Identification of novel peptides that stimulate human neutrophils

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.

Lysophosphatidylglycerol inhibits formyl peptide receptor like-1-stimulated chemotactic migration and IL-1beta production from human phagocytes

In this study, we observed that lysophosphatidylglycerol (LPG) completely inhibited a formyl peptide receptor like-1 (FPRL1) agonist (MMK-1)-stimulated chemotactic migration in human phagocytes, such as neutrophils and monocytes. LPG also dramatically inhibited IL-1beta production by another FPRL1 agonist serum amyloid A (SAA) in human phagocytes. However, LPG itself induced intracellular calcium increase and superoxide anion production in human phagocytes. Keeping in mind that phagocytes migration and IL-1beta production by FPRL1 are important for the induction of inflammatory response, our data suggest that LPG can be regarded as a useful material for the modulation of inflammatory response induced by FPRL1 activation.

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Efecto del levamisol en la actividad microbicida y quimiotaxis en células polimorfonucleares / The effect of levamisol on the functions of chemotaxis and bacterial activity

Antecedentes: la septicemia neonatal puede tener una mortalidad del 20 por ciento en recién nacidos pequeños para su edad gestacional. Esta predisposición a las infecciones se ha explicado por alteraciones inmunológicas. El propósito de este trabajo es conocer el efecto del levamisol en las funciones de quimiotaxis y actividad bactericida de las células polimorfonucleares en recién nacidos pequeños para su edad gestacional. Material y método: se estudiaron 20 recién nacidos de término. Diez eran pequeños para su edad gestacional y los otros 10 tenían peso adecuado. Se midió la actividad microbicida y la quimiotaxis; se hicieron comparaciones entre los grupos con pruebas estadísticas no paraméticas. Resultados: en el grupo de neonatos pequeños para su edad gestacional la actividad microbicida fue semejante a la del grupo testigo, pero la actividad quimiotáctica estuvo disminuida (p < 0.05)

In vitro effect of complete Staphylococcus aureus extract on chemotaxis of polymorphonuclear cells in children with bronchial asthma and chronic relapsing upper airways infections

Se midieron la quimiocinesis y quimiotaxis en células polimorfonucleares (PMN) obtenidas de 51 niños de 1 a 8 años de edad, de uno y otro sexo. Grupo de casos: 41 niños con asma no alérgica e infecciones crónicas recurrentes de las vías aéreas superiores; los criterios diagnósticos fueron antecedentes de sibilancias relacionadas con un episodio de infección de las vías aéreas superiores y pruebas cutáneas negativas. Grupo testigo: 10 niños sanos. Se obtuvo una muestra de 10 ml de sangre venosa periférica. Los PMN se incubaron con solución de Hank para medir la quimiocinesis y con C5a y extracto de Staphylococcus aureus para medir quimiotaxis. Los valores de quimiocinesis en los niños sanos testigos y en los pacientes con asma bronquial no alérgica fueron de 46.0 ñ 7.1 vs 23.8 ñ 6.1 µm (p < 0.01). Los valores de quimiotaxis estimulada con C5a en los niños sanos testigos y en los pacientes con asma bronquial no alérgica fueron de 91.0 ñ 21.3 vs 92.3 ñ 21.0 µm (ns), y los valores de quimiotaxis estimulada con extracto de Staphylococcus aureus fueron de 97.0 ñ 22.4 vs 92.0 ñ 23.0 µm (ns). Estos resultados sugieren que los PMN de niños con asma no atópica tienen quimiocinesis reducida. Después de un estímulo quimiotáctico con C5a y extracto bacteriano, la movilidad de los leucocitos se corige y alcanza valores similares a los coexistentes en niños sanos

Efecto de la cocaína y las catecolaminas sobre la quimiotaxis y la quimioluminiscencia de neutrófilos humanos in vitro

El objetivo fue determinar el efecto de la cocaína sola y mezclada con catecolaminas sobre la quimiotaxis y la explosión metabólica de los neutrófilos humanos y evaluar si la cocaína se une a estas células. La quimiotaxis se realizó en un gel de agarosa utilizando el péptido quimiotáctico FMLP. La explosión respiratoria se determino mediante la cuantificación de fotones (quimioluminiscencia) emitidos por las celulas activadas con PMA. Para cada prueba, las celulas se expusieron a concentraciones de cocaína de 1 a 1000ug/m y de catecolaminas 0.001 y 0.001 ug/mL y luego a la mezcla de 1000 ug/mL de cocaina con 0.001 ug/mL de catecolamina. La union de la cocaina a las celulas se evaluó mediante un ensayo de competición, con cocaína tritiada 10 nM y cocaína no radioactiva 500 uM. Los resultados no mostraron efecto de la cocaína quimiotaxis, pero sí aumento estadisticamente significativo (p=0.004), de la quimioluminiscencia de los neutrófilos tratados con 1000ug/mL de cocaína. Los valores de quimioluminiscencia sin cocaína fueron 1.2 x 10 a la 6 ñ 10 a la 4 cuentas por minuto (cpm) y con cocaina 1.7 x 10 a la 6 ñ1 x 10 a la 5 cpm. Las catecolaminas no modificaron los resultados obtenidos con la cocaina sola. La union de la cocaína tritiada a los neutrofilos tuvo un valor de 3.893 ñ 343 cpm, que disminuyó significativamente a 301 ñ 124 cpm en el ensayo de competición (p=0.008). Se demostró un incremento en la actividad metabólica de los neutrofilos inducido por cocaína, al parecer por su union a estas celulas. Estos resultados no son extrapolables a la clínica; sin embargo, el aumento de quimioluminiscencia debe ser tenido en cuenta, puesto que la exposición recurrente a la cocaína podría eventualmente conducir al mismo efecto

The inhibitory effect of methotrexate on PAF-induced neutrophil and eosinophil locomotion in asthmatic patients.

We have tested the effect of methotrexate (MTX) on platelet activating factor (PAF)-induced neutrophil and eosinophil locomotion, neutrophil leukotriene B4 (LTB4) generation and mononuclear cell DNA synthesis. Neutrophils from patients treated with low dose methotrexate showed reduced PAF-induced chemotactic responses (727.8 +/- 72.2/10 HPF vs 481.9 +/- 87.3/10 HPF, p < 0.05). Both MTX and the specific PAF antagonist BN-52021 significantly inhibited PAF-induced eosinophil and neutrophil locomotion in a dose-dependent manner. MTX also reduced calcium ionophore-driven LTB4 generation from the neutrophils of asthmatics (358.9 +/- 39.5 pg/10(6) cells vs 240.1 +/- 29.1 pg/10(6) cells, p < 0.05) and attenuated PHA-induced mononuclear DNA synthesis as shown by a reduction in 3H-thymidine uptake and propidium iodide staining. These findings support the view that the beneficial effects of MTX in asthma may be due not only to its anti-mitotic effects on the proliferation of mononuclear cells but also to direct effects on granulocyte locomotion and production of LTB4.

Chemotactic detection of incipient ovarian follicular atresia.

Chemotactic activity was measured in the follicular fluid collected from normal and atretic Graafian follicles isolated from the rat ovaries. The atresia of Graafian follicles was induced by pentobarbitone injections for 3 days beginning the day of proestrous. The chemotactic activity, as measured by direct morphological evaluation of cellular locomotion of individual cells and Boyden leading front assay, was significantly higher in follicular fluid from atretic follicles and it showed a progressive increase from day 1 to day 3 of blockade of ovulation. In vitro exposure of blocked follicles to PMSG and hCG on day 1, 2 and 3 failed to alter the chemotactic response of leukocytes towards follicular fluid of atretic follicles. Increased chemotaxis in the follicular fluid after 24 hr of blockade of ovulation appears to form an important criterion to identify atretic follicles well in advance, before the morphological symptoms of degeneration become apparent and the incipient change once induced in follicles is not reversed by gonadotropins.