Zvi Bar-Shavit

Enhancement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence.

Abstract The undecapeptide Substance P stimulates phagocytosis by mouse macrophages and human polymorphonuclear leukocytes. The activity of Substance P resides in its N-terminal tetrapeptide protion. Substance P and its N-terminal tetrapeptide are as active as tuftsin in their phagocytosis-stimulating activity and compete with tuftsin for its binding sites. The phagocytosis-enhancing activity of Substance P may play a role in inflammatory processes of neural origin where the involvement of the peptide has been implicated.

Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi

The attachment of Escherichia coli and Salmonella typhi to mouse peritoneal macrophages was inhibited by D-mannose, methyl α-D-mannopyranoside and yeast mannan, but not by any other sugar tested. D-Mannose and its derivatives also inhibited the attachment of E. coli to human polymorphonuclear leucocytes. Mannan inhibited phagocytosis when preincubated with E. coli, but not when preincubated with leucocytes. Attachment of opsonized bacteria to leucocytes was not inhibited by D-mannose or methyl α-D-mannopyranoside nor by any other sugar tested. Our results suggest that the surface of phagocytes, like that of epithelial cells, contains D-mannose residues which serve for the attachment of certain Gram negative bacteria.

On the mechanism of action of the phagocytosis-stimulating peptide tuftsin.

SummaryIncubation of human polymorphonuclear leukocytes (PMNL) or thioglycollate-stimulated mouse peritoneal macrophages with the phagocytosis-stimulating peptide, tuftsin (2.5 × 10−7 M, at 37 °C), caused an increase of 89–90% in intracellular cGMP levels, accompanied by a decrease of 20–25% in intracellular cAMP levels. Significant changes in cyclic nucleotide levels were detectable after 4 min of incubation, were maximal at 10–20 min and persisted for at least 60 min. The concentration dependences of the stimulatory effect of tuftsin on modulation of intracellular levels of cyclic nucleotides and on phagocytosis are similar, suggesting a cause and effect relationship between the two phenomena. This notion is further supported by the finding that 8-Br-cGMP and 8-Br-cAMP elicit stimulatory and inhibitory effects on macrophage phagocytosis, respectively. Measurement of 45Ca2+ influx into PMNL and macrophages in the presence and absence of tuftsin did not reveal any change in 45Ca2+ uptake from the media. However, tuftsin did enhance release of 45Ca2+ from cells preloaded with the isotope. Results suggest that modulation of both the amount of cell-associated 45Ca2+ and the intracellular levels of cyclic nucleotides are key steps in the mechanism by which tuftsin augments phagocytosis.

Neurotensin — macrophage interaction: Specific binding and augmentation of phagocytosis

Abstract Neurotensin (NT) was found to bind to thioglycollate-elicited mouse peritoneal macrophages and to modulate their phagocytic capability. A Scatchard analysis of the binding curve of [ 3 H]NT suggests the presence of two subclasses of binding sites having a.−a K D of 0.9 nM (4800 sites per cell) and b.−a K D of 28 nM (33500 sites per cell). NT as well as two of its partial sequences, NT(8–13) and NT(6–13) competed with [ 3 H]NT for its binding whereas NT(1–10) was rather ineffective. [ 3 H]NT was also competitively displaced by tuftsin, substance P (SP) and by SP (1–4). The K I values estimated for all the above competitive inhibitors of [ 3 H]NT binding (except for NT) suggest interaction with the relatively low affinity sites. NT exerts a biphasic effect on the phagocytic response of macrophages. At a concentration range of 10 −14 –10 −9 M NT had a dose dependent augmenting effect on the phagocytic response (up to 2 fold), further increase in concentration (>10 −9 M) of NT resulted in a gradual decrease of the augmented response which disappears at 10 −7 M NT. NT(8–13), NT(6–13) as well as NT(1–10) augment the phagocytic response of macrophages. However the maximal effect with these peptides was attained at about 10 −7 M and stayed at the same level at concentrations up to 10 −5 M. The phagocytosis augmenting dose-response curves of these peptides resembled that of tuftsin and SP, two unrelated peptides. It is suggested that NT-phagocyte interaction may be of relevance in the regulation of the functions of phagocytic cells.

Neurotensin modulates human neutrophil locomotion and phagocytic capability

Neurotensin (NT) was found to induce oriented locomotion and augment the phagocytic capability of human blood neutrophils over 10−11–10−7 M. The tridecapeptide also causes Ca2+ extrusion from neutrophils, very likely as a result of intracellular Ca2+ mobilization. It is suggested that the NT‐mediated functional modulation of neutrophils correlates with the capacity of NT to affect the intracellular compartmentalization of Ca2+. Peripheral NT‐elicited phenomena such as vasodilation, enhanced vascular permeability, mast cell degranulation and the enhancement of directional migration and phagocytosis of neutrophils described here, classify NT as a typical mediator of inflammation.

Enhancement of phagocytosis by neurotensin, a newly found biological activity of the neuropeptide.

Specific binding of neurotensin (NT) to mouse peritoneal thioglycollate-elicited macrophages and macrophages differentiated in vitro from bone marrow cells was demonstrated and characterized. NT binding to these phagocytes modulated their phagocytic capacity in a biphasic manner. At concentrations of 10(-14) to 10(-9) M NT, a dose-dependent augmentation of phagocytosis (up to 2-fold) was observed. Further increases in the concentration of NT resulted in a gradual decrease of the augmented response until the basal phagocytic activity (in the absence of NT) was reached. Three partial sequences of NT, NT (8-13), NT (6-13) and NT (1-10), were also effective in augmenting the phagocytic response of thioglycollate elicited macrophages, but the maximal effect was attained at about 10(-7) M and stayed at that level up to a concentration of 10(-5) M. The activity of the three NT partial sequences was comparable to that of substance P and tuftsin. Scatchard analysis of (3H)NT binding to macrophages suggested the existence of two populations of binding sites, a major population of relatively low affinity binding sites and a small population of high affinity binding sites. NT (8-13), NT (6-13), substance P and tuftsin competed with (3H)NT binding to the low affinity sites with a comparable KI to that of NT. NT (1-10) did not compete for the binding at the low affinity sites. It is suggested that NT binding to the high affinity sites leads to enhancement of phagocytosis, whereas its binding to the low affinity sites leads to inhibition of the augmented response. However, the low affinity sites are the sites of interaction of NT (8-13), NT (6-13), substance P and tuftsin with the phagocytes and their saturation with the peptides leads to augmentation of phagocytosis.

Functional tuftsin binding sites on macrophage-like tumor line P388DI and on bone marrow cells differentiated in vitro into mononuclear phagocytes

SummaryMouse bone marrow cells, differentiated in vitro into mononuclear phagocytes (BMDMP), possess functional tuftsin binding sites, i.e. both tuftsin binding capacity and augmented phagocytic response related to tuftsin binding. The binding capacity of BMDMP was shown to be higher by a factor of about three than that exhibited by mouse monocytes and by normal, thioglycollate and Corynebacterium parvum induced peritoneal macrophages. The relatively high binding capacity did not depend on experimental variations in the in vitro culture of these populations (i.e. length of in vitro cultivation, source of serum or presence of conditioned medium leading to cell proliferation).The macrophage-like line, P388D1, was also shown to possess functional tuftsin binding sites and its binding capacity was comparable to that of the peritoneal macrophage populations.

[15] Substance P and neurotensin

Publisher Summary This chapter describes two neuropeptides, substance P (SP) and neurotensin (NT). These two neuropeptides contain sequences analogous to tuftsin. Moreover, these peptides and their partial sequences containing the tuftsin-related domains displace labeled tuftsin from phagocytes and enhance their phagocytic capability. Both peptides exhibit a broad spectrum of pharmacological effects in the central nervous system as well as at the periphery. SP is widespread in both central and peripheral nervous systems. At the periphery, SP is recognized for its hypotensive, vasodilatory, and smooth muscle-contracting properties. In the central nervous system, SP is suggested to play a role in sensory nerve transmission. NT is distributed in various parts of the brain, gastrointestinal tissues, plasma, and in synovial tissue and fluid. SP is released from sensory nerves following injury, axon reflex, and antidromic stimulation, and thus is thought to be involved in neurogenic inflammation. Both SP and NT have properties and are located or released at sites that can enable them to act as mediators at various stages of inflammatory reactions.

Tuftsin and Substance P as Modulators of Phagocyte Functions

The basic tetrapeptide tuftsin, Thr-Lys-Pro-Arg, was described by Najjar and his collaborators as the entity responsible for the phagocytosis-enhancing activity of leukophilic γ-globulins. Since its discovery and synthesis, tuftsin was shown to affect many of the known functions of phagocytic cells. Tuftsin enhances phagocytosis, bactericidal activity, motility, cytotoxicity towards tumor target cells and immunogenic functions of macrophages. The tetra-peptide occupies the location 289–292 (Fc-region) in the amino acid sequence of all IgG subclasses and is released from the protein by a sequential two-step mechanism, involving a splenic carboxypeptidase and a surface leukokininase found on polymorphonuclear leukocytes (PMNL). The tetrapeptide exerts its activity only after its complete release from its parent molecule. (For reviews, see 1 and 2.)

Functional Similarity and Diversity in Peritoneal Macrophage Populations Induced in Vivo by Various Stimuli

Intraperitoneal injection of various foreign substances results in an augmentation of the size of the population of peritoneal macrophages. Various functional differences between stimulated populations and “normal” resident macrophage populations have been documented in recent years (2,4,8,11,13,16,20–22, 24,28,29). The terminology used by different workers in the field for description of the functional differences, i.e. stimulated, elicited, activated, is not consistent and depends to a large extent on the parameter chosen for comparison. We would, therefore, use the general term “stimulation” to describe populations induced by the various stimuli. Special emphasis has been given to differences in the phagocytic capacity of normal and thioglycollate (4) or endotoxin (22) stimulated mouse macrophages, since an alteration in complement receptor function upon stimulation was implicated. Another aspect that is currently intensively studied is that of macrophage-target cell interaction and the relevance of activated macrophages in combating neoplasia (2,11,13,21).