Guinea Pig Peritoneal Macrophages Research Articles

Assessment of Toxic and Immunoadjuvant Properties of Nanocomposites

Introduction. The current direction in medicine is the creation of immunobiological preparations to increase the effectiveness of specific immunotherapy and immunoprophylaxis, containing immunomodulators in their composition. These natural or synthetic substances can have a regulating effect on the immune system. At present, water-soluble organic-inorganic polymeric materials with nanoparticles of various chemical substances with bactericidal and immunomodulating properties can serve as such promising compounds.The aimof the work is to study the acute toxicity of polymer nanocomposites based on 1-vinil-1,2,4-triazole copolymer with N-vinylpyrrolidone with silver, gold and selenium nanoparticles and their effect on the functional state of immune system cells in vitro.Materials and methods.The study of acute toxicity was performed on outbred white mice. The activity of superoxide dismutase and glucose-6-phosphate dehydrogenase were studied in guinea pig peritoneal macrophages. The study of spontaneous and nanocomposite-induced production of pro- (interferon gamma and tumor necrosis factor alpha) and anti-inflammatory (interleukin-4) cytokines by blood cells was carried out using clinical material obtained from volunteers using the ELISA method. Results. It has been established that nanocomposites with silver and gold nanoparticles do not cause the death of white mice, their temperature increase and body weight decrease. The average lethal dose for a nanocomposite with selenium nanoparticles was determined as 1 gram per 1 kilogram of animal mass. It was shown that the tested nanocomposites have a stimulating effect on the production of cytokines by human blood cells in vitro. It was established that a nanocomposite with selenium nanoparticles increases the activity of superoxide dismutase and glucose-6-phosphate dehydrogenase. A comparative analysis of their actions with the actions of commercial preparations of biological origin, with immunomodulatory properties.Conclusion. The data obtained allow us to substantiate the need for further research on the effects of nanocomposites based on 1-vinyl-1,2,4-triazole copolymer with N-vinylpyrrolidone with silver, gold and selenium nanoparticles on the macroorganism in both in vitro and in vivo conditions.

Effects of in Vitro Exposure to Chrysotile Particles or Sodium Nitrite on the Production of Eosinophil Chemotactic Substance by Guinea Pig Peritoneal Macrophages

Effects of in Vitro Exposure to Chrysotile Particles or Sodium Nitrite on the Production of Eosinophil Chemotactic Substance by Guinea Pig Peritoneal Macrophages

Eosinophils adhere to and stimulate replication of lung fibroblasts ‘in vitro’

Eosinophils have been implicated in several disorders associated with the development of fibrosis. This led us to investigate the interactions between eosinophils and fibroblasts in vitro. Adhesion between purified guinea pig peritoneal eosinophils and monolayers of human fetal lung fibroblasts was assessed using the rose bengal dye staining assay. Fibroblast replication was assessed using a colorimetric assay based upon the uptake and subsequent release of methylene blue. Addition of phorbol myristate acetate induced a rapid, time-dependent increase in eosinophil adhesion (127% and 328% over basal adhesion after 10 and 30 min, respectively). Phorbol myristate acetate-induced adhesion was inhibited by the peptides RGDS and GRGDS (48% and 42%, respectively using 1 mM peptide) and by nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism (46% inhibition at 15 microM). In addition, 24 h culture of fibroblast monolayers with interleukin 1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha) resulted in enhanced adhesion (10 U/ml IL-1 alpha stimulated adhesion by 55% of control, 500 U/ml TNF alpha by 75% of control). Conditioned media from cultured eosinophils stimulated fibroblast replication in a time-dependent fashion with maximal stimulation at 3 h. In contrast, media from guinea pig peritoneal macrophages in culture did not show such an effect. This study indicates that eosinophils are capable of both adhering to and releasing mitogens for fibroblasts in vitro. These observations suggest that eosinophils have the capacity to play a role in the development of fibrosis in disorders where they have been shown to be present.

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig.

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig.

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.

Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant

Mucosal immune responses are known to play important roles in the establishment of protective immunity to microbial infections through mucosa. We examined the toxic effects of recombinant cholera toxin B subunit (rCTB) secreted by Gram-positive bacterium Bacillus brevis as a mucosal adjuvant. Incubation of guinea-pig peritoneal macrophages with cholera toxin (CT) or aluminium hydroxide gel (Al-gel) released a significantly higher activity of lactate dehydrogenase than did commercial natural CTB (CTB) or rCTB. Intraintestinal or intramuscular administration of CT, CTB or Al-gel caused severe histopathological reactions. CT also caused infiltration of neutrophils and irregular arrangement or partial loss of the respiratory epithelium. In addition, CT and CTB elicited vascular permeability-increasing effects. rCTB elicited no toxic effects to macrophages and no vascular permeability-increasing effects. Moreover, it is noticeable that no distinct local histopathological reactions were observed in the nasal cavity, the small-intestinal loop or the muscle given rCTB. These results suggest that, from a safety standpoint, rCTB is a useful candidate as mucosal vaccine adjuvant.

Influence of interleukin-1 beta on bradykinin-induced responses in guinea pig peritoneal macrophages.

The endogenous nonapeptide bradykinin is implicated in the pathogenesis of a number of inflammatory diseases. Recently, it could be shown that bradykinin is a potent stimulus for the generation of arachidonic acid, prostaglandin E2 (PGE2) and superoxide radical via the bradykinin B2 receptor from macrophages depending on their stage of maturation or activation. The present study was designed to characterize the effect of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) on responsiveness of macrophages to bradykinin. Guinea pig peritoneal macrophages were collected 7 days after i.p. injection of paraffinum subliquidum. The bradykinin-induced increase in cytosolic free calcium concentration ([Ca2+]i) and activation of the respiratory burst were significantly enhanced by interleukin-1 beta (100 U/ml) added to cultures of macrophages 24 h before stimulation with bradykinin. However, the EC50 values of bradykinin-induced calcium signal of 23 +/- 8 nM and 29 +/- 17 nM and superoxide radical formation of 64 +/- 22 nM and 34 +/- 29 nM in control and interleukin-1 beta-treated cells, respectively, were not changed. In contrast to the influence of IL-1 beta on these functions, saturation and competition experiments measured by binding of [3H]bradykinin ([3H]BK) to intact macrophages demonstrate that cell exposure to interleukin-1 beta for 24 h caused no change in the density and affinity of the bradykinin B2 receptors. The most likely explanation for the cytokine effects is an influence on the level of the receptor-effector coupling downstream of the agonist binding.

Interaction of IgG immunoglobulins with the guinea pig peritoneal macrophage Fcγ receptors. Effect on the association of the receptors with the membrane skeleton and the cytoskeleton

Binding of ligands to cell surface receptors may induce an interaction of the receptors with the cytoskeleton and/or membrane skeleton and decrease the solubility of the receptors in nonionic detergents. Cytochalasins, reagents affecting the structure of microfilaments, inhibit some cell functions induced by cross-linking of the receptors with ligands. Information concerning the function of the cytoskeleton in insolubilization of Fcγ receptors (FcγR) and in FcγR-mediated signal transmission is rather limited. The aim of this work was to investigate the effect of binding of homologous (guinea pig IgG1 and IgG2) and heterologous (rabbit IgG) immunoglobulins to guinea pig peritoneal macrophages on association of the macrophage Fcγ receptors with the membrane skeleton and cytoskeleton. Cross-linking the macrophage Fcγ receptors with immunoglobulin ligands induced insolubilization of the receptors in nonionic detergents suggesting association of the receptors with the membrane skeleton and the cytoskeleton. The ligands showed differential effects depending on a subclass and origin of the IgG used. The process of association of the Fcγ receptors with the skeletons was fast and did not depend on temperature. Treatment of insoluble complexes with cytochalasin D, DNAse I or colchicine showed that actin microfilaments and microtubules play a role, at least partially, in insolubilization of the cross-linked macrophage Fcγ receptors. Inhibition of insolubilization of the macrophage Fcγ receptors by genistein indicated that tyrosine kinases are involved in the process of insolubilization. The association with the skeletons might be a part of the process of transduction of a signal which depended on the subclass and origin of IgG used and on the type of the Fcγ receptor.

Characterization of phosphodiesterase 4 in guinea‐pig macrophages: multiple activities, association states and sensitivity to selective inhibitors

1. The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated. 2. Differential centrifugation of macrophage lysates revealed that approximately 90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC50s of 240 and 540 nM, respectively. 3. Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 microM), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 microM), but were markedly suppressed by RS-rolipram (10 microM). The two peaks of PDE activity were arbitrarily designated CPPDE4alpha and CPPDE4beta with respect to the order from which they were eluted from the column where the prefix, CP, refers to the species, Cavia porcellus. 4. The hydrolysis of cyclic AMP catalyzed by CPPDE4alpha and CPPDE4beta conformed to Michaelis-Menten kinetic behaviour with similar K(m)s (13.4 and 6.4 microM, respectively). 5. Thermal denaturation of membrane-bound PDE4 at 50 degrees C followed bi-exponential kinetics with t1/2 values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4alpha and CPPDE4beta each decayed mono-exponentially with significantly different thermostabilities (t1/2 = 2.77 and 1.15 min, respectively). 6. Gel filtration of CPPDE4beta separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a approximately 180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4alpha resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa. 7. Of ten PDE inhibitors examined, none distinguished CPPDE4alpha from CPPDE4beta with respect to their IC50 values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K(i)s of 2 microM and 1.5 microM for CPPDE4alpha and CPPDE4beta, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4alpha and CPPDE4beta. 8. In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive. 9. These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.

Recombinant guinea pig and human RANTES activate macrophages but not eosinophils in the guinea pig.

To characterize the biologic activities of potential mediators of allergic inflammation, we have cloned, expressed, and purified guinea pig RANTES (gpRANTES). cDNA for gpRANTES was cloned from Con A-stimulated guinea pig spleen cells. A high level of gpRANTES expression in Escherichia coli was achieved by mutation of a human RANTES (hRANTES) expression construct to obtain a 68-amino acid protein identical with the predicted guinea pig amino acid sequence, assuming an equivalent amino terminus as the human protein. Purified gpRANTES was an effective stimulus of human eosinophils as assessed by increases in intracellular free calcium in fura-2-loaded cells and chemotactic responses in vitro. gpRANTES exhibits similar potency and efficacy to hRANTES. In marked contrast, neither gpRANTES nor hRANTES was able to activate guinea pig peritoneal eosinophils in these assays, even in the presence of IL-5. However, gpRANTES was found to be a potent stimulator of guinea pig peritoneal macrophages. Following tracheal instillation of gpRANTES, a dose-dependent increase in macrophages, but not eosinophils, was observed in gpBAL. Macrophage accumulation was detectable by 6 h and sustained for at least 48 h. These results indicate that RANTES in the guinea pig may have a different cellular selectivity than that described in the human, which may be important in the use of animal models in the analysis of allergic disorders. These selectivities do not appear to be accounted for by differences in guinea pig and human RANTES sequences.

Role of Tyrosine Phosphorylation in the Signalling of Superoxide Anion Generation in Platelet-Activating-Factor-Stimulated Peritoneal Macrophages

The signalling pathway(s) involved in the activation of superoxide anion (O^–₂) generation by platelet-activating factor (PAF) are not well understood, But elevations of sn-l,2-diacylglycerol and intracellular calcium contribute to activation by stimulating protein kinase C activity. Since recent studies have suggested that tyrosine phosphorylation may also be a component of the signalling of O^–₂ generation in human neutrophils, we have investigated whether PAF stimulates tyrosine phosphorylation in guinea pig and rat macrophages. PAF (10 nM) stimulated tyrosine phosphorylation within 10–30 s, coinciding with the onset of the O^-₂ generation in guinea pig peritoneal macrophages. At 30 min after exposure of guinea pig macrophages to PAF, the phosphorylation levels were similar to those of unstimulated cells. In contrast, in rat peritoneal macrophages, PAF did not stimulate tyrosine phosphorylation of high molecular weight proteins, nor did it elicit the generation of O^–₂. PAF-induced O^–₂ generation was inhibited by pre-treatment of guinea pig peritoneal macrophages with the protein tyrosine kinase inhibitors methyl 2, 5-dihydroxymethylcinnamate and tyrphostin. The qualitative difference between PAF-induced phosphorylation patterns in rat and guinea pig macrophages and the inhibitory effects of protein tyrosine kinase inhibition are consistent with an important role for protein tyrosine kinase(s) in O^–₂ generation.

Negative correlation between maorophage proooagulant and migraion ability in the course of inflammation

The importance of macrophage procoagulant activity (PCA) to cell migration is presumed. In this study we assayed the relationship between the two functions in guinea-pig peritoneal resident macrophages and cells elicited by a sterile inflammation induction, which lasted up to 6 days. The findings pointed to an in vivo induction of PCA in macrophages, which declined with time during inflammation. A clear negative correlation between PCA and random migration ability was demonstrated. Our results suggest that the local induction of coagulation by macrophages may immobilize the cells at the site of inflammation.

Collagen-like complement component C1q is a membrane protein of human monocyte-derived macrophages that mediates endocytosis.

The collagen-like C1q molecule, a subcomponent of the first component of complement, C1, is synthesized by macrophages (M phi). Previously, we have demonstrated that C1q is a membrane protein of guinea pig peritoneal macrophages (M phi). To extend this observation as a general biologic characteristic of M phi, we investigated human (hu) monocyte-derived M phi. Interestingly, surface labeling with the biotin derivative sulfosuccinimidyl-6-(biotinamido)-hexanoate of M phi, freshly isolated monocytes, lymphocytes, granulocytes, and myelomonocytic U937 cells revealed that C1q occurs only on the surface of M phi and not on the surface of the other cells types. Therefore, C1q appears to be a marker for differentiation into M phi. FITC-labeled, fixed Staphylococcus aureus coupled to membrane C1q via a monoclonal alpha-hu-C1q Ab were used to demonstrate that membrane C1q is capable of mediating phagocytosis. Various detergents (Nonidet P-40, digitonin, lubrol, and Triton X-114) were used to solubilize membrane C1q. Membrane C1q of hu M phi is tightly bound to or located in the intact membrane, since treatment of cells with acidic buffers ("acid strip") failed to remove C1q from the cell surface. However, repeated freezing and thawing of cells and washing of segregated membranes with buffer containing 1 M KCl and 3 M urea brought about a marked release of membrane C1q.

Isoprenaline induction of cAMP-phosphodiesterase in guinea-pig macrophages occurs in the presence, but not in the absence, of the phosphodiesterase type IV inhibitor rolipram

The long-term effects of incubating freshly isolated, elicited guinea-pig peritoneal macrophages with the β-adrenoceptor agonist isoprenaline and the selective inhibitor of phosphodiesterase (PDE) IV rolipram, on adenosine-3′,5′-cyclic phosphate (cAMP)-specific PDE IV activity have been investigated. The level of cAMP PDE activity in macrophages was unaffected by long-term exposure of cells to rolipram alone. In contrast, in the presence of isoprenaline (10 μM), a concentration-related (0.05–50 μM) increase in cAMP PDE activity was observed in the cytosolic and paniculate fractions. Incubation with isoprenaline alone did not affect macrophage cAMP PDE. cAMP PDE activities in homogenates of control cells and macrophages treated with isoprenaline (10 μM) and rolipram (5 μM) (3-fold activation) were inhibited by the selective PDE IV inhibitor, rolipram, with similar potencies (IC 50: 2—3 μM). The increase in cAMP PDE activity in response to rolipram and isoprenaline was completely blocked by cyclohexamide (10 μg/ml). Incubating macrophages for 10 min with rolipram increased cAMP accumulation in the presence, but not in the absence, of isoprenaline (10 μM) over the same concentration range that induction of cAMP PDE activity was observed. cAMP levels remained elevated for at least 1 hr. Isoprenaline (10 μM) alone induced a transient elevation in cAMP levels that peaked at 2 min and had returned to basal levels by 10 min. Protein kinase A activity (PKA) was increased almost 10-fold (at 10 min) by exposing cells to rolipram plus isoprenaline and remained elevated for at least 4 hr. Isoprenaline alone induced a small (2-fold) increase in PKA activity and rolipram alone was without effect.

Bradykinin receptors and signal transduction pathways in peritoneal guinea pig macrophages

The presence of a bradykinin receptor on guinea pig peritoneal macrophages was evidenced by binding studies and by the effect of bradykinin on activation of the phospholipase C and the increase in intracellular calcium concentration ([Ca 2+] i). Binding studies demonstrated a specific, saturable binding for [ 3H]bradykinin inhibited by the bradykinin B 2 (HOE 140) but not bradykinin B 1 (des-Arg 9[Leu 8]bradykinin) receptor antagonist. Scatchard analysis revealed a single class of B 2 bradykinin binding sites with a binding affinity (k d) of 0.8 nM and a receptor concentration ( B max) of 35 fmol/5 × 10 6 cells, representing approximately 4000 bradykinin receptors per cell. Kinetic studies confirmed the presence of this single binding site by the determination of similar binding affinity. Activation of peritoneal macrophages by bradykinin resulted in a time-and dose-dependent release of inositol phosphates determined by anion exchang chromatography and intracellular calcium analyzed using fura-2/AM. The increase in [Ca 2+] i induced by bradykinin was blocked by the specific bradykinin B 2 receptor antagonist HOE 140 but not by the bradykinin B 1 receptor antagonist des-Arg 9[ These studies provide novel information regarding the nature of kinin receptors on guinea pig peritoneal macrophages and their signal transduction pathways.

Guinea pig peritoneal macrophages. Differential effects of lectins on interaction with IgG immunoglobulins

Guinea pig peritoneal macrophages have on their surface two receptors, one (Fc gamma 1/gamma 2 R) binding both guinea pig IgG1 and IgG2 and the second (Fc gamma 2R) binding only IgG2 immunoglobulins. We have previously shown that treatment of macrophages with neuraminidase or glycosylation inhibitors affects, in a different way, the binding of guinea pig IgG1, IgG2, and rabbit IgG. In the present study we have shown that pretreatment of guinea pig macrophages with lectins (Con A, WGA, and PNA) also has a different effect on the interaction of the cells with IgG. The lectins increased the binding of guinea pig IgG1, whereas rabbit IgG and guinea pig IgG2 were bound with a lower efficiency than in the case of control cells. Since sialic acid residues seem to modulate the activity of receptors and WGA interacts with sialylated oligosaccharides, we determined the IgG-binding characteristics for WGA-pretreated macrophages. We found that the increase in IgG1-binding ability was caused by an increase in the value of Kapp, but the number of IgG-binding sites was lower than in the control cells. In the case of rabbit IgG and guinea pig IgG2 we observed a decrease of both the value of Kapp and the number of IgG-binding sites. WGA did not interact directly with the Fc gamma receptor. The results of our former papers and the different effects of lectins of various specificities described in this paper suggest different positions of Fc gamma 1/gamma 2 and Fc gamma 2R in the plane of the macrophage membrane in respect to various membrane glycoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide generation by guinea-pig peritoneal macrophages is inhibited by rolipram, staurosporine and mepacrine in an agonist-dependent manner

Platelet-activated factor (PAF) ( ec 50−7.9±0.6 M ), formyl-methionyl-leucyl-phenylalanine (fMPL) ( ec 50 −7.7±0.1 M ), phorbol 12-myristate 13 acetate (PMA) ( ec 50−8.4±0.3 M ), opsonized zymosan (OPZ) (0.01–1 mg/ml) were potent stimuli to superoxide generated by guinea-pig peritoneal macrophages. Superoxide generation by low (≤ −8M) concentrations but not high (≥−7M) concentrations of PAF or fMLP were attenuated by rolipram (100 μM) in the presence of 1 μM prostaglandin E 2 (PGE 2). That stimulated by PMA or OPZ, however, was unaffected. At 1μM, staurosporine was a potent inhibitor of superoxide generation stimulated by both fMLP and PAF but was without effect on that stimulated by OPZ. Superoxide generation stimulated by fMLP, PAF and OPZ was inhibited by 100 μM mepacrine. We conclude that superoxide generation stimulated by the chemoattractants fMLP and PAF involves a cyclic AMP regulated and cyclic AMP independent process. The cyclic AMP independent process is mediated by protein kinase C. Although protein kinase C seems a central element in the respiratory burst stimulated by fMLP, PAF and PMA that stimulated by OPZ bypasses this mechanism. Phospholipase A 2 however, represents a common stage in the signal transduction pathway.

Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock.

Tumor necrosis factor-alpha (TNF) is a pluripotent cytokine that mediates many of the hemodynamic manifestations of endotoxic shock. To determine whether TNF is responsible for postburn myocardial dysfunction, we compared cardiac function (Langendorff preparation) in 49 guinea pigs 18 h after thermal injury. Group 1 (n = 15) was sham burned; all remaining animals received a 43% surface area burn under anesthesia. Group 2 (n = 15) received lactated Ringer solution (LR, 4 ml.kg-1.%burn-1). Group 3 (n = 9) received LR and drug vehicle. Group 4 (n = 10) received LR plus 1 mg of TNF inhibitor consisting of the human p80 TNF receptor linked to the Fc portion of human immunoglobulin G1, which was shown to specifically bind and neutralize TNF secreted by guinea pig peritoneal macrophages in vitro. Burn injury caused a significant fall in left ventricular pressure (LVP, from 86 +/- 2 to 62 +/- 3 mmHg, P < 0.05) and maximal rate of LVP rise (+) and fall (-) (+/- dP/dtmax) [from 1,365 +/- 42 to 1,109 +/- 44 mmHg/s (P < 0.05) and from 1,184 +/- 31 to 881 +/- 40 mmHg/s (P < 0.05), respectively], a decrease in time to peak systolic LVP (from 111 +/- 2 to 102 +/- 2 ms, P < 0.05), and a decrease in time to +dP/dtmax (from 57 +/- 1 to 48 +/- 1 ms, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity of extracellular Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaire's disease and Pontiac fever, is known to produce a cytopathic effect on macrophages. The capacity of extracellular L. pneumophila to mediate toxicity for guinea pig peritoneal macrophages and J774 mouse macrophages was assessed. Extracellular organisms were found to be capable of mediating toxicity; however, toxic activity appeared to require close proximity with the mononuclear cell surface. Serogroup 1 strains grown on supplemented Mueller-Hinton agar exhibited variable expression of toxic activity. One strain positive on supplemented Mueller-Hinton agar was cytotoxic and unable to replicate in J774 macrophages but remained virulent for guinea pigs at high doses.

Effect of sparfloxacin on Staphylococcus aureus adhesiveness and phagocytosis.

We investigated the effect of sparfloxacin, a new broad-spectrum fluoroquinolone, on the morphology, adhesiveness and phagocytosis of a clinical isolate of Staphylococcus aureus sensitive to this compound (MIC = 0.06 mg/L). The strain was tested for its adherence to human buccal epithelial cells, measured by interference contrast microscopy, and for phagocytosis by guinea-pig peritoneal macrophages, measured by fluorescence microscopy. Accumulation of sparfloxacin by macrophages was studied by means of a velocity-gradient centrifugation technique. The S. aureus strain, grown in the presence of sub-inhibitory concentrations of sparfloxacin, exhibited an increased cell diameter and a markedly reduced capacity to adhere to buccal epithelial cells. The phagocytic capacity and activity of macrophages were greater with the treated strain than with an untreated control. A reduction in numbers of intracellular cocci was also observed 2 h after postphagocytic treatment of macrophages with sparfloxacin at 10 x MIC. This intracellular bactericidal activity may result from accumulation of sparfloxacin in macrophages, evidenced by a high ratio of cellular to extracellular concentration. It was concluded that sparfloxacin reduces adherence to epithelial cells, increases phagocytosis and facilitates the intracellular killing of S. aureus.