Elliott Schiffmann

Role of secretory events in modulating human neutrophil chemotaxis.

The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.

Neuropeptides are chemoattractants for human tumor cells and monocytes: A possible mechanism for metastasis

Bombesin (BN), a tetradecapeptide neuropeptide growth factor, is shown to be a potent (ED50 of 5 X 10(-12) M) chemoattractant for human monocytes and small cell lung carcinoma cells (SCCL). These effects are BN receptor-mediated since potencies of several BN analogs to induce chemotaxis and to inhibit [125I-tyr4] BN binding activity correlate well (P less than 0.001). As has been demonstrated for other BN receptor-mediated effects, carboxy-terminal amino acids are required for optimum biological activity. BN is not an exclusive chemoattractant for SCCL cells but was also active in promoting migration of other, but not all, lung tumor cells. Other neuropeptides, such as beta-endorphin, substance P, and arg-vasopressin, are also shown to be chemoattractants for SCCL cells, with EC50s also in the 10(-12) M range. The ability of these ligands to effect monocyte and some tumor cell migration suggest a role for neuropeptides in inflammation and metastasis. In the latter case, tumor cells, in response to neuropeptide chemical gradients, may become localized at specific body sites. Neuropeptide release, in response to cognitive or other stimuli, may thereby modify cell migratory patterns. Additionally, such hormones may influence early developmental events such as tissue organization and histogenesis.

Insulin-like growth factors stimulate chemotaxis in human melanoma cells

Insulin and insulin-like growth factors stimulate motility in the highly metastatic human melanoma cell line, A2058. Insulin-like growth factor-I (IGF-I) is the most potent with a maximal response at a concentration of 10 nM compared to the activities of insulin and insulin-like growth factor-II (IGF-II) which peak at 300-400 nM. Using checkerboard analysis, the responses to IGF-I and insulin are predominantly chemotactic, although insulin had a significant chemokinetic component. Pertussis toxin does not inhibit the response to any of these polypeptides. However, in previous studies, it was shown that the motile response to autocrine motility factor from these same A2058 cells was markedly inhibited by pertussis toxin. 125I-labelled IGF-I binds saturably and specifically to the A2058 cells. Scatchard analysis indicates a high binding affinity (Kd approximately 3 x 10(-10) M) and an estimated 5000 receptors/cell. These studies indicate that in addition to their mitogenic properties, certain growth factors may profoundly enhance metastasis of tumor cells by their ability to induce motility.

Biochemistry of Phagocyte Chemotaxis

Publisher Summary This chapter presents the biochemistry of phagocyte chemotaxis. Chemoattractants induce profound changes in their target cells, such as increased adherence, spreading, sustained locomotion, and a markedly asymmetric morphology even in the absence of a gradient. Studies with the synthetic polypeptides suggest that certain general reactions occur during chemotaxis that underlies these phenomena. There are probably interactions between stereospecific receptors on the cell surface and the various attractants. This interaction triggers a series of reactions that amplify the signal. In the case of the migrating phagocyte, methylation reactions such as protein carboxyl-methylation of membrane proteins and alterations in the proportions of membrane phospholipids specifically stimulated by attractants may amplify chemotaxis. The respective neutralization of charge and the variation in the composition of essential components of the membrane could cause marked changes in the cell. In addition, both activation of surface proteolytic activity and changes in the availability of receptors occur, suggesting that an attractant can induce regulation of its own detection by the cell.

Inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor of blastocyst rejection

Uteroglobin, a steroid-dependent secretory protein first discovered in the rabbit uterus during early pregnancy, is a potent phospholipase A2 inhibitor. We found that uteroglobin also inhibited human and rabbit phagocyte chemotaxis in response to formyl peptide attractants in a dose-dependent manner. Half-maximal inhibition was at 1.2 microM. Uteroglobin did not compete with a formyl peptide for its receptor but inhibited internalization of radiolabeled formyl peptide. Uteroglobin appears to inhibit chemotaxis by a mechanism different from that of dansylcadaverine, a well studied inhibitor of endocytosis. Unlike dansylcadaverine, uteroglobin did not have any effect upon the synthesis of phosphatidylcholine or phosphatidylinositol. It is suggested that uteroglobin may protect trophoblastic cells from the defense system of the host not only by binding to antigenic determinants of embryonic cells but also by impairing migration of phagocytes, one of the primary components of the immune defense system. These results may explain why embryonic cells do not elicit an inflammatory response in the uterine endometrium during pregnancy.

Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells

The biochemical pathways through which tumor cell locomotion is mediated are poorly understood. Autocrine motility factor (AMF), which is produced by and stimulates motility in A2058 human melanoma cells, was used to characterize phosphoinositide (PtdIns) metabolism activated in association with tumor cell motility. AMF stimulated up to a 400% increase in de novo incorporation of 3H-myo-inositol into cellular lipids beginning 40 minutes after exposure. In cells prelabeled with 3H-myo-inositol, AMF stimulated a 200% increase in total inositol phosphates (inositol monophosphate, InsP1; inositol bisphosphate, InsP2; inositol trisphosphate, InsP3) after 90 minutes of exposure, with a 300% maximal increase in InsP3 at 120 minutes. InsP1 and InsP2 were maximally increased 130% of control values. Treatment with AMF stimulated a parallel dose-dependent increase in both motility and PtdIns levels. We have shown previously that the A2058 motile response to AMF is inhibited markedly by cell pretreatment with pertussis toxin (PT). Inositol phosphate production was inhibited by a 2-hour pretreatment of cells with PT (0.5 microgram/ml). PT treatment of A2058 membranes was associated with ADP-ribosylation of a 40-kDa protein consistent with the presence of an alpha subunit of a guanine nucleotide-binding protein (G protein). These data indicate that AMF elicits increases in cell motility and phosphoinositide metabolism via a PT-sensitive G protein signal transduction pathway.

Cell motility, a principal requirement for metastasis.

In studying the role of motility in the metastasis of tumor cells, we have described an autocrine motility factor. This agent, which stimulates random motility, probably contributes to the initial dissociation of the cells from the primary tumor mass. Extracellular matrix components, via several different mechanisms, may facilitate the crossing of biological barriers by the cells prior to the entry into the circulation. In locating at new sites, the tumor cells may be induced to exit from the circulation in response to attractants such as IGFs that could emanate from the target organ. These same growth factors could then stimulate cellular proliferation for another metastatic cycle. It is quite probable that detection of AMF may provide a new tool in cancer diagnosis. The complete characterization of AMF may also yield valuable therapeutic approaches: design of low molecular size antagonists of the attractants and antibodies that might be effective therapeutically as well as diagnostically. It seems clear, in any event, that immobilizing the tumor cell may be a crucial step in inhibiting metastasis.

Cyclocreatine inhibits stimulated motility in tumor cells possessing creatine kinase

Cyclocreatine (1‐carboxymethyl‐2‐iminoimidazolidine), an analog of creatine and a substrate for creatine kinase (EC 2.7.3.2), inhibits the stimulated motility of tumor cells which possess creatine kinase. A2058‐055 human melanoma cells, transfected with a creatine kinase gene, showed an 80–90% reduction in chemotactic response to type IV collagen when incubated overnight in the presence of 10 mM cyclocreatine (p < 0.0001 for n = 8 experiments). This inhibitory effect of cyclocreatine can be partially reversed by addition of creatine to the overnight cell treatment. Non‐transfected cells, with very low levels of creatine kinase, were not significantly inhibited. Further experiments utilizing type IV collagen as attractant demonstrated that cyclocreatine inhibited the chemokinetic (91%) and the haptotactic (73%) responses and the in vitro invasion of A2058‐055 cells through Matrigel‐coated membranes (88%). In addition, motility stimulation of A2058‐055 cells by either autotaxin or fibronectin was markedly inhibited by cyclocreatine. DU‐145 prostatic tumor cells, which express endogenous creatine kinase, also have a reduced motility response to either autotaxin or epidermal growth factor induced motility in the presence of cyclocreatine. Int. J. Cancer 78:46–52, 1998. Published 1998 Wiley‐Liss, Inc.

Antibiotics and peptides with agonist and antagonist chemotactic activity

Abstract It has been found that the polypeptide antibiotics gramicidin S, tyrocidin and bacitracin, containing Leu-Phe or Ile-Phe sequences, are chemoattractants for neutrophils. Related synthetic pentapeptides containing the sequence Leu-Phe have stronger biological activities, provided the N-terminal residue is acylated. The formylated peptide f-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe is a potent agonist (4 × 10 −9 M) whereas the t-butyloxycarbonyl analog is a good antagonist (8 × 10 −7 M).

A sensitive screening assay for secreted motility-stimulating factors.

Secreted motility-stimulating factors are often expressed and secreted at low concentrations that are difficult to detect by Northern or Western blot analysis. Autotaxin (ATX) is a tumor-secreted autocrine motility-stimulating factor that has been associated with tumor invasion and metastatic potential. Although ATX has a number of enzymatic activities, it is most sensitively detected by its induced chemotactic response. After transfecting ATX cDNA into NIH3T3 fibroblasts, we developed a motility-based method to screen the resulting cloned cells for secretion of active protein. We placed the cloned and transfected cells into the bottom wells of a modified Boyden chamber and placed responding cells (A2058 human melanoma cells) into the upper wells. After overnight incubation, the membrane that separated the two chambers was removed and stained. Simple densitometry measurements were sufficiently accurate to determine which clones secreted active protein. Utilizing this method, 4 positive cell lines were chosen out of 36 tested clones. Further tests on the expanded cell lines determined that all 4 were secreting ATX. Thus, this modified Boyden chamber assay appears to provide a rapid and highly adaptable means to identify cells that secrete motility-stimulating factors.