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PubMed1993

Differences in the activity of the alternative pathway of complement in BALB/c and C57Bl/6 mice.

The deposition of C3b on the surface of zymosan was assessed in inbred strains of mice. Mg(2+)-EGTA plasma from C57Bl/6 and BALB/c mice was incubated with zymosan and deposited C3b was eluted by methylamine. Eluted C3b was detected and quantified by Western blot and an enzyme-linked immunosorbent assay. 3- to 4-fold more C3b was deposited on zymosan incubated with C57Bl/6 plasma than on zymosan incubated with BALB/c plasma (p < 0.01). Data on the kinetics of deposition of C3b suggest a faster and greater activity of the alternative pathway of complement (ACP) in C57Bl/6 than in BALB/c. Higher C3b deposition in C57Bl/6 plasma occurs in the presence of higher plasma factor H concentration (1.3-fold of BALB/c concentration; p < 0.01) and is accompanied by higher factor H deposition on zymosan (3- to 10-fold of BALB/c concentration; p < 0.01). Demonstrable differences in the activity of the proteins of the ACP may provide a biological basis for the genetic variation in innate resistance to infectious organisms observed in inbred strains of mice.

Animals↗
PubMed1993

Role of xanthine oxidase and prostaglandins in inflammatory-induced bacterial translocation.

We have previously documented that a nonlethal dose of zymosan causes gut mucosal injury associated with increased xanthine oxidase activity and bacterial translocation. The current study was performed to investigate the role of xanthine oxidase activation and other potential mediators of intestinal injury in an LD50 zymosan model. Specific pathogen-free rats and mice were pretreated with saline, allopurinol, or ibuprofen prior to intraperitoneal injection of either saline or the LD50 dose of zymosan. Bacterial translocation to the mesenteric lymph node and systemic organs was measured at 6 or 24 hr following injection. In addition, separate animals in each group were followed for 7 days for survival. Pretreatment with allopurinol or ibuprofen reduced both the incidence and the magnitude of translocation at 6 hr in rats and mice (P < 0.05). In the rats pretreated with allopurinol or ibuprofen, no reduction in the incidence or magnitude of translocation occurred at 24 hr. In the mice pretreated with allopurinol or ibuprofen, although the incidence of translocation was not reduced at 24 hr, the magnitude of translocation was reduced (P < 0.05). Pretreatment with allopurinol or ibuprofen also resulted in an improvement in survival, when compared to zymosan alone (P < 0.01). Allopurinol and ibuprofen provide protection against bacterial translocation and improvement in survival following challenge with a lethal dose of zymosan.

Allopurinol↗
PubMed1998

Stem cell factor-deficient mice have a dysregulation of cytokine production during local inflammation.

We have studied the responses of stem cell factor-deficient (SCF-) mice in two models of local inflammation, turpentine-induced tissue damage and zymosan-induced peritonitis. These mice have a profound mast cells deficiency. Following administration of either zymosan or turpentine, SCF- mice developed a more severe body weight loss than wild type (SCF+) mice. An exaggerated systemic production of interleukin-1beta (IL-1beta) and IL-6 was observed in SCF- mice. However, systemic tumor necrosis factor-alpha (TNF-alpha) levels were undetectable in SCF- mice following zymosan administration. The peritoneal lavage fluid (PLF) of SCF- mice also showed a marked increase in IL-1beta following zymosan administration, whereas local IL-6 and TNF-alpha production were not affected. In addition, PLF levels for the chemokine MIP-1alpha were strongly reduced in SCF- mice. This was associated with a reduced number of infiltrating cells. A possible mechanism for these differences was a reduction in IL-4 production in SCF- mice. Both local and systemic levels for IL-4 were significantly lower in SCF- mice following either zymosan or turpentine administration. Our results suggest that mast cells and/or SCF are important regulators of the production of cytokines in the course of inflammatory responses.

Animals↗
PubMed1982

Chemotaxis in yersinia arthritis. In vitro stimulation of neutrophil migration by HLA--B27 positive and negative sera.

Chemotactic and chemokinetic migration of polymorphonuclear leukocytes in sera from patients with previous yersinia arthritis and from healthy subjects with or without HLA--B27 were studied by the leading front method. Irrespective of yersinia arthritis, zymosan-activated sera from subjects who were HLA--B27 positive were significantly more chemokinetic than were HLA--B27 negative zymosan-activated sera from healthy control subjects. The chemotactic activities of zymosan-activated sera that were HLA--B27 positive or negative, as determined by chemotactic increments, were much the same. The results suggest that zymosan-activated serum that is HLA--B27 positive stimulates random migration of polymorphonuclear leukocytes, but not their directional migration, more than does HLA--B27 negative serum that has been zymosan activated. This may contribute to accumulation of polymorphonuclear leukocytes at the site of inflammation in vivo and thereby to inflammatory symptoms in yersinia arthritis patients with HLA--B27.

Adult↗
PubMed2002

A low-morbidity murine model of peritonitis.

PURPOSE: Peritonitis continues to be a major source of mortality and morbidity in patients undergoing abdominal surgery. The aim of this study was to develop a nonfatal model of bacterial peritonitis in mice so that we could study aspects of the pathobiology and treatment of peritonitis in an in vivo model. METHODS: Mice were inoculated via a midline laparotomy with 0.5 mg of zymosan in 0.1 ml of saline into the subomental space. At 24, 48, and 96 hours after treatment, animals were killed, and analysis was performed to determine the degree of peritoneal inflammation. End points included intraperitoneal cellular influx, tumor necrosis factor-alpha concentrations, and myeloperoxidase activity. In addition, peritoneal lavage fluid was plated onto blood agar for analysis of bacterial colony-forming units. There were 40 mice in each group. RESULTS: There were no deaths in either group. Facultative Gram-negative bacteria were cultured from the peritoneal cavities of zymosan-treated animals at 24 and 48 hours after insult (colony-forming unit counts of 92+/-11 vs. 0 in control animals). In the zymosan-treated animals, there were significantly increased numbers of inflammatory cells (especially neutrophils) in the peritoneal cavity at 24 and 48 hours after treatment; these numbers returned to control levels by 96 hours. Myeloperoxidase activity was also elevated both in the peritoneal fluid (2.4 X 10(-4) units/ml compared with 1.6 x 10(-4) units/ml, P < 0.05) and in remote organs (i.e., lung, P < 0.05; liver, P < 0.001; and kidney, P < 0.05) at 24 hours after treatment. There was no significant difference in tumor necrosis factor-alpha levels between zymosan-treated and control mice in either serum or peritoneal fluid at any time point investigated. There was no mortality in either the zymosan-treated or control animals. CONCLUSION: In this model of bacterial peritonitis in mice, we have demonstrated how the peritoneal cavity can resolve a relatively localized inflammatory insult within 96 hours of induction. This response is characterized by a cellular influx of predominantly neutrophils and macrophages and by pronounced oxidative activity. We will use this in vivo model to characterize aspects of the pathobiology and treatment of peritonitis.

Animals↗
PubMed2003

Modulation of hepatic granulomatous responses by transgene expression of DAP12 or TREM-1-Ig molecules.

DAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-beta1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes. The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response. In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.

Acridines↗
PubMed1984

Chemiluminescence of macrophages depends upon their differentiation stage: dissociation between phagocytosis and oxygen radical release.

The in vitro differentiation and maturation of resident and activated mouse and human macrophages (M phi) from different anatomical sources was investigated with regard to their oxygen metabolism during zymosan phagocytosis. We found evidence that chemiluminescence (CL) of M phi depends upon their differentiation stage: a) In the absence of any phagocytic stimulus, the human M phi showed a lucigenin-dependent CL background that was approximately 10-fold higher than in mouse M phi and decreased to low levels in resident M phi (monocyte-derived human M phi). This background was reduced by SOD to about 50%. No relevant luminol-dependent background was observed in all mouse and human M phi during culture time. b) Resident and activated mouse and human M phi could be distinguished in terms of their lucigenin-dependent CL during zymosan phagocytosis, which was persistently high in activated M phi, but decreased to comparatively low levels in resident M phi during culture time. This zymosan-elicited CL was almost completely SOD-dependent during all culture time. c) A dissociation between phagocytosis and oxygen radical release is observed: the decrease of both minolul and lucigenin-dependent CL in resident phagocytizing M phi during maturation did not correspond to a decrease of their phagocytic activity. Phagocytosis occurred at a high rate also in the absence of a relevant CL-detectable generation of oxygen radicals. The oxygen radical release, as measured by SOD-inhibitable cytochrome c reduction, paralleled CL during zymosan phagocytosis and declined with maturation of monocytes into M phi. In contrast, the zymosan-induced nitro-blue-tetrazolium reduction increased in mature resident human M phi. Thus, it seems that different metabolic pathways are utilized during phagocytosis in young and mature M phi.

Animals↗
PubMed1996

Mitochondrial protein content and in vivo synthesis rates in skeletal muscle from critically ill rats.

1. Recently we reported decreased activities of two mitochondrial marker enzymes (citrate synthase and cytochrome c oxidase) in skeletal muscle from a rat model of critical illness (zymosan injection). In the present study we investigated (i) whether these decreases in enzyme activity reflect a reduction in mitochondrial content and (ii) whether this potential reduction in mitochondrial content was the result of decreased mitochondrial protein synthesis rates. 2. Mitochondrial protein content was calculated from the activities of cytochrome c oxidase in whole-muscle homogenates and purified mitochondria. Synthesis rates of mitochondrial protein in vivo were studied by measuring the incorporation of [3H]phenylalanine into mitochondrial protein using the flooding dose technique. 3. Mitochondrial protein content was reduced to 54% of that measured in the pair-fed rats and to 71% of that measured in control rats fed ad libitum 2 days after the zymosan treatment. The decreased mitochondrial protein content observed 2 days after zymosan challenge was preceded by a reduced rate of synthesis of mitochondrial protein 16h after treatment. Both changes were of greater magnitude than the general muscle wasting and the decreased rate of synthesis of mixed protein observed in the zymosan-treated rats. 4. We conclude that the acute phase of critical illness in zymosan-treated rats is characterized by a substantial reduction in muscle mitochondria that is at least in part caused by a decreased rate of synthesis of mitochondrial protein. This derangement in mitochondrial protein metabolism may be related to the impaired muscle function observed during and after critical illness.

Animals↗
PubMed1987

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils.

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.

Adult↗
PubMed1984

A selective defect in arachidonic acid release from macrophage membranes in high potassium media.

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.

Animals↗
PubMed1989

Measurement of complement activation in rabbit plasma or serum using monoclonal antibodies against C5a.

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan-induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen-antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan-induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat-inactivated plasma. A cross-reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme-linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan-induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan-activated serum. MoAb 2A5E3 was able to bind 125I-labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen-antibody complexes, or cobra venom factor.

Animals↗
PubMed1996

Acute inflammatory response in the mouse: exacerbation by immunoneutralization of lipocortin 1.

1. An immuno-neutralization strategy was employed to investigate the role of endogenous lipocortin 1 (LC1) in acute inflammation in the mouse. 2. Mice were treated subcutaneously with phosphate-buffered solution (PBS), non-immune sheep serum (NSS) or with one of two sheep antisera raised against LC1 (LCS3), or its N-terminal peptide (LCPS1), three times over a period of seven days. Twenty four hours after the last injection several parameters of acute inflammation were measured including zymosan-induced inflammation in 6-day-old air-pouches, zymosan-activated serum (ZAS)-induced oedema in the skin, platelet-activating factor (PAF)-induced neutrophilia and interleukin-1 beta (IL-1 beta)-induced corticosterone (CCS) release. 3. At the 4 h time-point of the zymosan inflamed air-pouch model, treatment with LCS3 did not modify the number of polymorphonuclear leucocytes (PMN) recruited: 7.84 +/- 1.01 and 7.00 +/- 0.77 x 10(6) PMN per mouse for NSS- and LCS3 group, n = 7. However, several other parameters of cell activation including myeloperoxidase (MPO) and elastase activities were increased (2.2 fold, P < 0.05, and 6.5 fold, P < 0.05, respectively) in the lavage fluids of these mice. Similarly, a significant increase in the amount of immunoreactive prostaglandin E2 (PGE2; 1.81 fold, P < 0.05) and IL-1 alpha (2.75 fold, P < 0.05), but not tumour necrosis factor-alpha (TNF-alpha), was also observed in LCS3-treated mice. 4. The recruitment of PMN into the zymosan inflamed air-pouches by 24 h had declined substantially (4.13 +/- 0.61 x 10(6) PMN per mouse, n = 12) in the NSS-treated mice, whereas high values were still measured in those treated with LCS3 (9.35 +/- 1.20 x 10(6) PMN per mouse, n = 12, P < 0.05). A similar effect was also found following sub-chronic treatment of mice with LCPS1: 6.48 +/- 0.10 x 10(6) PMN per mouse, vs. 2.77 +/- 1.20 and 2.64 +/- 0.49 x 10(6) PMN per mouse for PBS- and NSS-treated groups (n = 7, P < 0.05). Most markers of inflammation were also increased in the lavage fluids of LCS3-treated mice: MPO and elastase showed a 2.47 fold and 17 fold increase, respectively (P < 0.05 in both cases); TNF-alpha showed a 11.1 fold increase (P < 0.05) whereas the IL-1 alpha levels were not significantly modified. PGE2 was still detectable in most (5 out of 7) of the mice treated with LCS3 but only in 2 out of 7 of the NSS-treated mice. 5. Intradermal injection of 50% ZAS caused a significant increase in the 2 hoedema formation in the skin of LCS3-treated mice in comparison to PBS- and NSS-treated animals: 16.7 +/- 1.5 microliters vs. 10.8 +/- 1.2 microliters and 10.2 +/- 1.0 microliters, respectively (n = 14 mice per group, P < 0.05). ZAS-induced oedema had subsided by 24 h in control animals but a residual significant amount of extravasation was still detectable in LCS3-treated mice: 4.4 +/- 0.8 microliters (P < 0.05). 6. A recently described model driven by endogenous glucocorticoids is the blood neutrophilia observed following administration of PAF. In our experimental conditions, a single bolus of PAF (100 ng, i.v.) provoked a marked neutrophilia at 2 h (2.43 and 2.01 fold) in NSS- and PBS-treated mice (n = 11), respectively, which was significantly attenuated in the animals treated with LCS3: 1.26 fold increase in circulating PMN (n = 11, P < 0.01 vs. NSS- and PBS-groups). 7. Intraperitoneal injection of IL-1 beta (5 micrograms kg-1) caused a marked increase in circulating plasma CCS by 2 h, to a similar extent in all experimental groups. In contrast, measurement of CCS levels in the plasma of mice bearing air-pouches inflamed with zymosan revealed significant differences between LCS3 and NSS-treated mice at the 4 h time-point: 198 +/- 26 ng ml-1 vs. 110 +/- 31 ng ml-1 (n = 8, P < 0.05). 8. In conclusion, we found a remarkable exacerbation of the inflammatory process with respect to both humoral and cellular components in mice passively immunised agains

Animals↗
PubMed1982

Suppression of natural killing in vitro by monocytes and polymorphonuclear leukocytes: requirement for reactive metabolites of oxygen.

Natural killer cells spontaneously lyse certain tumor cells and may defend against malignancy. We have previously shown that natural killing (NK) by human peripheral blood mononuclear cells (PBMC) is suppressed in vitro by phorbol diester tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We here demonstrate that suppression of NK is mediated by monocytes or polymorphonuclear leukocytes (PMN) and that suppression is dependent on the generation of reactive forms of molecular oxygen (RO), particularly hydrogen peroxide (H2O2). NK was suppressed not only by TPA but also by opsonized zymosan (yeast cell walls), which, like TPA, was not toxic to PBMC. Both TPA and zymosan stimulated the production of superoxide anion (O2-) and H2O2 by PBMC. Production of RO correlated with suppression of NK. When PBMC were depleted of monocytes, the production of RO and the suppression of NK were both markedly reduced. Suppression could be restored by monocytes or PMN, both of which produced RO in response to TPA or zymosan. Suppression of NK was dependent on RO. Monocytes or PMN from a patient with chronic granulomatous disease, whose cells cannot generate RO, did not mediate suppression of NK. Suppression was also reduced in glucose-free medium, which did not support the generation of RO. Suppression of NK by TPA was inhibited by catalase. Bovine superoxide dismutase had a limited effect on suppression, even in high concentration, and tyrosine-copper (II) complex, which also enhances dismutation of O2- to H2O2, had almost no effect on suppression. When H2O2 was directly generated enzymatically from glucose oxidase and glucose, NK was suppressed and suppression was reversed by catalase. NK was also suppressed by the enzymatic generation of O2- from xanthine oxidase and xanthine, but suppression under these conditions was again inhibited by catalase and not by superoxide dismutase, indicating that suppression was due to the secondary formation of H2O2 from O2-. These results indicate that H2O2 is important in suppression of NK. Myeloperoxidase did not appear to play a role in suppression because inhibition of this enzyme by sodium azide, cyanide, or aminotriazole did not prevent suppression of NK. Suppression of NK was reversible; after exposure to zymosan, NK could be partially restored by the addition of catalase and superoxide dismutase or by the removal of zymosan. These studies demonstrate cellular regulation of NK by monocytes or polymorphonuclear leukocytes and indicate a role for RO in immunoregulation.

Cell Communication↗
PubMed2001

Superoxide anion generation in human milk macrophages: opsonin-dependent versus opsonin-independent stimulation compared with blood monocytes.

Macrophages are believed to play an important role within the immunoprotective effects of human breast milk. It was the purpose of this study to evaluate the capability of human milk macrophages (MMPhi) to generate superoxide anions (O2(-)) in comparison with peripheral blood monocytes (BMo) after stimulation with opsonized and unopsonized zymosan. Potential inhibitors of attachment and phagocytosis such as mannose and cytochalasin B were used. Expression of the mannose receptor on MMPhi was demonstrated by staining with MAb. BMo generated more O2(-) than MMPhi (417 +/- 79 versus 216 +/- 15 nmol O2(-)/mg protein, p < 0.05) after stimulation with opsonized zymosan. When unopsonized zymosan was used as a serum-independent stimulus, BMo generated slightly less O2(-) in comparison with MMPhi (150 +/- 34 versus 176 +/- 18 nmol O2(-)/mg protein, p < 0.05). These findings imply a higher proportion of opsonin-independent phagocytosis in MMPhi than in BMo (82 versus 36 %). Preincubation with mannose resulted in a significantly higher reduction of O2(-) generation in MMPhi compared with BMo stimulated with opsonized zymosan, whereas no difference was found when unopsonized zymosan was used. After addition of cytochalasin B, equal inhibition of O2(-) generation was observed regardless of the cell type or stimulus used. Thus, MMPhi are stimulated to a greater extent by serum-independent mechanisms than BMo. As opsonins like complement or IgG are rare in the colostrum and the neonatal intestinal environment, such a differentiation toward serum-independent phagocytic abilities could play an important role for protective functions of human MMPhi. Possible involvement of the mannose receptor and the beta-glucan receptor in this specialization are discussed.

Cells, Cultured↗
PubMed2000

Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins.

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or beta-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N:-acetyl-D-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.

Acetylglucosamine↗
PubMed1988

Systemic complement activation produces hemodynamic changes characteristic of sepsis.

Zymosan was administered intravenously in graded doses to Sprague-Dawley rats to investigate the hemodynamic effects of systemic complement activation. At two hours, thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, hematocrit, effective hepatic and renal blood flows, and percent change in total hemolytic complement activity were measured on all animals. Progressively increasing doses of zymosan produced characteristic hemodynamic changes of progressively deteriorating stages of hyperdynamic sepsis. In addition, complement activation resulted in a redistribution of systemic blood flow with hepatic hypoperfusion similar to that seen in sepsis. Renal blood flow was unaffected early after complement activation. Additional rats were studied from the control and a representative zymosan-treated group at 24 and 48 hours to determine if the hemodynamic changes observed at two hours persisted or resolved. All systemic hemodynamic measures returned to normal at both 24 and 48 hours. Liver blood flow, however, remained depressed and actually deteriorated over time. Renal perfusion, which was stable at both two and 24 hours, fell below control values in the zymosan-treated group at 48 hours. We conclude that complement may be a mediator of both systemic and visceral flow abnormalities that precede, and perhaps precipitate, organ failure in trauma and sepsis.

Animals↗
PubMed1989

Development of a bacteria-independent model of the multiple organ failure syndrome.

Criteria that allow definition of the multiple organ failure syndrome include pulmonary, hepatic, renal, and gut barrier dysfunction along with characteristic histopathologic changes. It has been difficult to study multiple organ failure due to lack of a satisfactory experimental model that would reproduce the pathophysiologic and histopathologic characteristics, would be stable enough to allow study over several days, and would be free of exogenous primary bacterial infection. We have studied pathophysiologic and histopathologic alterations in a potential model of multiple organ failure. Wistar rats received one of the following solutions by intraperitoneal injection: 4 mL of saline, 4 mL of mineral oil, or 1 mg per gram of body weight of zymosan A in 4 mL of mineral oil. Animals that received zymosan developed hypoxia, decreased creatinine clearance, and changes in hepatic microsomal cytochrome P450 content and aniline hydroxylase activity. Bacterial translocation occurred in the zymosan group. The lungs, liver, and kidneys of the animals that received zymosan exhibited histopathologic changes. We conclude that this model fulfills our criteria for a model of multiple organ failure.

Animals↗