Oxygen-independent antimicrobial action in sphingosine-treated neutrophils

Sphingosine is reported to inhibit the oxidative burst and superoxide anion production of human polymorphonuclear neutrophils (PMN) phagocytosing in atmospheric oxygen (Wilson et al., 1986). We have confirmed its effect on superoxide production and examined the antimicrobial phagocytic capacity of PMN treated with sphingosine, comparing them with PMN, untreated but phagocytosing either under anaerobic conditions or in atmospheric oxygen. Sphingosine just like anaerobiosis partially inhibited, but did not eliminate, the bactericidal activity of PMN when compared to non-treated aerobic cells. In fact, sphingosine-treated PMN mimicked killing of Staphylococcus aureus (S. aureus) and Serratia marcescens (S. marcescens) due to anaerobic PMN. Moreover, our results with Salmonella typhimurium and sphingosine-treated cells duplicated results this laboratory published previously about comparative killing of Salmonella in aerobic versus anaerobic neutrophils. In these studies sphingosine-treated PMN took up bacteria as avidly as untreated PMN and retained their viability, as assessed by trypan blue exclusion. While sphingosine should not be completely substituted for anaerobic studies, it is a convenient screening reagent for the study of non-oxidative killing mechanisms of PMN. Results achieved with anaerobic and with sphingosine-treated cells suggest that O2-independent antimicrobial action is substantially more powerful than has been generally acknowledged.     

Mechanism of antifungal action of kanosamine.

The antibiotic kanosamine inhibited growth of Saccharomyces cerevisiae and a range of human pathogenic fungi, including Candida albicans. Kanosamine was transported into C. albicans cells by the glucose transport system and subsequently phosphorylated. The product of its intracellular metabolism, kanosamine-6-phosphate, was an inhibitor of the enzyme glucosamine-6-phosphate synthase. Inhibition was competitive in respect to one of the substrates, D-fructose-6-phosphate, with Ki = 5.9 mM, and was non-competitive in respect to the second substrate, L-glutamine. On the other hand, kanosamine-6-phosphate had no effect on the enzyme catalysing the next metabolic step, namely glucosamine-6-phosphate N-acetylase. The action of kanosamine on C. albicans cells resulted in profound morphological changes, inhibition of septum formation and cell agglutination. Experiments with S. cerevisiae mutants showed that the presence of the Cdr1p drug efflux pump did not affect the antifungal activity of kanosamine.     

Modulation of keratinocyte-derived interleukin-8 which is chemotactic for neutrophils and T lymphocytes.

Interactions between T lymphocytes, neutrophils, and epidermal cells are believed to play a central role in the pathophysiology of psoriasis and other inflammatory cutaneous disorders. Although there is strong evidence that lymphocyte-function-associated antigen-1 (LFA-1) positive T cells are retained in the epidermis via intercellular adhesion molecule-1 (ICAM-1) expression induced on keratinocytes, the molecular basis for the directed migration of T cells or neutrophils towards the epidermis is not known. To investigate whether epidermal keratinocyte-derived products may be important in the migration of T cells and neutrophils into the epidermis, human keratinocytes were cultured in the presence of various cytokines and chemotactic activity of the supernatants were assessed. TNF-alpha stimulation produced directed migrational responses for both neutrophils and T-lymphocytes (both CD4 and CD8), but not B lymphocytes; 69% of T-cell movement and 80% of neutrophil migration induced by the TNF-alpha treated keratinocyte cell supernatants could be inhibited by anti-interleukin-8 (IL-8) serum. Using the same antibody, IL-8 was immunoprecipitated from the supernatants of TNF-stimulated 35S-labelled keratinocytes, and a single 7-kd band product detected by SDS-PAGE. In keeping with these biological activities and protein data, Northern blot analysis of total cellular RNA extracted from keratinocyte monolayers hybridized with a 32P-labelled 1-kb cDNA to IL-8 mRNA, revealed induction of the IL-8 gene in the presence of TNF-alpha and IL-1 beta, but not IFN-gamma. The protein kinase C agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known stimulator of psoriasiform cutaneous inflammation when applied directly to murine epidermis, strongly induced keratinocyte elaboration of IL-8 mRNA. These studies demonstrate that activated human keratinocytes are capable of producing biologically active IL-8, and provide evidence that keratinocytes can play a key role in mediating the influx of T cells and neutrophils into the epidermis.     

Mechanism of action of Moraxella bovis hemolysin.

Bovine erythrocytes (RBCs) exposed to Moraxella bovis culture supernatants exhibited rapid leakage of intracellular K+ (95% in 10 min), slower cell swelling (1.20-fold increase in mean corpuscular volume in 20 min), and subsequent lysis (76% leakage of hemoglobin in 25 min). Incubation media made hypertonic by the addition of 75 mM carbohydrates with molecular diameters of 0.72 to 1.32 nm prevented hemolysin-induced RBC swelling, but incubation media made hypertonic by the addition of carbohydrates with molecular diameters of less than 0.72 nm did not protect against hemolysin-induced RBC swelling. Raffinose (75 mM; molecular diameter, 1.14 nm) did not block hemolysin-induced K+ leakage but did block hemolysis. These findings support the hypothesis that hemolysin-induced lysis occurs by colloid-osmotic swelling and are compatible with M. bovis hemolysin acting as a pore-forming cytolysin. Assuming that M. bovis hemolysin acts as a transmembrane molecular sieve, then the functional size of the hemolysin transmembrane pores in bovine RBCs is approximately 0.9 nm, the molecular size of sucrose. Hemolytic activity was inhibited by the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), but hemolysin-induced K+ leakage was not affected by EGTA.     

Mechanism of action of Yersinia enterocolitica enterotoxin.

Enterotoxin derived from three clinical isolates of Yersinia enterocolitica was compared with the heat-stable enterotoxin of Escherichia coli. Both toxins were biologically active in infant mice examined at 2 h and in ligated rabbit ileal loops at 6 h. Neither substance, however, produced changes in ligated ileal loops at 18 h or in Chinese hamster ovary or Y1 adrenal tissue cultures. In addition, both Y. enterocolitica enterotoxin concentrated approximately 20 times by ammonium sulfate precipitation and ultrafiltration and a similarly prepared sample of E. coli heat-stable enterotoxin stimulated the activity of guanylate cyclase but not that of adenylate cyclase in infant mouse intestine. These findings suggest that the role of enterotoxin in the pathogenesis of intestinal Y.enterocolitica infection may be similar to that of heat-stable enterotoxin in E. coli diarrhea.     

Differential contribution of Yersinia enterocolitica virulence factors to evasion of microbicidal action of neutrophils.

The differential contribution of the virulence factors invasin, protein tyrosine phosphatase (YopH), cytotoxin (YopE), and adhesin (YadA) of Yersinia enterocolitica to evasion of the antibacterial activities of polymorphonuclear leukocytes (PMNs) (oxidative burst, phagocytosis, killing) was analyzed. We constructed virulence gene knockout mutants and a novel two-plasmid system allowing production and secretion of individual virulence factors. Wild-type Y. enterocolitica WA-314 harboring the virulence plasmid pYV08 resisted phagocytosis and killing by PMNs. Moreover, strain WA-314 was able to inhibit the neutrophil oxidative burst upon stimulation with opsonized zymosan independently on preincubation with normal human serum or YadA-specific serum. These phenotypic properties of strain WA-314 were differentially affected when mutants impaired in YadA production or Yop secretion were used. A more detailed analysis revealed that YopH plays the dominant role in suppression of the antibacterial action of PMNs without damaging the cells. The YopH suppressing effect could be enhanced by coproduction of YopE and YadA. The contribution of YadA is attributed to the adhesin function promoting interaction with PMNs under both opsonizing and nonopsonizing conditions. In contrast, invasin seems to mediate only opsonin-independent interaction with PMNs. Taken together, our results demonstrate that YopH, YopE, and YadA act in concert towards neutrophil attack to enable extracellular survival of Y. enterocolitica in host tissue.     

Autoradiographic detection of intracellular and extracellular bactericidal action of neutrophils

Department of Pathological Anatomy, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 11, pp. 621–623, November, 1988.     

Mechanism of action of aspirin on canine gastric mucosa.

Using an in vivo chambered canine stomach preparation, exposure of the gastric mucosa to 5, 10, and 20 mM aspirin(pH 3.0) resulted in a decrease in electrical potential difference (PD) and in an increase in resistance (R) within 30 min. In vitro, exposure of the mucosal side of the isolated canine gastric mucosa to 5, 10, and 20 mM aspirin (pH 3.0) for 1 h or of 1 mM aspirin (pH 3.0) for longer than 1 h resulted in marked permeability changes, i.e., increases in the undirectional fluxes of Na+ and Cl-, as well as inhibition of net ion fluxes. These concentrations of nonionized aspirin (pH 3.0) also reduced the R and PD. However, 1 mM aspirin (pH 3.0) or 20 mM ionized aspirin (pH 7.4) depresses the active transport of ion, increases R, but does not increase the ionic permeability. Mucosal adenosine triphosphate (ATP) content is reduced by mucosal instillation of aspirin (pH 3.0). These results demonstrate that the initial action of aspirin is inhibition of ion transport which is followed by an increase in permeability.     

Mechanism of action of colchicine

Colchicine suppressed the development of urate crystal-induced canine synovitis only at a dose (0.25 mg/kg) that produced a peripheral leukopenia; half that amount produced neither leukopenia nor inhibition of the inflammatory response. Colchicine in the lower dose range still had no antiinflammatory effect when the time of pretreatment was increased from 15 min to 5 h before the inflammatory challenge, and when an additional dose was given 24 h beforehand. Lower doses of Colchicine were not antiinflammatory when the drug was introduced directly into the joint, either along with the urate crystals or when the inflammatory response was present and increasing. Lumicolchicine (0.25 mg/kg) neither suppressed inflammation nor induced leukopenia. The canine model differs from human gouty inflammation and from urate crystal-induced human inflammation, both of which processes respond to small, nonleukopenic doses of colchicine.This work was supported in part by grants from the USPHS (AM-10493, AM-5639) and from the Arthritis Foundation. Dr. Malawista is the recipient of a Research Career Development Award of the NIH (AM-19864).     

Mechanism of action of pyrogen

1. In unanaesthetized rabbits the cerebral ventricles were perfused for 30-75 min from left lateral ventricle to cisterna magna with solutions of different composition, whilst rectal temperature was continuously recorded.2. Temperature did not rise during the perfusion when the perfusing fluid consisted of artificial c.s.f.; it did rise, however, when the perfusing fluid consisted merely of a 0.9% sodium chloride solution.3. Temperature fell, though not in all rabbits, during the perfusion when the calcium in the perfusing fluid was increased from 1.25 mM, the concentration in c.s.f. to 5 mM.4. Magnesium chloride had only a weak action, in comparison to calcium, in preventing the rise produced during perfusion with 0.9% sodium chloride solution. In a concentration of 1.25 mM it had no effect, but in a concentration of 5 mM it delayed and greatly reduced the rise.5. Temperature did not rise during perfusion with an isotonic sucrose solution.6. The rise in temperature produced by an intravenous injection of leucocyte pyrogen was not prevented when the injection was made during a perfusion with artificial c.s.f., but it was prevented when the calcium concentration in the perfusing fluid was raised to 5 mM or when the perfusing fluid consisted of isotonic sucrose solution. Again, magnesium had only a weak action in comparison to calcium.7. These results support the theory put forward recently (Feldberg, Myers & Veale, 1970) that the constancy of temperature depends upon the physiological balance of sodium and calcium ions in the anterior hypothalamus, that the calcium ions act as a kind of ;brake' preventing the sodium ions from exerting their temperature raising effect, and that pyrogen acts by removing the ;calcium brake', the pyrogen fever thus being a sodium fever.