Colchicine and pseudogout.
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Acute Disease↗
Biomedical subjects
I Spilberg
Publications and source records attributed to I Spilberg.
Chemotactic factor-induced generation of superoxide radicals by human neutrophils: effect of metabolic inhibitors and antiinflammatory drugs.
Human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was inhibited by 2-deoxy-D-glucose (ID50 4 X 10(-5)M), 2-iodoacetate (ID50 5 X 10(-5)M), and N-ethyl-maleimide (ID50 5 X 10(-6)M), suggesting a dependence on anaerobic glycolysis and sulfhydryl groups. Ouabain, microtubule-disrupting agents, inhibitors of respiration, oxidative phosphorylation, and protein and nucleic acid synthesis were without appreciable effects. Indomethacin (ID50 1 X 10(-4)M), ibuprofen (ID50 9 X 10(-4)M, and phenylbutazone (ID50 1 X 10(-5)M) all caused dose-dependent inhibition of superoxide generation at concentrations approximating those plasma and tissue levels obtained in human beings at therapeutic doses. Acetylsalicylic acid (125-500) microgram/ml) and aurothioglucose (10(-3)-10(-6)M) were without appreciable effects. Superoxide generation was inhibited only by relatively high concentrations of hydrocortisone (ID50 greater than 10(-3)M). Because superoxide radicals have been implicated in the pathogenesis of tissue injury in several forms of inflammation and arthritis in vivo, these studies suggest that the production of a potential cytotoxic factor may be subject to pharmacologic manipulation and that at least some of the antiphlogistic effects of the nonsteroidal antiinflammatory agents may be mediated through effects on superoxide production.
6-Aminonicotinamide↗
Demonstration of a specific neutrophil receptor for a cell-derived chemotactic factor.
The crystal-induced chemotactic factor, a cell-derived chemoattractant for neutrophils, binds specifically to a site on human neutrophils but not to erythrocytes or lymphocytes, suggesting a relationship between the presence of specific binding sites on the neutrophils and the ability to be chemotactically activated. The Scatchard analysis revealed an equilibrium dissociation constant at 37 degrees C of 0.446 micrometer and the presence of approximately equal to 6.44 x 10(5) binding sites for 125I-crystal-induced chemotactic factor per cell. Binding was not displaced by the synthetic chemotactic factors F-Met-Leu-Phe and Gly-His-Gly or by complement-activated plasma providing evidence of the specificity of the receptor.
Binding Sites↗
Mechanism of action of colchicine in acute urate crystal-induced arthritis.
Phagocytosis of urate crystals by human or rabbit neutrophils induces the synthesis and release of a glycoprotein, the crystal-induced chemotactic factor (CCF), which is chemotactically active both in vitro and in vivo. It has been proposed that CCF is a prime mediator of the acute gouty attack. Colchicine has been shown to decrease the production and release of this factor in vitro. In these studies, colchicine, at nonleukopenic doses, is shown to abrogate the acute arthritis induced by monosodium urate crystals in rabbits, but to have no effect upon the arthritis induced by the injection of the purified cell-derived chemotactic factor. Serum colchicine levels were 0.48-0.58 muM at 30 min and 0.12-0.3 muM at 90 min after intravenous injection of 0.2 mg/kg colchicine. Peripheral blood polymorphonuclear leukocytes obtained from colchicine-treated animals migrated normally towards a chemotactic stimulus but failed to produce CCF after phagocytosis of monosodium urate crystals. The dialyzed synovial fluid from rabbits injected with microcrystalline sodium urate contained chemotactic activity that was not present when animals were also given intravenous colchicine or injected intra-articularly with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Furthermore, the synovial fluid from rabbits injected with microcrystalline sodium urate significantly decreased (125)I-CCF binding to neutrophils. The binding of (125)I-CCF to its neutrophil receptor was not significantly reduced by the synovial fluid of colchicine-treated rabbits nor by the synovial fluid of control rabbits injected with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Colchicine (10 and 0.1 muM) was shown to have no effect upon the binding of (125)I-CCF to its cell receptor.
Animals↗
Generation of superoxide radicals by human peripheral neutrophils activated by chemotactic factor. Evidence for the role of calcium.
In response to activation by the synthetic chemotactic factor FMLP, human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction. A dose-dependent increase in the amount of superoxide induced by FMLP over the concentration range of 1 X 10(-8) M to 1.6 X 10(-7) M was observed. Examination of the kinetics of the response revealed large amounts of superoxide generated by 1 min of incubation at 37 degrees C at an optimal dose of FMLP and a plateau effect after 5 min of incubation. Divalent cations did not influence the binding of 3H-FMLP to the cell, but superoxide generation by FMLP-activated neutrophils was observed to be dependent on the presence of divalent cations in the medium. In the absence of Mg2+, increasing Ca2+ ion concentration in the medium led to progressive increases in superoxide generation up to 4 mM, after which the response declined slightly. Mg2+, 0.25 to 4 mM, increased FMLP-induced superoxide generation to a much lower extent than did Ca2+. Lanthanum ion, 0.1 to 1 mM, in the presence of 1 mM Ca2+ inhibited the production of superoxide by FMLP 4 X 10(-8 ) M. Over the concentration range 3.3 X 10(-5 M to 3 X 10(-4 M, verapamil, a drug which selectively blocks the calcium channel, caused a dose-dependent inhibition of superoxide production and calcium-45 uptake in response to FMLP. This effect of verapamil could be overcome by increasing the concentration of Ca2+ in the medium. These observations suggest that a calcium influx plays an important role in the superoxide-generating capacity of the neutrophil.
Calcium↗
Evidence for the role of superoxide radicals in neutrophil-mediated cytotoxicity.
Human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of lectins as assessed by release of 51chromium from labelled target cells. Phytohaemagglutinin (PHA) and concanavalin A (Con A), which caused time-dependent and dose-dependent cytotoxicity over a concentration range of 25--400 microgram/ml, also caused significant generation of superoxide radicals as measured by ferricytochrome C reduction. Pokeweed mitogen, which does not induce cytotoxicity over the same concentration range, was unable to promote superoxide generation by neutrophils. PHA-induced generation of superoxide paralleled and appeared to precede PHA-dependent cytotoxicity. Superoxide dismutase (SOD), which enzymatically destroys superoxide, caused moderate inhibition of PHA-dependent cytotoxicity over the concentration range of 100--500 microgram/ml whereas catalytically inactive enzyme had no effect. Incubation under oxygen-depleted conditions caused a marked decrease in both PHA-induced superoxide generation and cytotoxicity relative to that obtained with neutrophils incubated aerobically. These findings suggest a central role for superoxide radicals in causing target cell damage in this model of neutrophil-mediated cytotoxicity.
Catalase↗
Chemotactic factor-induced superoxide radical generation by human neutrophils: requirement for proteinase (esterase) activity.
The requirement for proteinase (esterase) activity in the generation of O2- by human peripheral neutrophils was investigated. Neutrophils were activated by exposure to the chemotactic peptide FMLP and superoxide generation was assessed by ferricytochrome C reduction. Inhibition of O2- generation was observed by pretreating cells with the chloromethyl ketone derivatives of tosyl-phenylalanine (TPCK, ID50 2.2 X 10(-5)M) and tosyl-lysine (TLCK, ID50 1.9 X 10(-4)M). Dose-dependent inhibition was also noted with synthetic proteinase substrates, especially those of chymotrypsin-like specificity, phenylalanine and tryptophan methyl esters (ID50S approximately 1.5 X 10(-4)M), as compared to derivatives of basic amino acids, arginine and acetyl-lysine methyl esters, which caused negligible inhibition at 10(-3M. The inhibition of O2- generation demonstrated by TryME was time- and temperature-dependent and reversible with washing of the cells, whereas the inhibition seen with TPCK was irreversible. DFP at high concentrations, 10(-3)M or greater, caused moderate inhibition of O2- generation that was unaffected by exogenous serine and almost completely reversible by washing the cells. TPCK and TryME, at concentrations that caused marked inhibition of O2- generation, had no effect on the fmlp-induced calcium-45 uptake by the cells. These studies suggest that intact proteinase function is required for O2- generation and that this step follows the calcium influx in the activation sequence induced by FMLP.
Calcium↗
Chemotactic factor-induced generation of superoxide radicals by human neutrophils: evidence for the role of sodium.
The role of sodium ion in superoxide (O2-) generation by human peripheral neutrophils was investigated. Cells were activated by exposure to the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and O2- release was assessed by ferricytochrome c reduction after 5 min of incubation at 37 degrees C in the presence of FMLP 4 X 10(-8) M. In the absence of monovalent cations (isotonic glucose), negligible O2- generation occurred. There was a progressive increase in the magnitude of FMLP-induced O2- generation with increasing Na+ concentration up to 90 mM, where the response was noted to plateau. Varying the K+ concentration (1 to 10 mM) had no effect on the amount of O2- produced in the presence of Na+ 140 mM. FMLP also stimulated 22Na+ and 48Ca2+ uptake by the cells in a dose- and time-dependent fashion. FMLP-induced 22Na+ uptake appeared to be independent of the external Ca2+ concentration ( to 4 mM). In contrast, there was a progressive decrease in themagnitude of the FMLP-induced increase in 45Ca2+ uptake as the Na+ concentration was reduced by replacement with choline+ or glucose. These studies support a requirement for Na+ in FMLP-induced O2- generation and suggest that a Na+ influx may underlie the nature of this requirement. The data are also consistent with the hypothesis that a Na+ influx may precede the Ca2+ influx in the FMLP-induced activation sequence.
Binding Sites↗
A proposed model for chemotactic deactivation: evidence for microtubule modulation of polymorphonuclear leukocyte chemotaxis.
Incubation of the CFs Gly-His-Glyc or CCF with PMNs in the absence of a gradient, resulted in a dose-dependent depression in chemotactic activity when, after washing, the cells were challenged with the CFs in a Boyden chamber. When the cells were preincubated with either CF and suitable concentrations of colchicine, the inhibition of chemotaxis that either of these agents induced when incubated with the cells alone was abolished. Deactivation reappeared when the optimal ratio between colchicine and CF was altered in either direction. Ultramicroscopic studies showed an increase in centriole-associated microtubules following incubation of cells with CFs. This increase was arrested by prior exposure of the cells to colchicine. Colchicine did not alter the specific binding of CCF to human neutrophils, and lumicolchicine had no effect on either chemotaxis or deactivation. Our data suggest that the control of PMN chemotaxis is predicted upon microtubule assembly evoked by cell interaction with a chemotactic gradient. Chemotaxis would be prevented by conditions that inappropriately organize responsive microtubules in either a polymerized or depolymerized configuration.
Chemotaxis↗
Dissociation of the neutrophill functions of exocytosis and chemotaxis.
The peptide Gly-His-Gly is shown to be chemotactic for human neutrophils in vitro and for rabbit neutrophils in vivo but to be unable to induce lysosomal enzyme release from human neutrophils at sublytic concentrations. The failure of this chemotactic peptide to elicit lysosomal enzyme release provides evidence that interactions with a chemotactic receptor does not necessarily activate chemotaxis and exocytosis in the human neutrophil, thus suggesting that the presumed common pathway of exocytosis and chemotaxis may be divergent at the cell receptor and/or postreceptor level.
Albumins↗
A mechanism of action for non-steroidal anti-inflammatory agents in calcium pyrophosphate dihydrate (CPPD) crystal induced arthritis.
The acute inflammatory response to calcium pyrophosphate dihydrate crystals follows the meeting of neutrophils and crystals. The ensuing phagocytosis leads to the generation of a glycoprotein chemotactically active for neutrophils and to the release of lysosomal enzymes. Indomethacin and phenylbutazone, at therapeutic concentrations, impaired phagocytosis of the crystals and generation of chemotactic factor activity. Colchicine had no effect upon phagocytosis but significantly impaired the appearance of chemotactic factor activity.
Arthritis↗
Induction of arthritis by purified cell-derived chemotactic factor: role of chemotaxis and vascular permeability.
The injection of monosidium urate-induced chemotactic factor into the joint cavities of rabbits induces an acute inflammatory response that resembles the one produced by monosodium urate crystals. The leukocyte accumulation induced by the factor was not accompanied by a measurable increase in vascular permeability as measured by appearance of 125I-albumin in the joint cavity. When histamine was injected into the joints, a marked increase in vascular permeability but no leukocytosis above control levels was observed. The above results suggest that the cell-derived factor is primarily responsible for the accumulation of cells seen in the acute inflammation induced by monosodium urate crystals.
Animals↗
Calcium pyrophosphate dihydrate (CPPD) crystal-induced chemotactic factor: subcellular localization, role of protein synthesis and phagocytosis.
A factor with chemotactic properties for polymorphonuclear leukocytes appears in their lysosomal fraction following phagocytosis of CCPD crystals. The factor, whose appearance was blocked by inhibition of protein synthesis, was estimated to have a molecular weight of 8,400 daltons. Inhibition of crystal phagocytosis by cytochalasin B was also shown to inhibit the generation of chemotactic factor activity, indicating that ingestion of the crystals by the cell is essential for the generation of the CPPD crystal-induced chemotactic factor. This latter finding provides a clue in the understanding of the development and termination of the acute pseudogout attack.
Calcium Pyrophosphate↗
Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor.
Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.
Cell Membrane↗
The roles of renal catabolism and uremia in modifying the clearance of fibrinogen and its degradative fragments D and E.
Elevated levels of fibrinogen/fibrin degradation products (FDP) occur in uremia, and have been thought to be in part related to intravascular coagulation in the kidney. More recent data indicated that delayed catabolism of fibrinogen fragment D occurred in anephric animals. To further evaluate FDP catabolism in the kidney, turnover studies of purified dog 131I-Fg-D and 125I-Fg-E were performed on dogs before and after acute subtotal nephrectomies, and later during chronic uremia. 131I-fibrinogen clearances were also perfomed. Slowed catabolism of Fg-D and Fg-E was observed in both the acute and chronic uremic stages. Altered urinary excretion was not a factor as only minimal amounts of Fg-D and Fg-E were excreted in the urine of the control animals. In the 131I-fibrinogen studies, there were significant changes in plasma volume, fibrinogen t 1/2, and intravascular/extravascular distribution, but not in fractional catabolic rate. To differentiate fully, the effects of uremia from those of loss of catabolic renal tissue, the Fg-D and Fg-E turnover studies were repeated on other animals with intact kidneys whose ureters were diverted into the peritoneum and compared to subsequent studies after total nephrectomy. The control and ureter-severed studies had the same clearance pattern, whereas decreased catabolism occurred in the nephrectomized dogs. The results demonstrate uremia per se does not have a major effect upon the catabolism of fibrinogen, Fg-D, and Fg-E. Loss of renal tissue does impair the clearance of Fg-D and Fg-E, indicating these proteins are normally catabolized in part by the kidneys. Thus elevated plasma FRA in uremic patients may reflect decreased Fg-D and Fg-E catabolism rather than increased FDP production from primary or secondary fibrinolysis.
Animals↗
Studies on crystal-induced chemotactic factor. II. Role of phagocytosis.
The interaction of polymorphonuclear leukocytes (PMN's) and urate crystal leads to the formation of a chemotactic factor. The purpose of this study was to determine the need for ingestion of crystals by the cells in the generation of the chemotactic factor. Our studies showed that when PMN's were incubated with cytochalasin B, an inhibitor of phagocytosis, and urate crystals, no chemotactic activity appeared in the lysosomal extract of the cells. Likewise, no chemotactic activity was present in the media of PMN's incubated with crystals although lysosomal enzyme activity was increased. These findings indicate that phagocytosis is required for chemotactic activity to appear. Our studies also show that cytochalasin B increases the release of lysosomal enzyme B-glucuronidase but not of cytoplasmic enzyme lactic dehydrogenase from PMN's when the cell encounters urate crystals.
Animals↗
A mechanism for decreased resistance to infection by gram-negative organisms during acute alcoholic intoxication.
The propensity of alcoholics to develop infections with gram-negative bacteria is well known. However, the known effects of ethanol on host-defense mechanisms do not explain this tendency satisfactorily. Since the principal defense mechanism against gram-negative bacteria is their destruction by complement in the presence of specific antibody, we investigated the effect of acute ethanol intoxication on serum complement concentrations and correlated these with serum bactericidal activity against a gram-negative organism. Serial measurements of total hemolytic complement (CH5O) and C'3, ethanol concentration, and serum bactericidal capacity were performed on the serum of 7 dogs infused with a 10 per cent ethanol solution. All animals showed a substantial decrease in total hemolytic complement activity which was inversely related to the increase in serum ethanol concentration. A decrease in serum bactericidal activity correlated with the decrease in CH50. Control animals showed no variation in either CH50 or serum bactericidal activity. Immunoprecipitation studies, using antibody to dog C'3, indicated that serum concentrations of this protein were not altered by infusing the animals with ethanol. Incubation of serum in vitro with both ethanol and acetaldehyde had no effect on CH50. We conclude that the effect of ethanol on serum complement is an in vivo phenomenon probably relating to decrease in the synthesis of one or more of the components of complement other than C'3, although the formation of an anticomplementary substance cannot be excluded. These findings are presented as a possible explanation for the development of infections with gram-negative bacteria in ethanol-intoxicated subjects.
Acetaldehyde↗
Current concepts of the mechanism of acute inflammation in gouty arthritis.
The acute gouty attack develops after free crystalline monosodium urate crystals appear in the joint cavity (1). Recent developments in the investigation of urate crystal-induced inflammation have led to a better understanding of the pathogenesis of acute gouty arthritis. The purpose of this review is to summarize present concepts concerning the mechanism of the acute gouty attack.
Acute Disease↗