Neutrophil neutral endopeptidase variation and its regulation by opioid peptides.

The opioid peptide methionine-enkephalin (MENK) has significant immunomodulatory ability in addition to its neurotransmitter function. Since neutral endopeptidase (NEP, CD10, enkephalinase EC 3.4.24.11) cleaves opioid peptides, the presence and activity of NEP in neutrophils from different persons might be responsible for the diverse, neuropeptide-induced, responses of neutrophils from different donors [Ann. N. Y. Acad. Sci. 650 (1992) 146]. The results obtained showed statistically significant differences in NEP activity among donors (high, medium and low). A 10-fold higher NEP activity in neutrophils (160-280 nmol/10(6) cells/h) and in their corresponding membrane preparations (550 nmol/mg protein/min) in our study, as compared to literature data, was a result of high specificity and affinity of Suc-Ala-Ala-Phe-pNA as substrate. In control nontreated neutrophils, the number of CD10 positive cells were not correlated with NEP activity. However, in neutrophils treated with a physiological (10(-10) M) concentration of MENK, two main events occurred; not only did the number of CD10 positive cells correlate with NEP activity, but contrary to control samples, MENK upregulated the expression of CD10 marker as demonstrated by an increase of mean florescence intensity (F-mean) in donors with low NEP activity. Taken together, these data add some clarity to the diverse activity of enkephalins in association with enzyme cleavage of those molecules.

The role of aminopeptidase N in Met-enkephalin modulated superoxide anion release.

We have previously shown that methionine-enkephalin (MENK) alters in dose-dependent fashion the capacity of human neutrophils to produce superoxide anion. The response of neutrophils from different donors was diverse and this effect could be due to variable activity of proteolytic enzymes involved in the degradation of the neuropeptide. In this study, we have demonstrated a highly individual aminopeptidase N (APN) activity of neutrophils from different donors. Preincubation of neutrophils with MENK, but not with the synthetic agonist of the mu (DAGO) or the delta (DPDPE) opioid receptor, down-regulated the APN activity. This was paralleled by a loss in cell surface expression of APN at physiological (10(-10) M) concentrations of MENK. The level of APN activity from different donors correlated with the effect of MENK on superoxide anion release. Neutrophils with low APN activity, if preincubated with MENK, released reduced amounts of superoxide anion. In contrast, neutrophils with high APN activity released increased amounts of superoxide anion after preincubation with MENK. Thus, the highly individual APN activity on the surface of neutrophils from different donors seems to be altered by MENK and to be related to the respiratory burst.

Influence of methionine-enkephalin on stress-induced parameters.

This study examines the influence of methionine-enkephalin (MENK) on stress-induced oxidative damage (lipid peroxidation; LPO) in mice liver homogenate, plasma corticosterone concentration (CS) and phagocytic activity of mouse splenocytes. The LPO value increased in the mice subjected to restraint stress and had no correlation to stress duration, while MENK had no effect. The CS concentration was enhanced after 6 h of stress and 6 h after injection of a low (2.5 mg/kg bw) dose of MENK. However, MENK and stress are adjunct modulators of LPO and corticosterone in vivo. LPO was additionally elevated when MENK (10 mg/kg bw) proceeded for 2 h after the onset of stress. However, corticosterone concentration seems to be regulated differently by the same dose of MENK depending on the duration of stress i.e. elevated in cases involving short periods of stress (2 h) and decreased in cases involving prolonged periods of stress (6 h). This modulation of LPO and corticosterone by 10 mg/kg bw of MENK and 2 h of restraint stress was paralleled with elevated phagocytosis.

In vitro modulation of preleukemic AKR mouse macrophage function by bacterial immunomodulators.

Spleen macrophages from 1- and 4-month-old preleukemic AKR mice were stimulated in vitro with the bacterial immunomodulators lipopolysaccharide (LPS), peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), in order to study their migration ability and mitochondrial enzyme activity. Macrophages from 1-month-old AKR mice, characterized by higher functional activity, failed to demonstrate any changes in the parameters studied after in vitro stimulation with the employed compounds. Conversely, the depressed macrophage function, spontaneously developed in 4-month-old AKR mice, most probably related to the preleukemic state, improved significantly and to about the same extent with all three immunomodulators.

Side-effects of peptidoglycan monomer (PGM) treatment in mice.

The influence of single (4 mg/mouse) and multiple (1 mg/mouse per day for 5 consecutive days) injections of PGM on some hepatic enzymes, lipid peroxide generation in serum and liver, sialic acid concentration in serum and spleen and hepatic lysosomal membrane permeability was investigated. The studies performed showed that a single injection of PGM in vivo changed temporarily the permeability of lysosomal membranes, lipid peroxidation products and sialic acid concentration, and when administered in vitro modulated superoxide anion production and did not affect the activity of lysosomal membrane enzymes. Multiple injections of PGM did not cause significant changes in the examined parameters. Although the metabolic changes were time-limited and from the toxicological point of view, provoked transient effects, the results obtained may be of importance when using PGM in combined chemo-immunotherapy.

Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin.

In the present study, we have examined the effect of opioid peptide methionine enkephalin (MENK) on production of factors with interleukin-1 (IL-1) and tumor necrosis factor (TNF) activity by mouse peritoneal macrophages and assessed whether modification in the production of those cytokines could be related to alteration of phagocytosis by MENK. None of the MENK concentrations examined altered IL-1 or TNF activity alone. However, peritoneal macrophages co-stimulated with 1 microgram of lipopolysaccharide (LPS) and 10(-10) M MENK potentiated IL-1 activity, compared to LPS alone, but abrogated TNF activity induced by LPS. While MENK alone slightly decreased phagocytosis of sheep red blood cells (SRBC) by mouse peritoneal macrophages, cells simultaneously incubated with 1 microgram of LPS and 10(-10) M MENK had increased phagocytosis compared to LPS alone. Moreover, phagocytosis of SRBC by cells incubated overnight with the supernatant of the respective cell culture was significantly augmented. These results provide additional evidence for the immunoregulatory role of neuropeptides and suggest that the modulatory action of MENK could be mediated, at least in part, through the up-regulation of cytokines, most probably IL-1 and TNF.

Pharmacokinetics of an immunomodulator peptidoglycan monomer in mice after intravenous administration.

A 14C labeled low molecular weight immunomodulator, peptidoglycan monomer (14C-PGM), was injected intravenously (i.v.) into mice. At various time intervals thereafter (15 min-6 h), radioactivity in the urine, whole blood, plasma, kidneys, liver, spleen, lungs, intestines and the brain of the mice was determined. Shortly after injection, 14C-PGM was very rapidly excreted from the organism, so that 1 h following administration, 80% of the radioactivity was found in the urine (62% as unchanged PGM and the rest as the metabolites pentapeptide and disaccharide). At the same time, around 2% of the injected material was found in the blood. Six hours after injection, equal quantities were found in the intestines, liver and blood (0.5%), slightly less in the kidneys, lungs and spleen (0.2-0.3%) and the least quantity in the brain (0.04%). However, the dynamics of retention in the organs was evidently different. In the kidneys, lungs and spleen, radioactivity steadily decreased over the studied period. In the liver following an initial decrease, radioactivity remained the same 3 and 6 h after injection. On the other hand, in the intestines and brain PGM seemed to accumulate rather than disappear following i.v. administration. This fact should be considered when explaining different biological activities of low molecular weight bacterial peptidoglycans.

The effect of Met-enkephalin on mice liver lysosomes.

The unsedimentable activities of acid phosphatase (AP) and beta-glucosidase (BG), from mice liver lysosomes significantly increased 6 h after a single i/p injection of Met-enkephalin (MENK). The activity of AP in the serum at the same time remained unchanged. Multiple injections of MENK (8 x 10 mg/kg) induced a significant decrease in AP activity in the serum and no change in the unsedimentable activities of AP or BG. MENK did not elicit any significant extracellular release of lactate dehydrogenase (LDH) either, indicating that, under the experimental conditions described, the cells remained intact. Other parameters, such as the activities of AP and BG in the liver and total sialic acid content in the serum and spleen remained unaltered. Moreover, MENK in concentrations of 10(-12) M, 10(-8) M, 10(-6) M or 10(-4) M did not change the activities of the lysosomal enzyme markers AP or BG in vitro. These data indicate far less pronounced transient effects of MENK on lysosomal membranes and enzymes compared to Leu-enkephalin which may be relevant for the use of MENK in combined chemo-immunotherapy.

Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leu-enkephalin.

The present work describes the ability of Met- and Leu-enkephalin to modulate the superoxide anion (O2-) release from unstimulated human polymorphonuclear cells (PMN) and from PMN stimulated with phorbol myristate acetate (PMA). The direction (stimulation or suppression) and the magnitude of change were dependent upon the baseline reactivity of the donor's PMN. Both opioid peptides stimulated O2- release by PMN from donors with low baseline reactivity in a concentration-dependent manner. PMNs collected from donors with medium baseline reactivity incubated with Leu-enkephalin regardless of concentration released less O2- than control, nontreated PMNs. Met-enkephalin stimulated O2- release but only at 2 X 10(-15) M concentration. Superoxide anion release from PMNs of individuals with high baseline reactivity was concentration dependent and suppressed by Met- and Leu-enkephalin. Leu-enkephalin induced baseline reactivity was dependent upon progressive increase in the magnitude of change on O2- release (i.e., the higher the baseline the higher the magnitude of change in O2- generation). Met-enkephalin data show this also, but to a lesser extent. In cells stimulated with PMA, Met-enkephalin caused additional O2- release, while Leu-enkephalin was ineffective in triggering already stimulated cells. The modulating effect of both opioid peptides on superoxide anion release by human PMN is a short phenomenon that lasts up to 10 min after the addition of the peptide.

Leu-enkephalin induced alteration of enzymes and sialic acid levels in vivo.

The influence in mice of 10 mg/kg Leu-enkephalin (LENK) on the activity of hepatic enzymes in serum and liver and sialic acid concentration in serum and spleen is described. The activity of acid and alkaline phosphatase and lactate dehydrogenase in serum was significantly enhanced 6 h after a single (10 mg/kg) LENK injection. This effect was not dose-dependent. The increase of acid phosphatase in serum was accompanied by a simultaneous decrease in the liver. While the level of serum sialic acid remained unchanged, it decreased in the spleen 6 h after a single LENK injection. The effect of LENK in the spleen and liver was dose-dependent; ie, the higher the dose the greater the effect. Multiple injections of Leu-enkephalin (6 x 10 mg/kg) induced the same degree of increase but an earlier rise in serum alkaline, acid phosphatase and lactate dehydrogenase levels than with a single injection. This was not accompanied by a change of activity in liver and spleen. These data may be relevant for use of LENK in combined chemo-immunotherapy, since liver enzyme changes may alter drug metabolism, and since sialic acid plays a regulatory role in immune processes.

Relationship of metabolism and immunostimulating activity of peptidoglycan monomer in mice after three different routes of administration.

[14C] Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid (omega NH2)-D-Ala-D-Ala (PGM) was administered to mice by the intravenous (i.v.), subcutaneous (s.c.) or peroral (p.o.) routes. The data on distribution of radioactivity and excretion of radioactive products, as well as the data on immunostimulating effects are presented on the comparative basis for PGM administered by three different routes. When injected i.v. or s.c., the major part of applied radioactivity was found excreted in urine, partly as unchanged original compound and partly as the corresponding pentapeptide, L-Ala-D-isoglutamine-meso-diaminopimelic acid (omega NH2)-D-Ala-D-Ala. If administered p.o., the major part of the radioactivity was retained in the stomach and intestinal tract for several hours. The drop in radioactivity in these organs was followed by exhalation of 14CO2 thus indicating extensive degradation of the original molecule. PGM stimulates the humoral immune response to sheep red blood cells in mice if administered i.v. or s.c., but is completely inactive if administered p.o.. Thus, absence of immunostimulating activity following p.o. administration might be explained by extensive metabolic degradation of peptidoglycan monomer.

The effects of immunomodulating peptidoglycan monomer and muramyl dipeptide on hepatic microsomal UDP-glucuronyltransferase and beta-glucuronidase.

The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT1) and testosterone for the second functional form (GT2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal beta-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of beta-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.

Antitumor and antimetastatic activity of the immunoadjuvant peptidoglycan monomer PGM in mice bearing MCa mammary carcinoma.

The antitumor and antimetastatic activities of the water-soluble peptidoglycan monomer GlnNAc-Mur-NAc-L-Ala-D-iso-Gln-meso-diamminopimelic acid (omega-NH2)-D-Ala-D-Ala (PGM), which has immunostimulant effects, have been evaluated in CBA mice bearing MCa mammary carcinoma. The antineoplastic effects of PGM depend strictly on the dosage and treatment schedule used. Though a significant inhibition of the primary tumor growth is observed over a wide range of dosage, only the IV administration of daily doses of 50 mg/kg/day on days 1, 5, 10, 15 inhibits spontaneous lung metastasis formation and in parallel prolongs the survival time of the treated mice. The magnitude of the antimetastatic effects of PGM depends on the degree of dissemination of the tumor, and is greater when the number of metastatic foci is low. Examination of the therapeutic potential of PGM in combination with surgery has further indicated that the timing of administration plays an important role in the overall effectiveness of this substance. A 5-day interval is necessary between two consecutive injections for the induction of significant increases of the survival times.

Immunotherapy of B-16 melanoma with peptidoglycan monomer.

B-16 melanoma-bearing mice received intravenously or intratumorally one or multiple injections of peptidoglycan monomer (PGM) derived from Brevibacterium divaricatum cell wall. Multiple injections of this non-toxic, water-soluble, low-molecular-weight peptidoglycan reduced the growth rate of tumor nodule on the leg, but did not significantly prolong the survival of tumor-bearing mice. One milligram of PGM administered 3 or 7 days after tumor inoculation inhibited formation of pulmonary metastases, induced either by intravenous injection of malignant cells or seeded spontaneously from tumor nodules in the legs before amputation. The inhibition reached about 50% of control values in saline-treated mice. Addition of PGM to in vitro cultures of B-16 melanoma cells did not change their growth rate. The phagocytic activity in the lungs, but not in the spleen and liver, was significantly augmented 3 and 7 days after treatment with PGM. These data indicate that the antimetastatic potency of PGM is probably due to activation of local (pulmonary) macrophages and not due to direct cytotoxic effects on B-16 melanoma cells or to activation of systemic antineoplastic defence.

Immunotherapy of Lewis lung carcinoma with hydrosoluble peptidoglycan monomer (PGM).

The water-soluble peptidoglycan monomer (PGM) isolated from the culture fluid of Brevibacterium divaricatum, which has immunostimulating activity, has been examined for its antitumor effects in C57BL mice bearing Lewis lung carcinoma. The formation of spontaneous lung metastases from SC tumor implants is significantly inhibited. The growth of SC primary tumors, including advanced ones, is also significantly inhibited, though to a less pronounced extent than the growth of metastases. The effects on metastases are evident with all treatment schedules used, whereas those on SC primary tumors are treatment schedule-dependent. The treatment with PGM was found to be therapeutically useful when combined with surgical removal of IM implants; in conditions where a single post-operative treatment was ineffective, combined post-operative and immediately pre-operative administration of PGM significantly increased (by 40%) the survival time of treated animals over that of controls undergoing surgery only.

Correlation of substance(s) immunologically cross-reactive with insulin, glucose and growth hormone in Hodgkin lymphoma patients.

The levels of substance(s) detectable by insulin specific radioimmunoassay (RIA), glucose and growth hormone (GH) were determined in the blood of patients suffering from Hodgkin lymphoma. In the relapse phase of the disease, the levels of substances immunologically cross-reactive with insulin (SICRI) were elevated and glucose concentrations were below normal. In these patients the basal and hypoglycemia-induced levels of GH in blood were strongly elevated. Contrary to this, both SICRI and glucose levels were normal in the blood of patients in remission, and GH levels were significantly reduced compared to those measured in patients in relapse.