Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet-derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.

https://rupress.org/jcb/article-pdf/92/2/584/1389555/584.pdf

Platelet-derived growth factor in chemotactic for fibroblasts.

Formyl peptide receptors (FPRs) are a small group of seven-transmembrane domain, G protein-coupled receptors that are expressed mainly by mammalian phagocytic leukocytes and are known to be important in host defense and inflammation. The three human FPRs (FPR1, FPR2/ALX, and FPR3) share significant sequence homology and are encoded by clustered genes. Collectively, these receptors bind an extraordinarily numerous and structurally diverse group of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. N-formyl peptides, which are encoded in nature only by bacterial and mitochondrial genes and result from obligatory initiation of bacterial and mitochondrial protein synthesis with N-formylmethionine, is the only ligand class common to all three human receptors. Surprisingly, the endogenous anti-inflammatory peptide annexin 1 and its N-terminal fragments also bind human FPR1 and FPR2/ALX, and the anti-inflammatory eicosanoid lipoxin A4 is an agonist at FPR2/ALX. In comparison, fewer agonists have been identified for FPR3, the third member in this receptor family. Structural and functional studies of the FPRs have produced important information for understanding the general pharmacological principles governing all leukocyte chemoattractant receptors. This article aims to provide an overview of the discovery and pharmacological characterization of FPRs, to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature, and to discuss unmet challenges, including the mechanisms used by these receptors to bind diverse ligands and mediate different biological functions.

https://pharmrev.aspetjournals.org/content/pharmrev/61/2/119.full.pdf

International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.

Cellular constituents of the alveolar-capillary wall may be key participants in the recruitment of polymorphonuclear leukocytes to the lung through the generation of the novel neutrophil chemotactic peptide interleukin-8 (IL-8). This interaction appears to occur via the ability of human alveolar macrophage (AM)-derived monokines, tumor necrosis factor (TNF), and interleukin-1 (IL-1) to induce gene expression of IL-8 from pulmonary type II-like epithelial cells (A549). Northern blot analysis demonstrated that steady-state IL-8 mRNA expression, by either TNF- or IL-1 beta-treated A549 cells, occurred in both a dose- and time-dependent fashion. Similarly, extracellular antigenic IL-8, as assessed by specific ELISA, was expressed from TNF- or IL-1 beta-stimulated epithelial cells in a time-dependent fashion with maximal IL-8 antigen detected at 24 h poststimulation. Immunohistochemical staining utilizing rabbit anti-human IL-8 antibody identified immunoreactive, cell-associated IL-8 antigen as early as 8 h post-TNF or IL-1 beta stimulation. A549-generated neutrophil chemotactic bioactivity paralleled IL-8 steady-state mRNA levels. Signal specificity was demonstrated in this system as IL-8 mRNA or protein expression by lipopolysaccharide (LPS)-treated A549 cells was not different from unstimulated cells. Although LPS did not serve as a direct stimulus for the production of IL-8 by type II-like epithelial cells, the condition media from LPS-challenged AM induced a significant expression of IL-8 mRNA by the A549 cells. 24-h conditioned media from LPS-treated cells was as potent as either IL-1 beta or TNF in generating steady-state IL-8 mRNA by A549 cells. Preincubation of LPS-treated AM-conditioned media with anti-human TNF or IL-1 beta neutralizing antibodies resulted in significant abrogation of IL-8 gene expression by A549 pulmonary epithelial cells. These findings demonstrate potential cell-to-cell communication circuits that may be important between AMs and pulmonary epithelial cells during the recruitment phase of acute lung inflammation.

/pdf/interleukin-8-gene-expression-by-a-pulmonary-epithelial-cell-2vyqxdafb7.pdf

Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung.

The HL-60 human promyelocytic leukemia cell line can be induced to terminally differentiate to mature myeloid cells sharing a number of functional characteristics with normal granulocytes including response to chemoattractants, development of complement receptors, phagocytosis, superoxide production, and nitroblue tetrazolium dye reduction. Hence the Me2SO-induced HL-60 cells provide a unique in vitro model for studying various important aspects of human myeloid cell differentiation.

/pdf/normal-functional-characteristics-of-cultured-human-4sa00g7y0d.pdf

Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide.

The biological properties of a neutrophil-activating factor (NAF), which was recently identified as a novel peptide of approximately 6,000 mol wt, are described. NAF is produced de novo by human blood monocytes upon stimulation with LPS, PHA, and Con A. It induces two main responses in human neutrophils, i.e., exocytosis (release from specific granules in normal, and from specific and azurophil granules in cytochalasin B-treated cells) and the respiratory burst (formation of superoxide and hydrogen peroxide). The action of NAF appears to be mediated by a surface receptor as shown by the following observations. (a) NAF induces a rapid and transient rise in cytosolic free Ca2+; (b) interaction with NAF results in desensitization, since the cells do not respond to a second NAF challenge; and (c) the respiratory burst elicited by NAF is similar in onset, and time course to that induced by C5a or FMLP. The NAF receptor can be distinguished from the receptors of C5a, FMLP, platelet-activating factor, and leukotriene B4 by the lack of cross-desensitization. Unlike C5a, the other host-derived neutrophil-activating peptide, NAF is not inactivated by serum and thus presumably accumulates in inflamed tissue.

https://rupress.org/jem/article-pdf/167/5/1547/1097992/1547.pdf

A novel neutrophil-activating factor produced by human mononuclear phagocytes.

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.

/pdf/the-structure-activity-relations-of-synthetic-peptides-as-2ph1qdrtu4.pdf

S H Zigmond

Papers

The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils.