Positive correlation between superoxide release and intracellular adenosine deaminase activity during macrophage membrane perturbation regardless of nature or magnitude of stimulus

Summary Tuftsin stimulates macrophages to release superoxide in direct proportion to intracellular adenosine deaminase activity over a concentration range of 125 to 625 nM tuftsin. This relation is comparable to that previously observed for stimulation by single concentration of several agents. This finding led to the conclusion that the relation between superoxide and adenosine deaminase is independent of the nature or magnitude of the stimulus. In absolute terms, tuftsin increases superoxide secretion up to 375 nM tuftsin; further increases in tuftsin concentration cause a rapid decrease in superoxide secretion to near base line at 500 nM tuftsin. In contrast, the phagocytic response to tuftsin remains maximal up to 10 M with no indication of inhibition at higher concentrations. Thus, tuftsin stimulation of phagocytosis and superoxide release may be at least partially independent phenomena.     

Peptide fragments from the tuftsin containing domain of immunoglobulin G synthesis and biological activity

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [3H-Arg4]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cell's capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsin's receptors.     

Bovine probursin tetradecapeptide contains amino acid sequence from somatostatin, tuftsin and bursin.

The B cell differentiating tripeptide bursin (lysyl-histidyl-glycyl-amide) is found in avian and mammalian bone marrow and in epithelial cells of the avian bursa of Fabricius and mammalian intrahepatic bile ducts. We now report the structure of probursin (Phe-Phe-Trp-Lys-Thr-Lys-Pro-Arg-Lys-His-Gly-Gly-Arg-Arg) isolated from bovine bone marrow and liver. Amino acids 1-5 correspond to the active site of somatostatin, 5-8 to tuftsin and 9-11 to bursin. Intact probursin has the biological activity of both somatostatin and bursin, and known enzyme cleavages could release free tuftsin, although intact probursin has low tuftsin activity. Probursin and its component peptides could regulate other bone marrow functions in addition to B cell differentiation, and, in mammals, could also regulate the function of hepatocytes and Kupffer cells after transport to the hepatic sinusoids via a local portal system involving the peribiliary capillary plexus.     

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.

     

Synthetic peptides from C-reactive protein containing tuftsin-related sequences

Peptides containing Lys-Pro-Arg or Thr-Lys-Arg segments corresponding to various regions of human C-reactive protein were synthesized. The peptides prepared were composed of amino acid residues, 37–58, 51–58, 173–187 and 181–187 of C-reactive protein. The relationship between C-reactive protein, its synthetic fragments and tuftsin (Thr-Lys-Pro-Arg) was investigated in binding studies, enhancement of phagocytosis and change in cyclic nucleotide levels of mouse macrophages. The peptides AA 51–58 and 181–187 did enhance macrophage phagocytosis capacity to a similar extent to that of tuftsin. They showed however only negligible binding to the cells. The effect of C-reactive protein and the synthetic peptides on metabolic activity of neutrophils was also investigated. It was shown that the peptides inhibited to some degree superoxide production, lysozyme release and Vitamin B₁₂ binding protein release from neutrophils in the absence and presence of the stimulants, PMA or Con A. Comparable activity with tuftsin was not found.     

A comparative study of [Leu1]Tuftsin and tuftsin, a natural phagocytosis-stimulating peptide

1. [Leu1]tuftsin was reported to have greater phagocytosis-stimulating activity than tuftsin (Thr-Lys-Pro-Arg). 2. However, a study on inactivation of tuftsin by polymorphonuclear leukocytes (PMNs) demonstrated that leucine aminopeptidase, an ecto-enzyme, located on PMN surface was responsible for this mechanism. 3. Since leucine aminopeptidase is known to cleave Leu more easily than Thr at the N-terminal position of peptides, this suggested to us that [Leu1]tuftsin might then be inactivated by PMNs more easily than tuftsin, and thus this analog might be less active than tuftsin. 4. In addition, many tuftsin preparations used in earlier studies were not fully active, as high-performance liquid chromatography was not available to separate out many contaminating diastereomers. 5. In view of this, we have synthesized and purified [Leu1]tuftsin and compared its phagocytosis-stimulating activity with tuftsin. 6. Our results indicate that [Leu1]tuftsin is not as active as tuftsin in stimulating phagocytosis.     

Tuftsin and some analogs: synthesis and interaction with human polymorphonuclear leukocytes

The phagocytosis-stimulating tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, was synthesized by both conventional and polymeric-reagent approaches. Using a combination of the two methods several analogs were prepared, including: [Ala1]tuftsin, [Lys1]tuftsin, [Ser1]tuftsin, [Val1]tuftsin, acetyl-tuftsin, p-aminophenylacetyl-tuftsin and tyrosyl-tuftsin. [Des-Thr1]tuftsin and [omega-NO2(4)]tuftsin were synthesized using a conventional procedure. The effects of synthetic peptides on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes were investigated. Tuftsin and to a lesser extent [Lys1]tuftsin and [Ser1]tuftsin were found to stimulate phagocytosis, whereas the other analogs synthesized as well as [Ser1]tuftsin exhibited inhibitory effects to tuftsin's action. Tuftsin alone has stimulated nitroblue tetrazolium reduction; [Des-Thr1]tuftsin and [Ala1]tuftsin repressed this stimulation, while the other peptides showed no effect.     

Substrate specificity of aminopeptidase M: evidence that the commercial preparation is contaminated by dipeptidyl aminopeptidase IV and prolidase

Commercial preparations of aminopeptidases M split Gly-Pro-beta-naphthylamide (Gly-Pro-2-NNap) into Gly-Pro and beta-naphthylamine, and Ala-Pro into Ala and Pro. The activities on Gly-Pro-2-NNap and Ala-Pro were completely inhibited by diisopropyl phosphorofluoridate (DFP) and p-chloromercuribenzoate (PCMB), respectively. When the substrate specificity was analyzed with tuftsin, Thr and Lys-Pro-Arg were released, and then Lys-Pro-Arg was split into Lys-Pro and Arg. Thereafter, slow liberation of Lys and Pro from Lys-Pro took place. The DFP-treated enzyme released only Thr from tuftsin and no hydrolysis of Lys-Pro-Arg was observed. With the enzyme treated with PCMB, tuftsin was converted into Thr and Lys-Pro-Arg, followed by the liberation of Arg, but no release of Lys and Pro was observed, contrary to the case of the untreated-enzyme. These results show that commercial aminopeptidase M contains dipeptidyl aminopeptidase IV and prolidase. Contamination by dipeptidyl aminopeptidase IV was confirmed by an immunological method.     

Cetp and exchangeable apoproteins. Common features in lipid binding activity

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of antigen-fed macrophages. The present study describes the mechanism of action of tuftsin in leprosy patients throughout the spectrum of the disease in vitro as a function of culture age in terms of (A) involvement of second messengers cAMP, cGMP and [Ca2+]i and (B) number of tuftsin binding sites/and their relative affinities on the monocytes/macrophages. There is apparently no direct involvement of either cAMP or cGMP while comparing the stimulated and unstimulated cultures during in vitro differentiation of monocytes (days 1, 3 and 7) or with the spectrum of the disease. Inhibition of superoxide anion release either by verapamil or with Quin 2 clearly demonstrated the involvement of [Ca2+]i as a second messenger during activation of monocytes/macrophages with tuftsin. Scatchard analysis of radiolabelled tuftsin binding data showed only one type of tuftsin receptor (low affinity) on BL/LL monoc ytes/ macrophages and normal and BT/TT cultures showed a gradual change in receptor number and affinities (low to high) with the maturation of monocytes to macrophages in contrast to BL/LL groups which displayed significantly less number of receptors. This study elicits a model which depicts that the biological responses/metabolic functions of early monocytes of normal and BT/TT gradually increase with the age of the culture till day 3 and tapers off thereafter in the older (day 7) cultures, whereas the monocytes/macrophages of BL/LL group are metabolically active only on day 1. The present study thereby implies that the clearance of leprosy bacilli from lepromatous leprosy lesions as a consequence of local or systemic immunotherapy (in the present study, the macrophage modulation by tuftsin) depends on the influx of new competent macrophages, rather than the local activation of resident lepromatous macrophages.     

Synthesis of an IgG fragment decapeptide exhibiting phagocytosis stimulating activity of polymorphonuclear leucocytes

Fragment 335-344 of human IgG was synthesized stepwise, using active esters of N-protected amino acids and benzotriazolyloxytris (dimethylamino)phosphonium hexafluorophosphate for coupling. This fragment could be liberated from the carrier molecule by the two specific enzymes responsible for the release of tuftsin from the same carrier molecule: tuftsin endocarboxy-peptidase cleaves at the Arg-Glu bond and leukokininase at the Lys-Thr bond. This decapeptide fragment, Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg showed significant activity in stimulating phagocytosis of human polymorphonuclear leucocytes.     

Tuftsin-macrophage interaction: specific binding and augmentation of phagocytosis.

The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22 degrees C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (K(D)) (calculated from a Scatchard plot) of 5.3 X 10(-8) M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a K(D) of 5.0 X 10(-8) M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollate-stimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide. Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding. Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.     

The phagocytosis stimulating peptide tuftsin: Further look into structure-function relationships

Summary Sixteen new analogs of the phagocytosis-stimulating peptide tuftsin have been synthesized. The biological activities of these synthetic peptides, in which either the C-terminal or both C- and N-terminals are chemically altered, were evaluated by studying their effects on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes. The results demonstrate that the integrity of the guanidine side chain of arginine at position four of tuftsin is crucial for maximal activity. Modification, even in side chain length, of the guanidine leads to decreasing activity. Preservation of the positive charge of position four of tuftsin yields analogs possessing considerable activity. Simultaneous alterations of both C- and N-terminal results in diminishing activites. The results of this study are discussed in relation to the structural features of tuftsin. It appears that interaction between the carboxyl of Arg⁴ and the amino group of Thr¹ which would indicate a specific conformation such as a 4 1 -turn are not favored.     

Comparative study of the psychotropic activity of tuftsin and its analogs

Tuftsin and its Leu1 and D-Arg4 analogs displayed stimulating activity in experimental behavioral despair in mice. In rats with different types of emotional reactions and with destroyed catecholamine terminals (6-OHDA treatment), tuftsin increased exploratory activity, with fear manifestations being decreased and avoidance behavior improved. This was shown while testing the rats in the "open field" and according to the ability to accomplish an extrapolation task of avoiding critical stress-situation. Leu1-tuftsin increased the emotional stress and sharply hindered the avoidance reaction, while D-Arg4-tuftsin modulated the behavior of the animals with increased emotional reactivity and made the avoidance behavior prompter. Pentapeptide, an inhibitor of tuftsin stimulation of phagocytosis, had no significant effect on the behavior. Modifications in the structure of tuftsin resulted both in the changes in phagocytosis-stimulating activity and the appearance of other psychotropic effects.     

Salt effects on peptide conformers: a dielectric study of tuftsin.

Four 1-ns molecular dynamics computer simulations of tuftsin, Thr-Lys-Pro-Arg, are analyzed: (1) cis tuftsin in water, (2) trans tuftsin in water, (3) cis tuftsin in 1 M NaCl, and (4) trans tuftsin in 1 M NaCl. Independently of the salt concentration, the trans conformer has a higher dielectric constant than the cis conformer because the former exhibits a more widely distributed charge distribution in space. Independently of the peptide conformation, the presence of salt reduces the dielectric constants of both the peptide and the solvating water molecules because ions, on binding, restrict the motion of other atoms. In contrast to the dielectric constants, neither the peptide conformation nor the salt concentration shows a significant influence on the dielectric relaxation time of water molecules.     

Synthesis and functional studies of tuftsin analogs containing isopeptide bond

In the present paper a new approach is reported, to increase the resistance of tuftsin toward enzymatic cleavage by the introduction of an isopeptide bond into the molecule. The tetrapeptides H-Lys(Thr)-Pro-Arg-OH and H-Lys(Ala)-Pro-Arg-OH, the pentapeptides H-Thr-Lys(Ala)-Pro-Arg-OH, H-Thr-Lys(Thr)-Pro-Arg-OH and H-Ala-Lys(Ala)-Pro-Arg-OH and their For- and Boc-protected derivatives were built up by stepwise elongation of the chain, using conventional solution-phase methods. Preliminary experiments confirmed that from the Lys residue in position 2 of tuftsin the alpha-peptide bond between the Thr and Lys is cleaved with a significantly higher rate by leucine aminopeptidase than the epsilon-peptide bond. Several of the isopeptide derivatives increased to a higher extent the interleukin (IL-1) secretion by monocytes than tuftsin or [Ala1]-tuftsin.     

Tuftsin: a naturally occurring immunopotentiating factor. I. In vitro enhancement of murine natural cell-mediated cytotoxicity

Tuftsin is a physiologic tetrapeptide, which has recently been shown to possess immunoadjuvant properties including the stimulation of macrophage and granulocyte phagocytosis, migration, bactericidal, and tumoricidal activities. Tuftsin has also been reported to possess in vivo immunologically mediated anti-tumor potential. To determine the potential role of tuftsin as an antineoplastic immunoadjuvant, the in vitro effects of tuftsin on murine natural cell-mediated cytotoxicity were studied. We observed that in vitro treatment of mouse splenic effector cells with synthetic tuftsin induced a pronounced enhancement of natural killer cell (NKC) cytotoxicity against the T cell lymphoma Yac-1. The magnitude of NKC enhancement was directly dependent upon the concentration of tuftsin employed, with maximum NKC stimulation observed at tuftsin concentrations of 50 to 100 microgram/ml. The tuftsin induced enhancement of NKC activity was not strain specific, since equivalent stimulation was seen in CBA/J, C56BL/10, and DBA/2 mice. Elimination of macrophages, monocytes, T cells, and immunoglobulin-bearing cells had no effect on the dose-dependent tuftsin stimulation of natural cell-mediated cytotoxicity; thus the characteristics of the effector cells activated by tuftsin were consistent with those reported for NKC. We also observed that treatment of splenic effector cells with tuftsin prolonged the cytotoxic capabilities of these cells beyond 18 hr.     

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

Summary Incubation 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.     

Excitatory effect of tuftsin tetrapeptide on the activity of white rats

Studies on male white rats have shown that tuftsin (trelys-pro-arg) enhances the locomotion in animals as disclosed by a series of the behavioral tests. The effect is dose-dependent: a dose of 50 microgram/kg did not change any of the test parameters, while that of 150 microgram/kg induced a short-term elevation of locomotion measured with an "Animeks". Administration of tuftsin in a dose of 300 microgram/kg led to the enhancement of the animals' locomotion as measured with the "Animeks", to the increased "open field" running time and to the reduced latent period of the reaction during the training in a T-shaped maze. Also, this dose of the peptide relaxed the reactions associated with fear. It is assumed that the effects observed are consequent on the stimulant action of tuftsin on the body of white rats.     

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.