Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19.

Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Poly-MVA attenuates 7,12- dimethylbenz[a]anthracene initiated and croton oil promoted skin papilloma formation on mice skin.

OBJECTIVE: Chemopreventive agents which exhibit activities such as anti-inflammation, inhibition of carcinogen induced mutagenesis and scavenging of free radical might play a decisive role in the inhibition of chemical carcinogenesis either at the initiation or promotion stage. Many synthesized palladium (Pd) complexes tested experimentally for antitumor activity are found effective. Poly-MVA is a liquid blend preparation containing B complex vitamins, ruthenium with Pd complexed with alpha lipoic acid as the major ingredients. The antitumor effect of Poly-MVA was evaluated against 7,12-dimethylbenz[a] anthracene-initiated croton oil-promoted papilloma formation on mice skin. Skin tumor was initiated with a single application of 390 nmol of DMBA in 20 µl acetone. The effect of Poly-MVA against croton oil- induced inflammation and lipid peroxidation on the mice skin was also evaluated. Topical application of Poly-MVA (100 µl, twice weekly for 18 weeks) 30 minutes prior to each croton oil application, significantly decreased the tumor incidence (11%) and the average number of tumor per animals. Application of Poly-MVA (100 µl) before croton oil significantly (p &#60; 0.05) protected the mouse skin from inflammation (36%) and lipid peroxidation (14%) when compared to the croton oil alone treated group. Experimental results indicate that Poly-MVA attenuate the tumor promoting effects of croton oil and the effect may probably be due to its anti-inflammatory and antioxidant activity.

A transporter of Escherichia coli specific for L- and D-methionine is the prototype for a new family within the ABC superfamily.

An ABC-type transporter in Escherichia coli that transports both L- and D-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds.

Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome.

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Human basophil releasability. VIII. Increased basophil releasability in patients with scleroderma.

We evaluated basophil releasability in 16 female patients with scleroderma (systemic sclerosis) and in 16 normal age- and sex-matched donors. Basophils from patients with scleroderma released significantly more histamine "spontaneously" than did those from normal donors (12.9 +/- 2.1% versus 4.5 +/- 0.7%; P less than 0.0005). Basophil reactivity (maximal percentage histamine release) to anti-IgE was higher in patients with scleroderma than in controls (57.0 +/- 7.5% versus 35.4 +/- 7.8%; P less than 0.05). Basophil sensitivity (the concentration of anti-IgE that causes 40% of maximal percentage histamine release) to anti-IgE in scleroderma patients was similar to that found in controls (4.6 +/- 2.8 x 10(-2) micrograms/ml versus 2.3 +/- 1.0 x 10(-1) micrograms/ml; P not significant). Scleroderma patients also showed enhanced releasability compared with that of the controls when challenged in vitro with interleukin-3 (8.3 +/- 1.7% versus 3.2 +/- 0.6%; P less than 0.01). Releasability induced by the formyl-containing tripeptide, f-met peptide, was significantly higher in the scleroderma patients than in the controls at the 2 lower concentrations used. No differences in basophil reactivity and sensitivity to f-met peptide and calcium ionophore A23187 were found between patients and normal donors. These results show that spontaneous basophil releasability and releasability in response to IgE cross-linking and activation of interleukin-3 receptors are increased in patients with scleroderma.

Partial purification and characterization of a formylmethionine deformylase from rat small intestine.

A formylmethionine deformylase from rat small-intestinal mucosa has been isolated, characterized and partially purified. The enzyme catalyses the release of equimolar amounts of formate and the free amino acid. The deformylase was active against formylmethionine (Km 7.1 mM) and formylnorleucine, but showed reduced activity against formyl-leucine. It was inactive against a range of other polar and nonpolar formyl-amino acids and against formyl di- and tri-peptides. The Mr of the native enzyme was between 45,000 and 66,000, as determined by h.p.l.c. gel permeation. Further purification of the enzyme either by h.p.l.c. ion-exchange chromatography and concanavalin A-Sepharose or by isoelectric focusing yielded a preparation with one predominant band of Mr 50,000 on SDS/polyacrylamide-gel electrophoresis. Bacteria in the intestine present the host with substantial amounts of formylmethionine (fMet) from proteinase and carboxypeptidase digestion of bacterial formyl-peptides in the intestinal lumen. fMet (0.01-1.0 mM) inhibited translation of a test RNA from brome mosaic virus in vitro, indicating that it could have adverse effects on cellular metabolism. Gut epithelial fMet deformylase may be required for deformylation of this exogenous (bacterial) and also endogenous (mitochondrial) fMet.

Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity.

Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium.

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.

Release of leukotriene B4 from human neutrophils and its relationship to degranulation induced by N-formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan and the ionophore A23187.

The release of leukotriene B4 (LTB4) from human neutrophils and its relationship to degranulation induced by the divalent cation ionophore A23187, serum-treated zymosan (STZ), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and arachidonic acid (AA) have been studied. Greatest release of LTB4, measured by specific radioimmunoassay, occurred in response to A23187 (5-10 ng/10(6) cells); lower concentrations were obtained after incubation with STZ (0.2-0.8 ng/10(6) cells) and AA (0.3-2.6 ng/10(6) cells) and low (0.02 ng/10(6) cells) or not detectable amounts from cells incubated with FMLP. Release of LTB4 induced by STZ, FMLP and submaximal concentrations of A23187 was potentiated by simultaneous addition of AA. Lower amounts (0.06-0.3 ng/10(6) cells) of thromboxane B2 (TXB2) were also released by these stimuli, however this release of TXB2 was not potentiated by exogenous AA. The secretion of beta-glucuronidase induced by A23187, STZ and FMLP was not quantitatively related to release of LTB4 or TXB2 and was not potentiated by exogenous AA. Furthermore, FMLP induced degranulation was cytochalasin B (Cyt B)-dependent, whereas LTB4 release in response to this stimulus was only marginally increased by pretreatment of the cells with Cyt B. These data indicate that LTB4 does not mediate degranulation induced by these stimuli.

Polymorphonuclear leukocyte function in psoriasis: chemotaxis, chemokinesis, beta-adrenergic receptors, and proteolytic enzymes of polymorphonuclear leukocytes in the peripheral blood from psoriatic patients.

Psoriatic patients, particularly those with psoriatic arthritis, have neutrophilic and eosinophilic leukocytosis. Isolated polymorphonuclear leukocytes (PMNLs) from psoriatic patients have normal concentrations of proteolytic enzymes and they have beta-adrenergic receptors of normal density and affinity. PMNLs from psoriatic patients responded normally to the synthetic chemotactic peptide, f-Met-Leu-Phe (formyl-methionine-leucine-phenylalanine). The chemotactic activities of sera from psoriatic patients were similar to those of normal sera. Sera from psoriatic patients enhanced chemokinesis of PMNLs more than normal control sera at a final concentration of 1%; no difference in chemokinetic response between psoriatic and normal sera was found at serum concentrations greater than 2.5%. This study suggests that the peripheral PMNLs from psoriatic patients are normal, but the sera of psoriatic patients has more chemokinetic activity for PMNLs than does normal serum.

Is cytosolic ionized calcium regulating neutrophil activation?

The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.

Decrease in apparent Km for oxygen after stimulation of respiration of rat polymorphonuclear leukocytes.

The respiratory burst of polymorphonuclear leukocytes, induced by the addition of chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) and cytochalasin B was found to consist of two phases. The first phase of very rapid oxygen uptake lasted 1-3 min. and was followed by a second more prolonged phase of lower magnitude. The apparent Km for oxygen of unstimulated cells was 9.6 +/- 0.67 microM, while that of the second phase of stimulation was 3.7 +/- 1.6 microM oxygen. The possibility that lowered oxygen concentrations may regulate polymorphonuclear leukocyte activity in some pathological conditions is discussed.

Enhancement of human neutrophil adherence by synthetic leukotriene constituents of the slow-reacting substance of anaphylaxis.

The functionally predominant constituents of the slow-reacting substance of anaphylaxis (SRS-A), designated leukotrienes C4 and D4 (LTC4 and LTD4), as well as the leucocyte chemotactic factor leukotriene B4 (LTB4) enhance the adherence of human neutrophils to Sephadex G-25. Enhancement of neutrophil adherence was significant at leukotriene concentrations of 3 X 10(-9) M -3 X 10(-7) M, and reached a maximum level for each of the leukotrienes that was similar in magnitude to that evoked by the neutrophil chemotactic peptide N-formyl-methionyl-leucylphenylalanine (FMLP). The leukotrienes and FMLP elicited optimum increases in neutrophil adherence within 1-2 min at 37 degrees. Indomethacin inhibited the increase in neutrophil adherence evoked by LTC4 and LTD4 and the concurrent elevation in the concentration of endogenous thromboxane B2. The smooth muscle contractile and vasoactive factors LTC4 and LTD4, which lack chemotactic activity for leucocytes, are as active as LTB4 in stimulating human neutrophil adherence, and the effect may be mediated in part by neutrophil-derived thromboxane A2.

Selective defect in human neutrophil superoxide anion generation elicited by the chemoattractant N-formylmethionylleucylphenylalanine in pregnancy.

Pregnancy has been associated with alterations of polymorphonuclear neutrophil (PMN) function. Superoxide anion production was studied in pregnant women paired with nonpregnant women of childbearing age. There was a significant decrease in the amount of cytochrome c reduced in response to 1 microM N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) but not to phorbol myristate acetate, 4 or 20 ng/ml. Chemotaxis was also depressed. Binding of tritiated fMet-Leu-Phe to PMNs from pregnant women was not defective. Incubation of normal cells in up to 10(-6) M estradiol or progesterone did not mimic the defect, but 10(-7) M progesterone caused a decrease in chemotaxis. Serum pooled from women with the defect had no effect on superoxide anion production by normal PMNs. PMN rosetting with IgG-sensitized human erythrocytes was normal. Defective production of superoxide anion may contribute to the amelioration of connective tissue disease and increased susceptibility to infection often seen during pregnancy.

Modulation of polymorphonuclear leukocyte function by cetiedil.

Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose-dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.

Activation of the respiratory burst enzyme in human polymorphonuclear leukocytes by chemoattractants and other soluble stimuli. Evidence that the same oxidase is activated by different transductional mechanisms.

Chemoattractant-receptor coupling triggers several biologic responses in phagocytic cells including activation of the respiratory burst. Prior evidence in intact cells implied that stimulation of the respiratory burst by chemoattractants was by a mechanism different from other soluble agents suggesting the possibility that different oxidative enzymes were responsible. We now show that the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and a split fragment of the fifth component of complement (C5a) stimulate an NADPH oxidase activity, measured in the 50,000-g particulate fraction from human polymorphonuclear leukocytes (PMN). Levels of oxidase activity stimulated by the chemoattractants were both time and dose dependent and required the presence of cytochalasin B during stimulation. In contrast, activation by two nonchemotactic stimuli, the ionophore A23187 and phorbol myristate acetate (PMA), did not require cytochalasin B. Temporal patterns of oxidase activation suggested that different stimuli follow different transductional pathways. Chemoattractant-mediated activation was immediate (no lag); peaked by 45 s and declined rapidly to approximately 50% of maximal by 2 min. In contrast, activation by A23187 or PMA had a 15-30-s lag and increased more slowly. Stimulation by A23187 peaked at 5 min, then declined. Stimulation by PMA plateaued at 20 min and did not decline by 90 min. Comparison of Km values for NADPH and NADH obtained by Lineweaver-Burk analysis of the oxidase activity stimulated by N-formyl-methionyl-leucyl-phenylalanine, A23187, and PMA suggested that the same enzyme was activated by all stimuli. Thus, chemoattractants and other soluble stimuli appear to activate the same respiratory burst enzyme in PMN but they utilize different transductional mechanisms and are regulated differently.

Altered polymorphonuclear leukocyte responses in psoriasis: chemotaxis and degranulation.

Chemotactic activities of circulating polymorphonuclear leukocytes (PMN) were determined in twenty patients with psoriasis and twenty healthy control persons. After serial dilution of the complement split product C5a and the formylated tripeptide f-met-leu-phe (FMLP), chemotaxis profiles showed that PMN migration toward both chemotaxins was significantly increased in psoriasis. In addition, PMN from psoriatic patients responded to chemotaxins at much lower concentrations compared with controls. The liberation of (lysosomal) beta-glucuronidase was also determined in cytochalasin B-treated cells confronted with increased concentrations of the chemotaxins. Secretion of this marker enzyme started at lower concentrations in PMN derived from psoriatic patients. Our observations demonstrate migratory and secretory hyper-responsiveness of PMN from psoriatic patients. This may play a role in perpetuating the psoriatic tissue reaction.

Enhancement of chemotactic migration by the local anesthetic tetracaine.

The local amine anesthetic tetracaine added to a suspension of guinea pig or human neutrophilic granulocytes inhibited their random migration in Boyden chambers, but increased their chemotactic migration towards the chemotactic tripeptide f-Met-Leu-Phe, complement-activated normal guinea pig serum, and the eosinophil chemotactic factor ECF. Tetracaine not only increased the distance migrated by the leading cells, it also caused more cells to leave the upper filter surface and to migrate into the filter. The effect required the presence of the drug; cells preincubated with tetracaine and washed did not differ from control cells. It is suggested that tetracaine specifically enhanced a mechanism operative in a cell's response to a concentration gradient of a chemotactic factor.