling of the joints and histologic features of the synovium were assessed. Th1 and Th2 cytokines were quantified by enzyme-linked immunosorbent assay. RESULTS: Both PLIF and C48 significantly inhibited the in vitro immunoreactivity of mouse splenocytes in MLCs. Treatment of ZIA mice and AIA rats with C48 effectively reduced joint swelling. C48 treatment reduced synovial lining thickening, numbers of mononuclear cells and histiocytes, as well as cartilage destruction and bone erosions. In vitro, activated splenocytes from C48-treated ZIA and AIA animals produced significantly higher levels of interleukin-10 (IL-10). In animals with ZIA, this was accompanied by lower levels of tumor necrosis factor and IL-2. CONCLUSION: Human PLIF and C48 were shown to exert cross-species immunosuppressive activity in vitro. The in vivo suppression of articular inflammation in the experimental models of ZIA and AIA was the result of treatment with the antiinflammatory human C48. These results suggest that treatment with C48 may offer an effective immunotherapeutic means of controlling inflammatory polyarthritis.

Adjuvants, Immunologic↗
PubMed1989

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils--analysis by chemiluminescence counting and its microscopic imaging.

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca2+ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.

Free Radicals↗
PubMed2005

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells.

BACKGROUND: The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)-labeled zymosan particles by human leukocytes. METHODS: FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence. RESULTS: The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equations were presented. From 28 control samples, numerical values of mean fluorescence intensities and percentages of phagocytotic subpopulations inside phagocytotic gates before and after quenching were inserted into phagocytotic equations and corrected phagocytotic parameters were calculated. Calculated parameters were surprisingly constant across individuals. CONCLUSIONS: Essential elements of the present method appeared to be partial quenching of extracellular fluorescence with trypan blue and distinguishing between overlapping populations of binding and ingesting cells. Corrections using derived phagocytotic equations proved necessary for accurate kinetic phagocytotic measurements. Corrections were less necessary when the ingestion process was finished.

Buffers↗
PubMed1984

Cell surface events which may initiate lysosomal enzyme secretion by human monocytes.

Freshly isolated and subsequently matured human monocytes secreted lysosomal hexosaminidase in response to exposure to IgG-Sepharose, but not certain derivatized control Sepharoses. The cells bound selectively to the surface of IgG-Sepharose (and not the control Sepharoses) but because of the large size of the particles, could not ingest them. Since the soluble IgG was covalently linked to the Sepharose and free soluble IgG was not an inducer of secretion, the secretion was thus induced directly at the cell surface. Zymosan, a yeast cell wall particle which contains a mannan, was also able to induce secretion in the various monocyte stages under study. It could even bind to the cell surface of fresh monocytes which lacked the receptor for mannose-terminated glycoproteins, and induce secretion in these cells. The mannose receptor appeared as monocytes matured, and the Ir number on the surface was increased by the action of lymphokines. Although zymosan-induced secretion could be inhibited by mannose and certain other sugars, these seemed to have some complex metabolic effects in human monocytes (which previous work with mouse macrophages has not revealed). Thus, it was not possible to demonstrate whether zymosan could initiate secretion directly by interaction at the monocyte surface mannose glycoprotein receptor.

Cell Membrane↗
PubMed1987

Potentiation of neutrophil function by recombinant DNA-produced interleukin 1a.

The effect of interleukin 1a (IL-1a) produced by E. coli-derived recombinant DNA was evaluated on various parameters of human neutrophil function. IL-1a alone stimulated neutrophil hydrogen peroxide production in a dose-dependent manner, but the rate was much lower than that of opsonized zymosan. IL-1a induced release of specific granule contents, but not azurophilic granule contents. Cytochalasin B did not augment the rate of release. IL-1a was chemotactic for neutrophils at the optimal concentrations of 0.1-10 ng/ml. Pretreatment of the neutrophils with IL-1a augmented neutrophil oxygen radical production induced by opsonized zymosan, and this synergistic effect was evident as early as 10 min after IL-1a was added to the neutrophil culture. Phagocytosis of opsonized particles by neutrophils, and degranulation induced by opsonized zymosan were also enhanced by IL-1a in a dose-dependent manner. The present results suggest that IL-1a is weak as a direct activator of neutrophil function and that IL-1a in vivo may augment the response of neutrophils to other stimulators such as foreign bodies.

Chemotaxis, Leukocyte↗
PubMed1988

An assessment of the respiratory burst and bactericidal activity of alveolar macrophages from adult and senescent mice.

To assess the effects of advanced age on the nonspecific antimicrobial activity of resident alveolar macrophages (AM), superoxide anion (O2-) release and the phagocytic and bactericidal capacity of cells from three genetically distinct murine strains were evaluated. In initial experiments, resident AM, obtained by bronchoalveolar lavage of pathogen-free adult female CD-1 mice and studied in suspension, were found to produce O2- spontaneously and in response to phorbol myristate acetate (PMA) snd unopsonized zymosan. Maximum quantities of O2- were released following stimulation with 1 microgram/ml PMA and by a particle-to-cell ratio of 100:1 with zymosan; responses to the agonists peaked between 60 and 90 min. Resident AM obtained from pathogen and disease-free senescent (18-26-month-old) female C57BL/6, BALB/c, and DBA/2 mice released significantly more O2- in response to both PMA and zymosan than did cells secured from adult (4-8-month-old) animals. In vivo, the capacity of AM from adult and senescent animals to phagocytose Streptococcus pneumoniae (unencapsulated strain) and Staphylococcus aureus was comparable, and although the cells from the senescent mice tended to be more efficient in their ability to kill internalized bacteria, statistically significant differences between the two groups were not observed. The results of these studies indicate that the enhanced susceptibility of the senescent host to infection of the lower respiratory tract cannot be attributed to age-related changes in the nonspecific antimicrobial activity of resident AM.

Aging↗
PubMed1990

Suppression of late phase enhanced vascular permeability in rats by selective depletion of neutrophils with a monoclonal antibody.

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophils using a monoclonal antibody, RP-3. An intraperitoneal injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by in situ injection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected.

Animals↗
PubMed1991

Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages.

Mouse peritoneal macrophages were labeled with [1-3H]ethanolamine, and the presence of radioactive [3H]phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3[H]PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3[H]PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3[H]PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.

Animals↗
PubMed1993

Particle-induced in vivo priming of alveolar macrophages for enhanced oxidative responses: a novel system of cellular immune augmentation.

A novel system for priming adult rabbit alveolar macrophages (AMs) in vivo for markedly enhanced oxidative responses is described. When adult rabbits were injected intravenously (i.v.) with 1- to 5-microns particles such as zymosan, latex particles, or heat-killed bacille Calmette-Guérin, AMs were primed in 1-3 days for greatly enhanced phorbol myristate acetate (PMA)- or opsonized zymosan (Op-zym)-elicited chemiluminescent (CL) responses. Intratracheal (i.t.) injection of zymosan particles also primed AMs for enhanced PMA- or Op-zym-elicited CL responses. AMs obtained from particle-injected rabbits showed up to 100-fold higher levels of PMA-elicited CL responses than AMs from normal rabbits. In contrast, Op-zym failed to prime normal AMs in vitro for enhanced CL responses. Whereas AMs could not be primed in vivo with an i.v. injection of particles of approximately 24 microns diameter. AMs could be primed if the particles were administered by the i.t. route. The priming appears to be independent of particle types. The priming effect was of short duration and declined after 5 to 7 days. The possibility that this system represents the primitive cellular immune response found in invertebrates is discussed. The potential use of this system as a means of immune augmentation prompts further investigation.

Animals↗
PubMed1995

Role of YopH in the suppression of tyrosine phosphorylation and respiratory burst activity in murine macrophages infected with Yersinia enterocolitica.

Tyrosine phosphorylation is an important component of the signaling pathways responsible for the activation of the macrophage respiratory burst. Because the virulence plasmid of Yersinia enterocolitica encodes a phosphotyrosine phosphatase, YopH, it is possible that the pathogenic strategy of Y. enterocolitica involves the disruption of tyrosine phosphorylation in the macrophage leading to inhibition of respiratory burst activity. We have investigated the effects of Yersinia infection on tyrosine phosphorylation and respiratory burst activity in murine bone marrow-derived macrophages. Infection of macrophages with virulent [Ye(pYV+)] but not avirulent [Ye(pYV-)] strains of Y. enterocolitica was found to suppress both tyrosine phosphorylation and respiratory burst activity in response to zymosan. Mutational inactivation of YopH reversed the suppressive effect of Ye(pYV+) on zymosan-induced tyrosine phosphorylation, indicating that YopH is responsible for the dephosphorylation of macrophage phosphotyrosine-containing proteins observed in macrophages infected with the virulent strain of Y. enterocolitica. In contrast, mutational loss of YopH failed to reverse the inhibitory effect of Ye(pYV+) on the zymosan-triggered respiratory burst. We conclude that the inhibition of the macrophage respiratory burst by Y. enterocolitica involves a plasmid-encoded virulence protein(s) other than, or in addition to, YopH.

Animals↗
PubMed2005

Skeletal Muscle wasting and contractile performance in septic rats.

We investigated the temporal effects of sepsis on muscle wasting and function in order to study the contribution of wasting to the decline in muscle function; we also studied the fiber-type specificity of this muscle wasting. Sepsis was induced by injecting rats intraperitoneally with a zymosan suspension. At 2 h and at 2, 6, and 11 days after injection, muscle function was measured using in situ electrical stimulation, Zymosan injection induced severe muscle wasting compared to pair-fed and ad libitum fed controls. At 6 days, isometric force-generating capacity was drastically reduced in zymosan-treated rats. We conclude that this was fully accounted fo by the reduction of muscle mas. At day 6, we also observed increased activity of the 20S proteasome in gastrocnemius but not soleus muscle from septic rats. In tibialis anterior but not in soleus, muscle wasting occurred in a fiber-type specific fashion, i.e., the reduction in cross-sectional area was significantly smaller in type 1 than type 2A and 2B/X fibers. These findings suggest that both the inherent function of a muscle and the muscle fiber-type distribution affect the responsiveness to catabolic signals.

Animals↗
PubMed1975

Comparison of the effect of various antisera and cobra venom factor on inflammatory reactions in guinea-pig skin. II. The Arthus reaction and the local Shwartzman reaction.

The ability of antisera to guinea-pig C3 to inhibit the Arthus and local Shwartzman reactions was studied. They were found to reduce the non-haemorrhagic component of the active and reversed passive Arthus reactions and to delay the appearance of the haemorrhage in the active Arthus reaction. Cobra venom factor, however, had no effect on the non-haemorrhagic components of these reactions and only delayed the appearance of the haemorrhage of the active Arthus reaction. There appeared to be a correlation between the serum complement level and the time taken for the haemorrhage to appear, and between the circulating platelet count and the extent of the non-haemorrhagic, oedematous component of the reaction. The haemorrhagic component of the local Shwartzman reaction was not affected by decomplementation with cobra venom factor. The ability of the antisera to inhibit the haemorrhage of the Shwartzman reaction was not dependent on lowering the serum complement titre. However, the haemorrhage was inhibited if the circulating platelet count was also reduced to very low numbers. Antiserum to zymosan alone had the same effect as anti-beta1C/beta1A globulin (zymosan) in blocking the reaction, although it did not alter the complement levels or the platelet counts. The possibility of an immunological cross-reactivity between zymosan and endotoxin in this action is discussed.

Animals↗
PubMed1979

Anti-inflammatory effects of blood platelets in the rat.

A selective accumulation of blood platelets occurs at the sites of intradermal injection of zymosan and carrageenan in the rat. The inflammatory response to zymosan, assessed by the local accumulation of systemic radioactive human serum albumin, was significantly enhanced in platelet-depleted rats. Similar enhancements of the paw oedemas, caused by the subplantar injection of either carrageenan or zymosan, were also observed in thrombocytopenic animals. It is concluded that blood platelets may exert protective, i.e., anti-inflammatory, effects in some models of acute inflammation in the rat.

Animals↗
PubMed1996

The mechanism of inhibitory effect of eicosapentaenoic acid on phagocytic activity and chemotaxis of human neutrophil granulocytes.

Free eicosapentaenoic acid (EPA) was found to inhibit dose dependently the chemiluminescence of human neutrophil granulocytes phagocytosing zymosan and their chemotaxis induced by C5a-containing zymosan-activated serum (ZAS) and platelet-activating factor. Rigidification of plasma membranes in the ZAS-treated cells could be observed by measuring the fluorescence anisotropy. The cells were labeled by 3-[p-(6-phenyl-1,3,5-hexatrienoil) phenyl] propionic acid, reporting plasma membrane for determination of membrane fluidity. In resting, nonstimulated neutrophils, EPA dose dependently increased the fluidity of plasma membrane. In zymosan-activated cells, however, after a short fluidization, the basic effect of EPA was a rigidification compared to very low fluorescence anisotropy values of activated control cells. This diminished fluidity, increased membrane stability of plasma membranes can be one of the reasons for the decreased functions (phagocytosis and chemotaxis) of human EPA-treated neutrophils.

Cell Membrane↗
PubMed1999

Internal defenses of the snail Biomphalaria glabrata.

The three populations of hemocytes and the fixed phagocytes of Biomphalaria glabrata show different affinities for the following injected foreign materials: ferritin, horseradish peroxidase, zymosan particles, latex beads, and live bacteria. Ferritin and horseradish peroxidase, which are internalized by micropinocytosis, induce a moderate activation of hemocytes. Particles trigger a strong activation that consists of mobilization of the cytoskeleton, with formation of pseudopodia and filopodia and modifications of the plasma membrane that enable hemocytes to adhere together, as in encapsulations. Phagocytosis of zymosan and live bacteria induces alterations in mitochondria that may reflect changes in the respiratory activity. Phagocytosis of particles follows different processes: fixed phagocytes ingest latex beads of two different sizes according to the "zipper model", which implies sequential binding between phagocyte receptors and opsonized ligands; zymosan particles are ingested by medium-size hemocytes by a variant of the zipper model in which a limited close contact between cell and particle resembles focal contacts in cell spreading; bacteria or large latex beads are ingested by large hemocytes without apparent close contact following a "trigger model" which resembles macropinocytosis.

Animals↗
PubMed1996

Distinct signal transduction pathways for activation of rabbit alveolar macrophages in vitro by cotton bract tannin.

These experiments were designed to study signal transduction pathways in alveolar macrophages stimulated by condensed tannin or zymosan. Condensed tannins, present in cotton mill dust, alter the host-defense function of alveolar macrophages and may contribute to the pathogenesis of byssinosis. We tried to determine the early steps in signal transduction mechanisms of cell activation by tannin. With the quantification of 51Cr release, we determined that tannin was cytotoxic for the cells after 30 min activation with 130 micrograms for 2 x 10(6) cells. 51Cr release was similar for control cells and zymosan- or 30 micrograms tannin-activated cells. Using the luciferine luciferase reaction, we showed that tannin markedly depleted ATP cell content. In inositol-labeled cells, tannin increased inositolphosphate release in a dose-dependent manner. In lysoPAF-labeled cells, tannin induced synthesis of phosphatidic acid and diglycerides. In the presence of ethanol, the level of tannin-induced phosphatidic acid was slightly reduced, and phosphatidylethanol was synthesized. No phosphatidylethanol was found in alveolar macrophages stimulated by zymosan in the presence of ethanol. GF 109203X, a specific inhibitor of protein kinase C decreased only tannin-induced phosphatidylethanol synthesis. In conclusion, tannin (at 30 or 130 micrograms/ml) activated an inositol phospholipase C in alveolar membranes. Phosphatidylcholine phospholipases C and D were found only at the higher concentration of tannin.

Adenosine Triphosphate↗
PubMed1990

Extracellular phospholipase A2 activity in two in vivo models of inflammation.

Two "in vivo" models of inflammation have been used to investigate the role of phospholipases A2 (PLA2) in inflammation. These models are casein-induced peritonitis in the rat and zymosan-induced peritonitis in the mouse. The extracellular PLA2 activities from peritoneal lavage fluid in these two models are similar: they are calcium dependent and have broad neutral pH optima. However, the relationship between extracellular PLA2 activity and cell influx in these models are not identical. In zymosan peritonitis, PLA2 activity preceded peak cell influx, reaching a maximum within 15 min after zymosan injection, while cell influx peaked by 8 hr. In casein-induced peritonitis, the PLA2 activity peaked at 24 hr, while cell influx continued through 48 hr. The origins of the PLA2 activities in both models remain unclear; one potential source is the plasma. Understanding the role of extracellular PLA2 activity in "in vivo" models, and investigating specific inhibitors in these models may aid in our understanding of the role of extracellular PLA2 in diseases such as rheumatoid arthritis, endotoxin shock and pancreatitis.

Animals↗
PubMed1996

[Modification of phagocytic microbicide function by antifungal agents--measuring luminol enhanced chemoluminescence in full blood].

Fungus infections are becoming more important in surgical intensive medicine, and various preparations are now available to treat them. The goal of our investigations was to determine the influence of the antimycotics in current use on the microbicide phagocytic function by measuring chemiluminescence. To this end the luminol-enhanced chemiluminescence in whole blood samples from a total of 29 healthy donors was measured with a LKB 1251 Luminometer after stimulation with zymosan or a Canadida albicans preparation. We tested the substances amphotericin B, flucytosin (Ancotil), fluconazol (Diflucan) and itraconazol (Sempera), each in three different concentrations within the recommended dose range and compared the results with those in an untreated sample. For the insertions of amphotericin B or intraconazol after zymosan stimulation no significant differences in the measurements were found (50.84 vs 47.99 mV and 46.10 vs 47.89 mV) compared with the blank test. Similar situations revealed by the tests with C. albicans administration (15.21 vs 12.35 mV and 11.16 vs 11.91 mV). However, the tests with flucytosin in the higher concentration range, after stimulation with either zymosan or C. albicans, evidenced a significant reduction in the measurements (34.70 vs 52.74 mV, P<0.005, and 10.98 vs 14.57 mV, P<0.01). The tests with fluconazol showed a decrease of the chemiluminescence exclusively for the highest concentration in the C. albicans group (14.36 vs 17.20 mV, P<0.005). Our results indicate a negative influence of the phagocytes on the oxidative metabolism especially with flucytosin in the higher concentrations. This emphatically confirms demands for exact indications and dosage of antimycotics and their correct administration.

Antifungal Agents↗