Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin. O Vindrola, … , S Finkielman, V Nahmod J Clin Invest. 1990;86(2):531-537. https://doi.org/10.1172/JCI114740. Research Article In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met- enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect. […] Find the latest version: https://jci.me/114740/pdf Proenkephalin System in Human Polymorphonuclear Cells Production and Release of a Novel 1.0-kD Peptide Derived from Synenkephalin Osvaldo Vindrola,* Maria Rosa Padros,t Aida SterinnPrync,* Ariel Ase,* Samuel Finkielman,* and Victor Nahmod* *Instituto de Investigaciones Medicas, Seccion de Sustancias Vasoactivas, 1427 Buenos Aires, Argentina; and tHospital de Clinicas "Jose' de San Martin, " Immunogenetica, 1120 Buenos Aires, Argentina Abstract possible exception of a distinct gelatinase-containing popula- tion (8), those granules and their contents have not been well In the hematopoietic system a pluripotent stem cell generates defined. precursors for lymphoid and myeloid lineages. Proenkephalin- Ligand binding to neutrophil surface receptors such as derived peptides were previously detected in differentiated chemotactic peptides, C5a, and phorbol myristate acetate, as lymphoid cells. We have studied whether the proenkephalin well as perturbation of the membrane with calcium ionophore system is expressed in a typical differentiated cell of the my- A23 187, have been shown to cause the release of the granular eloid lineage, the neutrophil. Human peripheral polymorpho- content (9-1 1). nuclear cells contain and release proenkephalin-derived pep- Opioid peptides are involved in several events of the im- tides. The opioid portion of proenkephalin (met-enkephalin- mune response (12, 13). These molecules induce changes on containing peptides) was incompletely processed, resulting in the activity of lymphocytes, macrophages, mast cells, and the absence of low molecular weight products. The nonopioid polymorphonuclear cells. On the other hand, opioid peptides synenkephalin (proenkephalin 1-70) molecule was completely may induce a local analgesic effect in acute inflammatory pro- processed to a 1.0-kD peptide derived from the COOH-termi- cesses through the activation of peripheral receptors (14, 15). nal. This molecule was characterized in neutrophils by bio- These studies have been mainly focused on peptides arising chemical and immunocytochemical methods. The chemotactic from the nervous system or endocrine glands. peptide FMLP and the calcium ionophore A23187 induced the The proenkephalin gene is expressed in cells of the nervous release of the proenkephalin-derived peptides, and this effect (16), reproductive (17), and hematopoietic (18) systems. In the was potentiated by cytochalasin B. The materials secreted latter, a pluripotent stem cell generated precursors for lym- were similar to those present in the cell, although in the super- phoid and myeloid lineages. In this system proenkephalin-de- natant a higher proportion corresponded to more processed rived peptides were detected in differentiated lymphoid cells products. The 1.0-kD peptide was detected in human, bovine, (18-20). Peripheral blood lymphocytes produced and released and rat neutrophils, but the chromatographic pattern of synen- met-enkephalin and synenkephalin (proenkephalin 1- kephalin-derived peptides suggests a differential posttransla- 70) containing peptides when activated with a mitogenic tional processing among species. These findings demonstrate agent (20). the existence of the proenkephalin system in human neutro- In this work we have analyzed whether the proenkephalin phils and the production and release of a novel 1.0-kD peptide system is expressed in one typical differentiated cell from my- derived from the synenkephalin molecule. The presence of eloid lineage, the neutrophil. Production and release of met- opioid peptides in neutrophils suggests their participation in enkephalin- and synenkephalin-containing peptides induced the inflammatory process, including a local analgesic effect. (J. by the chemotactic peptide FMLP and by calcium ionophore Clin. Invest. 1990. 86:531-537.) Key words: polymorphonu- A23 187 were studied. The production and release of a novel clear cell- proenkephalin system- synenkephalin-derived pep- 1.0-kD peptide derived from the synenkephalin molecule 'was tide detected. Introduction Methods Polymorphonuclear leukocytes play a major role in host de- Cell isolation. Human neutrophils were prepared from fresh blood fence against microbial infections (1-3) and are one of the samples (100 ml) obtained from healthy volunteers. Neutrophils were primary mediators of the acute inflammatory response (4, 5). purified by dextran sedimentation followed by centrifugation through Basically, the neutrophil contains two distinct populations of a layer of Ficoll-Hypaque as described by Boyum (21). The granulo- secretory granules, the primary (azurophil) and the secondary cyte pellet was washed in saline, and the contaminating erythrocytes (specific) granules (6). In addition, tertiary and quaternary were eliminated by hypotonic shock. Preparations obtained in this granule populations have been suggested (7, 8), but with the manner contained 95% or more neutrophils. Cell viability was tested by the ability to exclude trypan blue. Bovine neutrophils were purified from fresh blood (250 ml) by Address correspondence to Dr. Osvaldo Vindrola, Instituto de Investi- differential centrifugation and a gradient of Ficoll-Hypaque (7), com- gaciones Medicas, Seccion de Sustancias Vasoactivas, Donato Alvarez bined with the hypotonic lysis of contaminating erythrocytes. This 3150, 1427 Buenos Aires, Argentina. preparation also contained 95% or more neutrophils. Receivedfor publication 29 January 1990. Rat bone marrow cells were obtained from the femur. After cutting off the epiphyses, the marrow was extruded by forcing a cold salt J. Clin. Invest. solution through with a small needle and syringe. Cell clumps were © The American Society for Clinical Investigation, Inc. broken up by repeated gentle aspiration through a 25-gauge hypoder- 0021-9738/90/08/531/07 $2.00 mic needle. Neutrophils from cell suspensions were isolated as de- Volume 86, August 1990, 531-537 scribed for human blood. Proenkephalin System in Human Neutrophils 531 Exocytosis experiments. 2 X 107 polymorphonuclear cells sus- Cells were fixed with 3% glutaraldehyde in PBS for 60 min at 4VC, pended in 1 ml of Krebs Ringer phosphate (KRP),' containing (in washed three times in PBS, and immunocytochemically stained as millimolar) 131 NaCi, 5.2 KC1, 1.3 MgSO4, 0.9 CaC12, and 15.7 follows: (a) Exhaust of endogenous peroxidase activity. Incubation in NaPO4 buffer (pH 7.4), were incubated at 370C for 0-30 min in the 3% H202 (in ethanol) for 10 min at room temperature, then in 9% presence or absence of stimuli at the indicated concentrations. Incu- H202 (in anhydrous methanol) for 20 min at 4VC; rinsed three times bation was terminated by rapid cooling on ice and centrifugation at with PBS. (b) Incubation with primary antiserum against COOH-ter- 10,000 g for 5 min. The cell-free supernatant was immediately incu- minal synenkephalin-recognizing antiserum (1:200) in PBS with bated in boiling water for 15 min. The pellet was stored at -70'C until 0.01% BSA for 18 h at 4VC; rinsed three times with PBS. (c) Incubation used. Cells that were treated with cytochalasin B (5 Mg/ml) were prein- with peroxidase-labeled goat anti-rabbit IgG (1:50) in PBS-BSA buffer cubated with this agent for 5 min at 370C before addition of the for 10 min at room temperature; washed three times with PBS. (d) stimulus. The chemotactic peptide, FMLP, and calcium ionophore Incubation with diaminobenzidine and H202 as peroxidase reaction A23 187 were used as exocytotic agents. substrates for 5 min at room temperature. (e) Cells were finally stained Total and free met-enkephalin