Met-enkephalin induced alterations of macrophage functions.

Met-enkephalin /Met-enk/ was found to stimulate IgG2a-mediated antibody dependent cytotoxicity /ADCC/ of thioglycollate elicited rat peritoneal macrophages /PM/ through naloxone-sensitive opiate receptors in concentrations ranging from 10(-9) - 14(-7) M. Phagocytosis of IgG2a coated 51Cr-sheep red blood cell /SRBC/ was suppressed by M-enk in the same concentration range. In the same range of concentrations, M-enk was observed to induce a significant increase in the generation of luminol dependent chemiluminescence /LDCL/. The observed stimulation of ADCC was abolished by calmodulin inhibitor triflouroperazine /TFP/ in 10(-6) M concentration. The involvement of cyclic nucleotides in the M-enk induced functional alterations is indicated by finding cGMP accumulation to be augmented in M-enk treated PMs.

Age related impairment in phosphatidylinositol breakdown of polymorphonuclear granulocytes.

It is well known that with aging the immune response decreases. Most of the effector functions occur through specific receptors. Thus, we investigated the effects of various stimulants, acting through receptors or directly through the GTP-binding Gi protein, on phosphatidylinositol breakdown in PMNLs of young and elderly subjects and try to modulate it. A marked decrease in inositol phosphate (IP1, IP2, IP3) formation in PMNLs of elderly was found under FMLP stimulation when compared to that of young subjects. Neither GTP gamma S, nor AIF4- could induce an increase of IP3 in PMNLs of elderly comparable to that of young subjects. The results suggest that at least an alteration exists at the GTP-binding Gi protein level, as well as in the mechanism of linkage of the receptor to the G protein.

Age-related changes in cAMP and cGMP levels during phagocytosis in human polymorphonuclear leukocytes.

Alterations of cyclic nucleotides have been studied during the incorporation of opsonized yeast cells into human polymorphonuclear leukocytes (PMNL) from both young and aged subjects. In PMNLs obtained from healthy young males the cAMP level rose to a maximal value during the first 15 min and returned to the starting level at the 60th min of phagocytosis. The cGMP level started to rise only at the 30th min of incorporation and enhanced progressively up to the 120th min. In contrast, the elevated cAMP level remained increased during the 120 min of phagocytosis in PMNLs obtained from aged subjects, whereas the cGMP level was not altered during the same period. The basic level of cAMP diminished, while the cGMP level was found to be elevated with ageing in PMNLs. It is concluded that phagocytosis was not impaired with age yet cytotoxic functions were diminished and that the data presented support the idea that cyclic nucleotides may be directly involved only in the latter process.

Altered post-receptorial signal transduction mechanism under various stimulation in polymorphonuclear granulocytes of Alzheimer's disease.

The cyclic nucleotide changes were studied under 10(-6) M isoproterenol (IP), 10(-6) M carbachol and 10(-8) M Met-enkephalin (Met-enk) stimulations in polymorphonuclear granulocytes (PMNLs) of middle-aged (aged 35-52 years) and elderly (aged 61-97 years) healthy subjects, as well as of patients with Alzheimer's disease (AD) (aged 58-65 years). From our results we can conclude that in the case of middle-aged healthy subjects only the IP caused a marked cAMP elevation while in elderly and AD all the applied substances stimulated the cAMP at different degrees. Concerning the cGMP levels in PMNLs, we observed a marked increase under carbachol and Met-enk stimulation, in middle-aged subjects, while in the elderly a weak change was obtained by carbachol. In AD practically no change of cGMP levels could be obtained. Thus, the main features of AD are a cAarP response to Met-enk and an abolition of a GarP response to carbachol. We can conclude that in PMNLs of elderly and patients with AD we assist to an altered post-receptorial signal transduction mechanism, which seems to be even more marked in the case of AD comparing to normal aging.

Interrelation between body composition and endocrine system in healthy elderly people.

The body composition of 40 healthy aged and 20 healthy young people was determined using a multi-isotope method. At the same time their hormonal status was assessed. A correlation between the body composition and the endocrine status was investigated. Our results concerning the body composition and the hormonal values correlate with the findings of the literature. A strong correlation was found between the plasma volume and the renin-angiotensin-aldosterone system. The interrelation could explain the reciprocal age related changes of plasma volume and the renin-angiotensin-aldosterone system. No or weak correlation could be found between the body composition and other measured hormones.

Age related variations of some polymorphonuclear leukocyte functions.

The age-related alterations of some biochemical processes in polymorphonuclear leukocytes (PMNL) obtained from 20 healthy aged male and 20 healthy aged female (age: 60-94 years) subjects were investigated. The basic level of luminol dependent chemiluminescence (LDCL) was increased, whereas the basal value of reduced/oxidized glutathione (GSH per GS-SG) was decreased in the aged groups. The enhancement of LDCL was more significant by PMNLs of aged males than of females. The change of both GSH and GS-SG levels during phagocytosis were diminished in the PMNLs of aged subjects. The L-alanine beta-naphthylamide specific elastase like protease (ELP) activity measured in living cell suspension was markedly increased with aging in male subjects. Therefore, it was concluded that the aging of PMNLs is partly a sex related process. The intensive release of both beta-glucuronidase (beta-G) and ELP by the PMNLs of aged subjects after in vitro treatment with calcium ionophore A23187, Cytochalasin B or low density lipoprotein (LDL) suggests that they could play an important role in some age-related disorders.

Effect of lymphokine on the activation and Fc receptor expression of rat macrophages.

Lymphocyte supernatant (LS) containing lymphokine (LK) activated rat peritoneal macrophages (PM) and at the same time enhanced their Fc receptor (FcR) activity. The signs of the early activation could not be observed on alveolar macrophages (AM) but the augmented FcR expression occurred under the effect of lymphokine. The LK-induced macrophage activation and the enhancement of erythrocyte-antibody (EA) rosette formation were found to be independent of each other. In the presence of soybean trypsin inhibitor (STI) the crude lymphokine-induced augmented EA rosette formation was only slightly diminished. However, it did not seem likely that the LK components of molecular weight over 15,000 could enhance the number of rosettes under the effect of protease inhibitor. These data indicate that the macrophage proteases play an important role in the generation of EA rosette formation enhancing breakdown product(s) during the macrophage-lymphokine interaction.

The effect of oligopeptides on the C3b receptor-mediated functions of rat macrophages.

Effects of various substances known to influence macrophage functions on the adherence of C3b-coated sheep erythrocytes (E-C3b) and their phagocytosis by elicited rat peritoneal macrophages (PM) has been studied. Different oligopeptides (OP) having a chemotactic effect, such as tuftsin, angiotensin II (At II) and the OP-fraction of a lymphokine preparation (LK-OP) increased both adherence and phagocytosis of E-C3b in a concentration-dependent fashion. In contrast, neither substances influencing the membrane cation transport nor prostaglandins (PG) and cyclic nucleotides significantly changed the C3b receptor (C3bR)-dependent effector functions of macrophages. Enhanced functions of the C3bR were not associated with an increased capability of PMs for intracellular killing of Candida albicans.

Alterations of the FMLP-induced Ca2+ efflux from human monocytes with aging.

The Ca2+ with the calmodulin system plays a major role in the receptor-induced intracellular biochemical events. Thus, as in our previous studies, we found an altered postreceptorial coupling in the case of elderly, we were interested to elucidate the Ca2+ transport in the case of healthy young (less than 25 years) and aged (greater than 65 years) subjects. The chemotactic peptide FMLP was used for receptor stimulation. Our data show that both spontaneous and FMLP-triggered Ca2+ extrusion measured on monocyte monolayers is decreased with aging. The FMLP-triggered Ca2+ extrusion nevertheless did not occur through the TFP-sensitive pathway. These alterations of Ca2+ transport seem to indicate an impaired Ca2+-calmodulin function in monocytes with aging.

Immunology of elastin: study of anti-elastin peptide antibodies by DOT immunobinding assay.

In order to further investigate the role of the immune system in the arteriosclerotic process, we investigated the anti-elastin peptide antibodies (AEAb) of the IgG and IgM types by DOT immunobinding assay in the sera of patients suffering from various arteriosclerotic diseases. In total 232 control and pathological sera were studied. In obliterative arteriosclerosis of the legs 90%, ischemic heart disease 67% and hypertension 60% of sera were positive for AEAb of the IgG type independent of age. In the case of diabetes mellitus, however, the duration of the disease was determinant. In rheumatoid arthritis, the results were negative. No clear-cut positivity could be demonstrated in stroke patients either. These results indicate that AEAb can be detected in some diseases and DOT appears to be an appropriate method for the AEAb screening in various diseases.

Ionophore-induced functional changes in macrophages.

Ca ionophore A 23187 and valinomycin known to induce Ca2+ influx, i.e. K+ efflux, appeared to induce activation-related changes in rat peritoneal macrophages (PM) at 10(-5)-10(-7) M concentrations as revealed by aggregation, nitro blue tetrazolium (NBT) reduction, superoxide anion production as well as beta glucuronidase release; however, lactate dehydrogenase (LDH) activity was measured in supernatants only after treatment with 10(-5) M of A 23187. The PM-activating effect of A 23187 was abolished in the presence of 5 mM EGTA, whereas the activating effect of K ionophore did not depend on the extracellular Ca2+. Fc receptor (FcR) mediated incorporation, intracellular killing as well as the antibody dependent cytotoxicity (ADCC) were markedly inhibited upon incubation with Ca ionophore or valinomycin. C3bR mediated phagocytosis was found to be more resistant to the cation transport affecting drugs. The impairment of effector functions was elicited by nontoxic concentrations of ionophores, however, their inhibiting effects did not require extracellular Ca2+ at the time of ionophore-exposition.

Regulation of Fc mu receptor-mediated functions of resident and provoked peritoneal macrophages.

Modifying and/or regulating effects on Fc mu receptor (R) mediated phagocytic and ADCC activity of both resident (r) and provoked (p) peritoneal macrophages (PM) was studied applying drugs affecting the cytoskeleton and cation transport. In addition, the effects of exogenous PGE2 and cyclic nucleotides were also examined. Fc mu-receptors appear to be functionally less significant in provoked PMs than in resident ones since both Fc mu-receptor mediated phagocytosis and ADCC were lower in the formers. The cytoskeletal system is important in the regulation of both Fc mu-receptor-mediated functions. Phagocytosis through Fc mu-receptor is decreased by Cytochalasin B and by Vinblastine treatment, whereas ADCC is enhanced by Cytochalasin B. Extracellular PGE2 and cAMP induced a higher phagocytic activity and suppressed ADCC, whereas cGMP displayed an opposite effect. The sensitivity of Fc mu R-mediated activities to ionophoric and to cytoskeleton-damaging drugs was lower in provoked than in resident PMs. In addition, the regulatory role of PGE2 and of cyclic nucleotides on the same activities was less marked on provoked PMs. In contrast, ouabain inhibits Fc mu R-dependent antigen incorporation and ADCC on provoked PM monolayers only. These findings suggest differing regulatory mechanisms for Fc mu R-mediated functions in provoked PMs as compared with resident ones.

Age-dependent variations of intralysosomal enzyme release from human PMN leukocytes under various stimuli.

The intralysosomal beta glucuronidase and elastase release from polymorphonuclear leukocytes (PMNL) of young and aged male subjects were determined after 60-min incubation with 10 micrograms/ml Cytochalasin B (CB), 10(-6) M of Ca ionophore A 23187 and various concentrations of human low density lipoprotein (LDL). The beta glucoronidase secretion was triggered by both A 23187 and LDL; however, no significant differences were found between the enzyme release from PMNLs of young and aged subjects. In contrast, a marked elastase release was triggered in the young group only by LDL, whereas in the aged group, all of the applied drugs induced a significant elastase release. LDL caused the most dramatic enzyme release from PMNLs of aged males. It was concluded that the release of PMNL-elastase after LDL incorporation as well as by CB and Ca ionophore stimulation may be an age-related process.

Heterogeneous signal pathways through TSH receptors in porcine thyroid cells following stimulation with Graves' immunoglobulin G.

OBJECTIVE: We compared different signal transduction pathways through thyroid stimulating hormone receptor (TSH-R) in porcine thyroid cells (PTC) following stimulation with thyroid stimulating hormone (TSH) and 11 thyroid stimulating immunoglobulin samples (TSI) obtained from patients with Graves' disease. DESIGN: Following stimulation with TSI, the level of inositol trisphosphate (IP3) and [Ca2+]i, as well as the membrane bound protein kinase C (PKC) activity and the intensity of the arachidonic acid (AA) cascade, were determined in PTC. RESULTS: Seven out of eleven TSI samples activated PTC through IP3 generation, elevated [Ca2+]i from the intracellular pools, exhibited verapamil-insensitive membrane-bound PKC activation, and enhanced release of [14C]AA derivates (however, one of the samples was also able to take up Ca2+ from the extracellular space). Four out of eleven TSI samples did not activate the phospholipase C (PLC) system in which case the Ca2+ signal occurred only in the presence of extracellular Ca2+, the membrane bound PKC activation was verapamil sensitive, and in two of these four TSI samples, the AA release was extremely high. CONCLUSIONS: The simultaneous examination of the majority of the known signal pathways using TSI samples showed that TSI samples from different patients activate thyroid cells through different pathways. Their effects differ from that of TSH and, to a certain extent, from each other. The results give a certain new insight into the intracellular mechanisms exerted by TSI.

Possible correction of defective polymorphonuclear cell functions in type-2 diabetes mellitus by met-enkephalin.

In vitro pretreatment with Met-Enk of human PMNLs obtained from patients suffering from type-2 diabetes mellitus resulted in the normalization of the ROS generating system. It is assumed that Met-Enk has a modulating effect on the arachidonic acid metabolism, with a consecutive increase of the LTB4 release.

Concentration-dependent effect of met-enkephalin on human polymorphonuclear leukocytes.

Met-enkephalin in 10(-9)-10(-7)M concentrations exerts an ADCC-stimulating effect on human PMNLs through naloxone-sensitive opiate receptors, elevating the cytoplasmic free Ca2+ and cGMP levels. In higher, 10(-6)-10(-5)M concentrations ME has a cAMP-elevating effect and a rapid 45Ca2+ influx was observed. This latter effect of ME, which is also associated with the suppression of ADCC activity, was abolished by the enkephalinase inhibitor, puromycin. A strong relationship is suggested between the suppressing effect of metenkephalin, enkaphalinase and the protein kinase C system.

Disturbed regulation of cholesterol synthesis in monocytes of obese patients with hypercholesterolemia.

The aim of the present study was to clarify the influence of obesity on the functions of low-density lipoprotein receptors (LDL-R) and 3-hydroxy-3-methylglutarate-coenyzme A (HMG-CoA) reductase both in healthy control subjects and in patients with hypercholesterolemia (HC). Experiments were performed on monocytes of 15 non-obese (C I) and 11 obese (C II) healthy control subjects and on 22 non-obese (HC I) and 26 obese (HC II) patients with HC. [(125)I]LDL was used to determine LDL-R activity by measuring binding and intracellular degradation. The rate of endogenous cholesterol synthesis was measured using [(14)C]acetate incorporation into the cholesterol fraction of monocytes. The binding ability of [(125)I]LDL was identical across all groups. The [(14)C]acetate incorporation in resting monocytes was increased only in obese HC group. The 50-microg/mL LDL protein-induced inhibition of [(14)C]acetate incorporation was significantly diminished (P <.001) in the same group. A strong positive correlation was detected between the [(14)C]acetate incorporation by resting cells and LDL-induced inhibition in all groups except the obese HC group, in which their correlation was negative (P <.001). Furthermore, in the obese HC group, a significant positive correlation was detected between body mass index (BMI) and the basal level of [(14)C]acetate incorporation, whereas a negative correlation was found between BMI and LDL-induced inhibition of [(14)C]acetate incorporation. The present data suggest that in patients with HC the concomitant obesity results in dysregulation of cholesterol homeostasis, which may contribute to the accelerated atherosclerosis.

Specific and scavenger low-density lipoprotein receptors involved in the disturbed lipid metabolism of patients with non-insulin-dependent diabetes mellitus are independent of obesity.

Comparative studies were performed on monocyte-derived macrophages (MDMs), prepared by a 72-hour incubation of blood monocytes obtained from patients with non-insulin-dependent diabetes mellitus (NIDDM) and age-matched obese and non-obese controls. The MDMs, after a 72-hour culturing, expressed both specific and scavenger low-density lipoprotein (LDL) receptors on their surfaces. To study the binding capacity of both receptor types, [125I]LDL and [125I] acetylated LDL (acLDL) were applied to cells and the labeled ligands were then monitored to estimate the rate of intracellular degradations. The LDL-induced inhibition of endogenous cholesterol synthesis and the acLDL-triggered apolipoprotein (apo) E secretion were also studied, as the biological marker of receptor activation. The results indicate that the binding capacities of both specific and scavenger LDL receptors were not reduced in MDMs of diabetic patients. However, the intracellular degradation after LDL incorporation was decreased. The LDL-induced inhibition of cholesterol synthesis and the acLDL-transmitted apo E secretion were also found to be decreased in the MDMs of patients with NIDDM as compared with the obese and non-obese control groups. The NIDDM-induced impaired signal transduction of both specific and scavenger LDL receptors suggests an unclarified functional alteration of both receptor structures.

Disturbed intracellular calcium-related processes of hepatocytes and neutrophils in human alcoholic liver disease.

Isolated human hepatocytes and separated neutrophils of 11 patients with alcoholic liver disease (ALD) were used to study some aspects of cellular calcium-related processes compared to nonalcoholic controls. 45Ca2+ efflux from the cells decreased in ALD and the calmodulin-inhibitor trifluoperazine did not influence it further. The intracellular free calcium concentration [( Ca2+]i) of nonstimulated hepatocytes and neutrophils proved to be higher in ALD with the Quin2/AM loading technique. However, the [Ca2+]i rise in hepatocytes and neutrophils, with stimulation by low density lipoprotein (LDL) and N-formyl-methionyl-leucyl phenylalanine (FMLP), respectively, was diminished in ALD compared to appropriate controls. The slower 45Ca2+ extrusion rate, higher basal [Ca2+]i levels, and the diminished [Ca2+]i elevation of activated hepatocytes and neutrophils, suggest disturbed calcium-related intracellular processes in ALD, in particular, impaired regulation of the plasma membrane Ca2(+)-ATPase.

Activation of the lipoxygenase pathway in the methionine enkephalin induced respiratory burst in human polymorphonuclear leukocytes.

In comparative studies of f-met-Leu-Phe (FMLP) and methionine enkephalin (ME) induced polymorphonuclear leukocyte (PMNL) stimulation the following results were obtained: (i) both FMLP and ME increased the intracellular killing (IK) capability of human PMNLs probably through NADPH oxidase activation, (ii) the ME-induced respiratory burst (RB) differed from the chemotactic peptide FMLP-triggered superoxide generation because the former was not accompanied by the activation of the glutathione system and the duration of the superoxide production was prolonged. The reaction was dependent on lipoxygenation, was potentiated by indomethacin (IM) and was inhibited by nordihidro-guairetic acid (NDGA), (iii) both 14C-arachidonic acid (14C-AA) release and leukotriene B4 (LTB4) synthesis of ME-treated PMNLs were elevated as compared to those of FMLP triggered cells. Our results suggest that lipoxygenation and even an increased LTB4 synthesis are involved in the ME-induced RB of leukocytes.