E Schiffmann

Leucocytes such as neutrophils are attracted by N-formylmethionine, but not by methionine. Di- and tripeptides containing formylmethionine are strong attractants for both neutrophils and macrophages, whereas the corresponding nonacylated compounds are not chemotactic. The formylated peptides may be related to an incompletely characterized chemotactic material normally produced by bacteria which attract the same animal cells.

When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [1-14C]arachidonic acid previously incorporated into phospholipids. The inhibitory potencies of glucocorticoids on phospholipase A2 activity correlated well with their anti-inflammatory activities and their abilities to bind to glucocorticoid receptors. Inhibitors of RNA and protein synthesis suppressed the inhibitory effect of glucocorticoids on phospholipase A2 activity. Digestion of the glucocorticoid-treated cells by Pronase overcame the inhibitory activity. Phospholipase A2 activity induced by Ca2+ ionophore A23187 was not affected by Pronase treatment. Gel filtration of proteins from neutrophil membranes labeled with [3H]lysine showed an induction of protein(s) (about 40,000 daltons) after glucocorticoid treatment. This protein inhibited a partially purified pancreatic phospholipase A2 and reduced the peptide-initiated chemotactic response of neutrophils.

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.

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.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.