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PubMed1981

The role of immunoglobulins in alternative complement pathway activation by zymosan. I. Human IgG with specificity for Zymosan enhances alternative pathway activation by zymosan.

Prior absorption of normal human serum (NHS) or C2-deficient human serum (C2D) with zymosan at 0 degrees C results in diminished consumption of C3 and factor B during subsequent incubation of the sera in Mg-EGTA buffer with zymosan at 37 degrees C for 30 min. An acid eluate from the zymosan restores the defect of absorbed NHS and C2D, and also enhances C3 and factor B utilization in hypogammaglobulinemic serum (H gamma S) in a dose-dependent fashion. The activity is specific in that the eluate from zymosan fails to enhance C3 and B depletion in H gamma S or absorbed NHS by lipopolysaccharide or Sepharose. The active component of th zymosan eluate emerges from both Sepharose 4B and Sephacryl S-200 in the region of molecules with m.w. of 150,000. Absorption with protein A-Sepharose removes the activity, demonstrating that it is IgG. Digestion of the IgG with pepsin fails to diminish activity, indicating that the Fc region is not required for activity; reduction to monovalent Fab' fragments, however, abrogates activity. When IgG antibody is bound to Protein A-Sepharose, it fails to enhance C3 depletion in H gamma S by Sepharose, indicating that binding of IgG antibody by the Fab region is necessary for enhancement of alternative pathway activity in human serum.

Antibody Specificity↗
PubMed2003

Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A.

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.

Animals↗
PubMed1994

Activation of a potassium outward current by zymosan and opsonized zymosan in mouse peritoneal macrophages.

The effects of zymosan and human serum opsonized zymosan on membrane currents of adherent mouse peritoneal macrophages which had been cultured for 5 to 20 days were investigated with the whole-cell voltage-clamp technique. Both stimuli activated an outward current. The outward current activation was transient and lasted about 5 min. In solutions with 10 or 50 mmol/l extracellular potassium concentration the activation of an outwardly directed current occurred at test potentials positive to the respective potassium equilibrium potential. This particle-induced current resembled a calcium-activated potassium current which could be activated with the calcium ionophore A 23187 and with platelet activating factor. The order of maximal responses (test potential + 55 mV, amplitude given as percentage of the respective control) was: 0.1 mumol/l platelet activating factor (222 +/- 36%, n = 8, P < 0.01) > 1 mumol/l A 23187 (190 +/- 24%, n = 11, P < 0.01) > 900 micrograms/ml opsonized zymosan (134 +/- 7%, n = 22, P < 0.01) > 900 micrograms/ml zymosan (116 +/- 5%, n = 21, P < 0.01). The lower efficiency of zymosan as compared to opsonized zymosan is explained in part by a lower percentage of responding cells which was 48% for zymosan and 73% for opsonized zymosan. Macrophages which were pretreated with particles showed a greater reactivity to calcium as compared to untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Pharmacological studies on zymosan inflammation in rats and mice. 2: Zymosan-induced pleurisy in rats.

Injection of zymosan in rat pleural cavity provokes an exudate which is already detectable at 15 min and which is maximum at 24 h. The leucocyte count (mostly neutrophils) increases at 2-4 h and is maximum at 48 h. In this paper the reaction has been studied up to 6 h. Evidence of histamine release, of mast cell degranulation and of reduction of the exudate by anti-H1 compounds, as well as by sodium cromoglycate, proves the active role played by histamine in the early stage of pleurisy. Serotonin (whose role was studied exclusively using antagonists) seems to have only a minor part in the early phase of the reaction. Some metabolites of arachidonic acid were determined in the pleural exudate at 1 h and 6 h. The concentration of leukotriene B4 was high at 1 h and decreased at 6 h. The thromboxane B2 level was already high at 1 h and was neatly augmented at 6 h while the amount of prostaglandin F1 alpha was high at both times. The non-steroidal anti-inflammatory substances studied all reduced the pleural exudate at 1 h but their activity then varied from each other at 6 h. Cyclooxygenase and lipoxygenase inhibitors (phenidone, BW755C) induced a reduction of the exudate at both times. Zymosan-induced pleurisy seemed thus to be an excellent model for the investigation of antiallergic and anti-inflammatory compounds active on histamine and cyclooxygenase and lipoxygenase pathways.

Animals↗
PubMedDate not supplied

Pharmacological studies on zymosan inflammation in rats and mice. 1: Zymosan-induced paw oedema in rats and mice.

Injections of zymosan in mouse and rat paws provoke inflammatory reactions, the kinetics of which are different. In both models, inflammation occurs at an early stage but oedema is maximal at 30 min in rat paw and 6 h in mouse paw. In this study the two reactions have been studied up to 6 h. The reduction of oedema by anti-H1 compounds, as well as by disodium cromoglycate, proves the active role played by histamine in rat paw oedema. In mouse its role appears to be minor or non-existent. Serotonin seems to be clearly implicated in the early stages of the oedema in mouse, somewhat less in rat. In the two species, non-steroidal anti-inflammatory compounds only reduce the 4-6 h phase. BW755C and phenidone reduce the early and late phase of paw oedema in both species, with the exception of phenidone which is inactive on the 4-6 h phase in the mouse. We can hypothesize that in the two species some leukotrienes seem to be implicated principally in the early phases, while derivatives of cyclooxygenase play a more important role in the late phases. Theophylline reduces inflammation in the two models, hydrocortisone acetate, however, is only active on the late phases. These results indicate that there are important differences in the participation of the various mediators studied in the two models.

Animals↗
PubMed1992

Different preparations of zymosan induce glycogenolysis independently in the perfused rat liver. Involvement of mannose receptors, peptide-leukotrienes and prostaglandins.

Zymosan (non-boiled) induced glycogenolysis biphasically, with no lag time, in the perfused rat liver. After the zymosan was boiled, it could be separated into two fractions, both of which stimulated glycogenolysis independently. The soluble fraction of boiled zymosan (zymosan sup) showed homologous desensitization, indicating that zymosan sup-induced glycogenolysis is a receptor-mediated event. Mannan (polymannose), which is known to be a biologically active component of zymosan, induced a glycogenolytic response similar to that produced by zymosan sup, and desensitized the response to the latter. Preinfusion of platelet-activating factor (PAF, 20 nM) or isoprenaline (10 microM) did not extinguish the glycogenolytic response to zymosan sup, while the response to a secondary infusion of PAF was blocked. The glycogenolytic response to zymosan sup was completely inhibited by nordihydroguaiaretic acid (NDGA, 10 microM), a lipoxygenase inhibitor, and by ONO-1078 (100 ng/ml), a leukotriene (LT) D4 receptor antagonist. On the other hand, the glycogenolytic effect of zymosan pellet (the particulate fraction of boiled zymosan) was not affected by preinfusion of zymosan sup, and was inhibited by ibuprofen (20 microM), a cyclo-oxygenase inhibitor. Prostaglandins (PGs) detected in the perfusate were augmented with infusion of zymosan pellet. Opsonization of the zymosan pellet by serum (complement) enhanced the glycogenolytic response without a lag period, and with a concomitant enhancement of PG output. Correlations between glucose production and PGs were r = 0.832 (PGD2), r = 0.872 (PGF2 alpha), r = 0.752 (PGE2) and r = 0.349 (6-oxo-PGF1 alpha). The glycogenolytic response to non-boiled zymosan was delayed and the biphasic glycogenolytic response was not observed when mannan was infused first. NDGA mimicked the effects of the preinfusion of mannan, while ibuprofen had no effect on the non-boiled-zymosan-induced glycogenolysis. These results suggest: (1) that non-boiled zymosan stimulates glycogenolysis through a mannose receptor-dependent, but unidentified, pathway, (2) that zymosan sup induces glycogenolysis via mannose receptor activation through the production of peptide-LTs but not PAF, and (3) that zymosan pellet causes glycogenolysis through the production of prostanoids, which is enhanced in the presence of complement.

Animals↗
PubMed1997

Responses of primary afferents and spinal dorsal horn neurons to thermal and mechanical stimuli before and during zymosan-induced inflammation of the rat hindpaw.

Intraplantar administration of zymosan produces inflammation and results in behavioral evidence of hyperalgesia to mechanical and thermal stimuli in the rat. In the present studies, responses of primary afferents and spinal dorsal horn neurons to mechanical and thermal stimuli were examined before and during zymosan-induced inflammation of the hindpaw. In tests of responses of primary afferents to mechanical stimuli, group mean mechanical response thresholds of C-mechanonociceptor (CMN) units significantly decreased after zymosan administration. The group mean mechanical response thresholds of low threshold mechanoreceptor (LTM) units, A-mechanoheat (AMH) units, high threshold mechanoreceptor (HTM) units, and C-mechanoheat (CMH) units showed either no change or were increased significantly by intraplantar administration of zymosan. The group mean total discharges evoked during the 10 s mechanical stimulus were significantly increased after zymosan administration in CMN units. The group mean total discharges were either significantly decreased or unchanged in LTM, AMH, HTM, and CMH units. In tests of responses of spinal dorsal horn neurons to mechanical stimuli, the group mean mechanical response threshold of nociceptive specific (NS) units decreased significantly 1 h following administration of zymosan, whereas no significant changes occurred in the mechanical response thresholds of wide dynamic range (WDR) neurons in zymosan-injected rats, WDR neurons in saline-injected rats, or NS neurons in saline-injected rats. The group mean total discharges of only NS neurons were significantly increased during the 10 s mechanical stimulus 3 and 4 h after zymosan administration. In tests of responses of primary afferents to thermal stimuli, intraplantar administration of zymosan resulted in significant decreases in group mean response thresholds of CMH units and significant increases in group mean response thresholds of AMH units. The group mean total discharges of CMH units was either unchanged or significantly increased during thermal stimuli depending on both the time of testing and the temperature of the test stimulus. The group mean total number of discharges of AMH units was significantly decreased during tests of all thermal stimuli. In tests of responses of spinal dorsal horn neurons to thermal stimuli, intraplantar administration of zymosan resulted in significant decreases in thermal response thresholds of both WDR and NS units of zymosan-injected rats, but no changes in WDR and NS units of saline-injected rats. The group mean total discharges evoked by the 15 s thermal stimuli also increased significantly in both WDR and NS units after zymosan administration. Zymosan administration resulted in increased background activity only in CMH units. These increases occurred immediately following the injection and dissipated by the first hourly test period. Significant changes in background discharges of both WDR and NS units occurred at some hourly test intervals following administration of zymosan, but these changes were not consistent with respect to either unit type or modality of the test stimulus. These data suggest that the zymosan-induced hyperalgesia to mechanical stimuli observed in behavioral studies reflects decreases in response thresholds of peripheral CMN units and spinal NS neurons. Hyperalgesia to thermal stimuli reflects decreases in response thresholds of peripheral CMH units, spinal WDR neurons, and spinal NS neurons. These data support the view that different physiological substrates mediate hyperalgesia to either thermal or mechanical stimuli following intraplantar administration of zymosan.

Afferent Pathways↗
PubMed1999

Protective effect of N-acetylcysteine on multiple organ failure induced by zymosan in the rat.

BACKGROUND AND METHODS: In the present study, we evaluated the effect of N-acetylcysteine treatment in a nonseptic shock model induced by zymosan in the rat. Animals were randomly divided into eight groups (ten animals in each group). The first group was treated with ip administration of saline solution (0.90% NaCl) and served as the sham group. The second group was treated with ip administration of zymosan (500 mg/kg suspended in saline solution). In the third and fourth groups, rats received ip administration of N-acetylcysteine (40 mg/kg; 1 and 6 hrs after administration of zymosan or saline). In the fifth and sixth groups, rats received ip administration of N-acetylcysteine (20 mg/kg; 1 and 6 hrs after zymosan or saline administration). In the seventh and eighth groups, rats received ip administration of N-acetylcysteine (10 mg/kg; 1 and 6 hrs after zymosan or saline administration). After zymosan or saline injection, animals were monitored for the evaluation of systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy), loss of body weight, and mortality for 72 hrs. Exudate formation, leukocyte infiltration, nitrate/nitrite production, lung and intestine myeloperoxidase activity and lipid peroxidation, and histologic examination were evaluated at 18 hrs after zymosan administration. RESULTS: Administration of zymosan in the rat induced acute peritonitis, as assessed by a marked increase in the leukocyte count in the exudate, as well as by an increase in the exudate nitrate/nitrite concentration. Lung and intestine myeloperoxidase activity and lipid peroxidation was significantly increased in zymosan-treated rats. This inflammatory process coincided with the damage of lung and small intestine. Peritoneal administration of zymosan in the rat also induced a significant increase in the plasma levels of nitrite and nitrate and stable metabolites of nitric oxide and in levels of peroxynitrite, as measured by the oxidation of the fluorescent dihydrorhodamine 123 at 18 hrs after zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the lung of zymosan-shocked rats. Pretreatment of zymosan-shocked rats with ip administration of N-acetylcysteine (40, 20, and 10 mg/kg, 1 and 6 hrs after zymosan) prevented the development of peritonitis and reduced peroxynitrite formation in a dose-dependent manner. In addition, ip administration of N-acetylcysteine (40 mg/kg, 1 and 6 hrs after zymosan) was effective in preventing the development of lung and intestine injury and neutrophil infiltration, as determined by myeloperoxidase evaluation. CONCLUSIONS: Taken together, the present results demonstrate that N-acetylcysteine exerts potent anti-inflammatory effects.

Acetylcysteine↗
PubMed1999

Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages--the role of prostanoids and interleukin-1.

OBJECTIVE: To study the effect of zymosan on the release of osteoclast stimulating activity from macrophages. MATERIALS: Calvarial bones from neonatal mice and peritoneal macrophages were incubated in the absence and presence of zymosan for 72 h and supernatants harvested for subsequent analysis of bone resorbing activities and prostaglandin concentration. METHODS: Bone resorption was assessed in vitro by analysing the release of 45Ca and 3H from neonatal mouse calvarial bones prelabelled in vivo by injections of [45Ca]CaCl2 or [3H]-proline. Prostaglandin E2 (PGE2) and I2 (PGI2) were analyzed using radioimmunoassays. RESULTS: Supernatants from macrophages treated with zymosan stimulated the release of 45Ca and 3H. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan (0.1-100 microg/ml), as well as the number of macrophages present. The 45Ca release induced by zymosan treated macrophages was inhibited by three different inhibitors of osteoclastic bone resorption (calcitonin, acetazolamide, amino bisphosphonate). The bone resorbing activity released by the zymosan-activated macrophages was lost after ultrafiltration using a filter with a molecular weight cut off of 30,000 Daltons, but retained when using a filter with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased during the first hour of exposure to zymosan and then reached a plateau for 96 h. PGE2 and PGI2 release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together with bone resorbing activity, was abolished by indomethacin. The bone resorbing activity present 3-72 h after zymosan exposure, however, was not inhibited by indomethacin. Bone resorption stimulated by conditioned media from zymosan treated macrophages after 3 h was inhibited by 60-75% in the presence of anti IL-1alpha, 0-20% by anti IL-1beta, and completely by antisera neutralizing both IL-1alpha and IL-1beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymosan treated macrophages. CONCLUSIONS: These data indicate that treatment of mouse peritoneal macrophages with zymosan results in production of activities capable of stimulating bone resorption in vitro. The activity released initially appears to be due to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1alpha.

Animals↗
PubMed1993

Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles.

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced CL reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.

Animals↗
PubMed1998

Effects of hyperbaric oxygen exposure on a zymosan-induced shock model.

OBJECTIVE: To evaluate the effects of hyperbaric oxygen (HBO) therapy on zymosan-induced shock in rats. Zymosan, a cell wall component of the yeast Saccharomyces cerevisiae, induces inflammation by causing the production of various cytokines and pro-inflammatory mediators. The administration of zymosan to rats represents a new experimental shock model by inducing acute peritonitis, severe hypotension, and signs of systemic illness. However, it has been recently proposed that the zymosan-induced shock, like septic shock, may be mediated by overproduction of nitric oxide. DESIGN: Experimental study. SETTING: Institute of Pharmacology and Toxicology, 2nd University of Naples, Naples, Italy. SUBJECTS: Male rats were treated with zymosan (500 mg/kg) by intraperitoneal route, with HBO (2 Absolute Atmosphere) or with zymosan and HBO (2 Absolute Atmosphere). MEASUREMENTS AND MAIN RESULTS: Peritoneal exudate, plasma, and peritoneal nitric oxide metabolites (NOx) and zymosan determined a time-dependent increase in peritoneal and plasma NOx concentrations, and peritoneal leukocytes were determined. Moreover, symptomatology was observed. The administration of zymosan caused the appearance of a severe illness in the rats characterized by ruffled fur, lethargy, conjunctivitis, diarrhea, and a significant loss of body weight. All zymosan-treated rats developed an acute peritonitis, producing turbid exudate. Zymosan determined a time-dependent increase in peritoneal, plasma NOx, and tumor necrosis factor (TNF)-alpha concentrations. Morbidity of zymosan shocked rats has been attenuated and no mortality was observed by treatment with HBO. These findings were associated with a significant reduction either of peritoneal leukocytes and exudate, or plasma and peritoneal NOx concentrations. Moreover, TNF-alpha levels were significantly reduced in animals shocked by zymosan and treated with HBO.

Animals↗
PubMed1999

Role of melatonin in reduction of lipid peroxidation and peroxynitrite formation in non-septic shock induced by zymosan.

Zymosan, a non-bacterial agent, causes inflammation by inducing the production of a variety of cytokines and pro-inflammatory mediators, wherein reactive oxygen species including nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. The current study was designed to investigate the protective effect of melatonin, a radical scavenger and antioxidant, on non-septic shock induced by zymosan in the rat. Four groups of rats (controls, melatonin-injected [5 mg/kg x 6], zymosan-injected [500 mg/kg], and zymosan + melatonin) were used in this experiment. Thiobarbituric acid reactive substances (malondialdehyde [MDA] + 4-hydroxyalkenals [4-HDA]), as an index of lipid peroxidation, were measured in the liver, lung, small intestine (ileum), kidney and pancreas. Twenty-four hours after zymosan administration, MDA + 4-HDA levels were significantly increased in the liver, lung, small intestine, and kidney while the increase in the pancreas was not statistically significant compared to levels in control rats. The percentage increases in lipid peroxidation products were 34.3%, 39.2%, 48.5%, 32.5%, and 17.4% for the liver, lung, small intestine, kidney, and pancreas, respectively. In animals given melatonin 30 minutes before zymosan, and 5 more times after zymosan (i.e., every 4 hours), the increase in MDA + 4-HDA levels was reduced in all organs studied. There was also a significant increase in the volume of peritoneal exudate in zymosan-treated rats that was reduced when the zymosan-shocked rats received melatonin. After zymosan administration, immunohistochemical and histological examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, and tissue damage in the liver, lung, and small intestine of zymosan-shocked rats. Again, melatonin treatment reduced both nitrotyrosine immunoreactivity and tissue damage associated with zymosan administration.

Animals↗
PubMed1990

Tumour necrosis factor alpha (TNF-alpha) and interleukin 6 in a zymosan-induced shock model.

TNF plays a central role in septic shock induced by endotoxin or Gram-negative bacteria. Zymosan can elicit a septic shock-like syndrome in rodents in the absence of endotoxin. TNF and IL-6 release in mice treated with zymosan was investigated. One hour after intraperitoneal zymosan injection, maximal TNF levels were measured in serum, followed by IL-6 peak levels 1 h later. Treatment with a monoclonal antibody against TNF lowered zymosan-induced mortality from 63 to 11.6%, while maximal IL-6 levels were lowered by about 40%. Mechanisms triggering zymosan-induced cytokine release in murine macrophages were analysed in vitro. Cytokine release was only slightly triggered by uncoated zymosan particles. Thirty-nine per cent of TNF release by macrophages appeared to be triggered by zymosan-bound activated complement. Maximal TNF release also required the presence of natural antibodies against zymosan and zymosan-activated serum. In contrast, maximal IL-6 release was reached upon stimulation with zymosan-activated serum only, while the presence of zymosan particles lowered this response. We conclude that TNF is a crucial mediator in zymosan-induced shock. TNF release can be induced by different immunological pathways, without the need for the direct presence of endotoxins. Although IL-6 release during septic shock is partly dependent on TNF, in vitro trigger mechanisms for IL-6 and TNF differ remarkably.

Animals↗