Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator.

Granulysin, a cationic protein produced by activated human CTL and NK cells, is cytolytic against microbial and tumor targets. In this study we show that granulysin also functions as a chemoattractant and activates monocytes to produce cytokines/chemokines. Although granulysin-mediated cytotoxicity occurs at micromolar concentrations, chemoattraction occurs in the nanomolar range, and immune activation occurs over a wide range of concentrations (nanomolar to micromolar). Granulysin causes a 2- to 7-fold increase in chemotaxis of monocytes, CD4(+), and CD8(+) memory (CD45RO) but not naive (CD45RA) T cells, NK cells, and mature, but not immature, monocyte-derived dendritic cells. Pertussis toxin treatment abrogates chemoattraction by granulysin, indicating involvement of G-protein-coupled receptor(s). At low concentrations (10 nM), granulysin promotes a 3- to 10-fold increase in MCP-1 and RANTES produced by monocytes and U937 cells, while a 2-fold increase in TNF-alpha production by LPS-stimulated monocytes requires higher concentrations of granulysin (micromolar). Taken together, these data indicate that the local concentration of granulysin is critical for the biologic activity, with high concentrations resulting in cytotoxicity while lower concentrations, presumably further from the site of granulysin release, actively recruit immune cells to sites of inflammation.

Folic acid, ascorbic acid and sodium selenite restore the motility of Dictyostelium discoideum inhibited by triethyllead.

The effect of triethyllead (TriEL) on motile activity, structure of cytoskeleton and chemotaxis of Dictyostelium discoideum amoebae in developing concentration gradients of folic acid (FA) and cAMP has been studied. It was observed that 3 microM TriEL had little or no effect on locomotion and chemotactic response of cells, whereas 5 microM TriEL strongly reduced the motile activity of Dictyostelium discoideum amoebae and inhibited their chemotaxis towards cAMP, but not towards FA. FA was found to restore the motile activity of Dictyostelium discoideum, inhibited by TriEL. A similar effect was observed in the presence of other antioxidants, i.e. ascorbic acid and sodium selenite, suggesting that oxidative stress may be involved in the action of TriEL. Moreover, the treatment of Dictyostelium amoebae with 5 microM TriEL caused disruption of microtubules while 3 microM TriEL had little effect on their structure. FA caused restoration of microtubules only in some cells within 1 h of incubation, i.e. when the directional movement of cells towards this chemoattractant was already observed. However, their organization was significantly different from that observed in the untreated cells, suggesting that microtubule undisturbed organisation may be not necessary for Dictyostelium discoideum amoebae locomotion and chemotaxis

The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1.

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.

High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

IL-8 is involved in homologous TNF alpha-, but not in IL-1 beta-induced neutrophil infiltration in rabbits.

OBJECTIVE AND DESIGN: We examined the involvement of IL-8 in TNF alpha and IL-1 beta neutrophil infiltration, using homologous reagents. SUBJECTS: Female New Zealand white rabbits (n = 211). METHODS: After injection of TNF alpha (1 microgram) or rrIL-1 beta (300 pg) into rabbit knee joints, the leukocyte influx and the production of IL-8 were estimated. Anti-TNF alpha mAb, rrIL-1Ra, and anti-IL-8 IgG (10 micrograms each) were administrated simultaneously with either TNF alpha or IL-1 beta. RESULTS: Administration of TNF alpha or IL-1 beta induced IL-8 production; both of which peaked at 1 h after the injection. Maximum neutrophil infiltration induced by TNF alpha was inhibited by 38-56% by anti-IL-8 IgG, 22-43% by rrIL-1Ra, and 77-83% by anti-IL-8 IgG plus rrIL-1Ra. In contrast, maximum neutrophil infiltration induced by IL-1 beta was not inhibited by anti-IL-8 IgG whereas anti-TNF alpha mAb reduced the infiltration by 68-75%. CONCLUSIONS: The involvement of IL-8 in TNF alpha- and IL-1 beta-induced neutrophil infiltration is limited. Factor(s) other than IL-8 appear to function as major neutrophil chemo-attractants.

Saturated and monofluoro analogs of the oriental fruit fly attractant methyl eugenol show reduced genotoxic activities in yeast.

Methyl eugenol, is a commercially used fruit fly attractant and a suspected carcinogen. Several phenylpropenes, including methyl eugenol and the known carcinogen safrole, score negative in the Salmonella assay but score positive in the yeast DEL assay that selects for intrachromosomal recombination events in the yeast Saccharomyces cerevisiae. In an attempt to dissociate the beneficial properties of methyl eugenol from its genotoxic properties, saturated or fluorinated analogs were evaluated for their ability to induce intrachromosomal (DEL) recombination in yeast. Field tests have previously shown that all of the analogs used have appreciable properties as fruit fly attractants. The analogs 1,2-dimethoxy-4-ethylbenzene, 1,2-dimethoxy-4-(2-fluoro-2-propenyl)benzene, 1,2-dimethoxy-4-(2-fluoroethyl)benzene and 1,2-dimethoxy-4-(3-fluoro-2-propenyl)benzene all showed reduced toxicity and reduced recombinagenicity in yeast compared to methyl eugenol. These results confirm the validity of fluorination and/or removal of the 2-propenyl moiety in reducing the toxicity and recombinagenicity of methyl eugenol derivatives.

Inhibition of PAF-, LPS-, and cytokine-induced granulocyte accumulation in guinea pig lung by dexamethasone: evidence that inhibition of IL-5 release is responsible for the selective inhibition of eosinophilia by glucocorticoids in guinea-pigs.

The potency of dexamethasone has been determined as an inhibitor of intratracheally administered platelet activating factor- (PAF), or interleukin (IL)-5-induced eosinophilia, and of lipopolysaccharide-(LPS), tumour necrosis factor alpha-(TNF alpha) or cytokine-induced neutrophil chemoattractant- (CINC) induced neutrophilia in guinea-pig lungs. Dexamethasone was a potent inhibitor of PAF- induced eosinophil accumulation, but higher doses of dexamethasone were required to inhibit IL-5-induced eosinophilia. LPS-induced neutrophilia was less sensitive to the inhibitory effects of dexamethasone, than PAF-induced eosinophilia. Both LPS- and TNF alpha-induced neutrophilia were inhibited by the same doses of dexamethasone. In contrast, higher doses of dexamethasone were required to inhibit CINC-induced neutrophilia. Since data in the literature show that PAF-induced eosinophilia in guinea-pig lungs is dependent on the generation of IL-5, it is concluded that inhibition of this response, by dexamethasone, is due to inhibition of release of IL-5. Similarly, although data in the literature show that LPS-induced neutrophilia is dependent on the generation of TNF alpha, it is concluded that inhibition of this response, by glucocorticoids, is due to an action on an event which occurs after the release of TNF alpha, possibly through inhibition of chemokine release.

Chemotactic peptide-induced acute colitis in rabbits.

Bacterial chemotactic peptides from the intestinal lumen could potentially induce inflammation if they reached the mucosa. We tested several peptides chemotactic for different inflammatory cells, as well as a nonchemotactic peptide, bradykinin, for their ability to induce colitis in vivo in rabbits. These peptides were also assessed for their ability to stimulate release of the eicosanoids leukotrienes B4 and C4 and prostaglandin E2 from normal rabbit colons perfused ex vivo. Intracolonic administration of n-formyl-methionyl-leucyl-phenylalanine (chemotactic for neutrophils); its methyl ester (chemotactic for monocytes), and alanyl-glycyl-seryl-glutamic acid (chemotactic for eosinophils) all produced colitis (assessed grossly and histologically) within 4 days. Bradykinin did not induce colitis although it did release prostaglandin E2. n-Formyl-methionyl-leucyl-phenylalanine methyl ester induced the greatest degree of colitis in vivo and released prostaglandin E2 and leukotrienes ex vivo. n-Formyl-methionyl-leucyl-phenylalanine and alanyl-glycyl-seryl-glutamic acid induced comparable degrees of inflammation, but alanyl-glycyl-seryl-glutamic acid produced no eicosanoid release while n-formyl-methionyl-leucyl-phenylalanine released both prostaglandin E2 and leukotriene B4 and leukotriene C4 products from normal ex vivo perfused colons. Thus alanyl-glycyl-seryl-glutamic acid produces colitis independent of proinflammatory eicosanoids while eicosanoid release could contribute to colitis produced by n-formyl-methionyl-leucyl-phenylalanine methyl ester. This experimental model of colitis may reflect one possible etiology of inflammatory bowel disease in humans, when bacterial chemotactic peptides breach mucosal defenses in susceptible individuals.

Effect of chemotactins released by Staphylococcus aureus and Pseudomonas aeruginosa on the murine respiratory tract.

Staphylococcus aureus, the Pseudomonas aeruginosa temperature-sensitive (ts) mutant A/10/25, and the P. aeruginosa parental wild type were aerosolized to C5-deficient mice, and the total number of polymorphonuclear leukocytes (PMN) recovered by lung lavage was determined 4 h after aerosol exposure. S. aureus induced a slight but significant recruitment of PMN, as compared with the effect of a saline aerosol. Both wild-type P. aeruginosa and the ts mutant induced a significant PMN recruitment of a magnitude ca. 180 times higher than that produced by S. aureus. Gentamicin-killed ts P. aeruginosa induced a PMN recruitment of a magnitude similar to that produced by live ts P. aeruginosa. Thorough washing of the bacteria, however, removed ca. 90% of the chemotactic activity. Exposure of the animals to a ts P. aeruginosa culture supernatant aerosol induced significant PMN recruitment into the lower airways. The same culture supernatants were chemotactic for mouse PMN in a dose-dependent fashion when tested in vitro in the absence of serum. Culture supernatants of S. aureus exhibited weak chemotactic activity in vitro and did not induce PMN recruitment in the lungs when aerosolized to DBA/2J mice. The results suggest that chemotactins released by P. aeruginosa may be an important virulence factor and play a significant role in lung tissue damage.