Binding sites for angiotensin II in human mononuclear leucocytes.

Angiotensin II binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.     

Expression of protease and protease-inhibitory activity in human mononuclear leukocyte cultures : Effect of conA stimulation

Trypsin-activated protease activity was observed in the supernatants of conA-treated unfractionated or monocyte-depleted cultures at 1 h of incubation and trypsin-inhibitory activity was detected at 24 h. In contrast, supernatants from wheat germ agglutinin (WGA)-treated cultures or untreated cultures exhibited trypsin-inhibitory activity at 1 h and trypsinactivated protease activity after 24 h. The expression of proteases and protease inhibitors may be early events that occur during the activation of quiescent lymphocytes to a proliferative state.     

Lymphocyte chemotactic activity of human interleukin 1

In the cellular immune response, there is an accumulation of mainly nonantigen-specific mononuclear cells that presumably is dependent on the local secretion of chemotactic factors. In view of the presence of large numbers of macrophages early in the delayed hypersensitivity response, the possible role of these cells in the chemotaxis of lymphocytes was investigated by studying the chemotactic activity of purified human interleukin 1 (IL 1) on T and B cells. Chemotactic activity for T and B cells was observed, the effect on B cells being greater than on T cells. At low concentrations (less than 1 U/ml), IL 1 had predominantly chemotactic activity for B cells and chemokinetic activity for T cells. At high concentrations (10 to 20 U/ml), IL 1 had pure chemotactic activity for both cell types. A relationship was found between levels of migration of T and B cells and mouse thymocyte proliferation induced by purified IL 1 and by lipopolysaccharide-stimulated monocyte supernatants. The principal peaks of both activities were found in 16,000 to 18,000 m.w. fractions. In additional studies, the chemotactic response to IL 1 was inhibited by preincubation of T and B cells with IL 1 or stimulated monocyte supernatant, demonstrating the role of binding of IL 1 in the chemotactic response.     

Angiotensin II augmentation of tyrosine kinase activity in human adherent mononuclear cells

The relationship of angiotensin converting enzyme activity and angiotensin II to the inflammatory process in diseases such as sarcoidosis remains unclear. We hypothesize that granuloma macrophages regulate inflammation by release of angiotensin converting enzyme, which produces angiotensin II, and that angiotensin II in turn modulates monocyte/macrophage activity. Since tyrosine kinase catalyzes phosphorylation of tyrosine residues in proteins and is important in signal transduction and cellular activation, we further postulated that monocyte tyrosine kinases may play a role in the regulation of this process. Mononuclear cells from 11 healthy subjects were assayed for tyrosine kinase activity in the presence and absence of angiotensin II. In addition, tyrosine-specific phosphorylation of cellular proteins was also determined. Angiotensin II increased tyrosine kinase activity in a concentration-dependent manner. The maximal stimulation, which varied from 31 to 506%, was achieved following incubation of cells with 10(-4) M angiotensin II. Angiotensin II also increased the tyrosyl-phosphorylation of three proteins with molecular weights of 57, 62, and 63 kDa. We conclude that tyrosine kinase activity of adherent mononuclear cells and tyrosine phosphorylation of certain protein(s) may be involved in angiotensin II regulation of inflammatory processes.     

Constitutive activity of human angiotensin II type-1 receptors by Gq overexpression

We have developed an inducible HEK293/Tet-On cell line that transiently expresses both FLAG-tagged human angiotensin II type-I receptors (FLAG-hAT(1)R) and G(q)alpha G protein subunits in response to doxycycline. High and tightly regulated levels of FLAG-hAT(1)R (740+/-57 fmol/mg protein) and G(q)alpha (36-fold increase compared with non-induced cells) overexpression were consistently achieved. We investigated the possibility of using an inducible system to increase the proportion of constitutively active wild-type FLAG-hAT(1)Rs by overexpressing G(q)alpha. Following doxycycline treatment, we observed no significant change in the apparent binding affinity or potency (coupling efficiency) of angiotensin II, though significant increases in the intrinsic activity of several partial agonists were observed, indicative of constitutive activity. DUP753 (10 microM), a suggested inverse agonist, did not inhibit the enhanced level of basal (agonist-independent) activity. The data suggest that the resting equilibrium of hAT(1) receptors between the inactive (R) and active (R*) forms is predominantly weighted towards the inactive conformation.     

The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states

The binding of the chemoattractant N-formyl-methionylleucyl-[³H]phenylalanine to intact polymorphonuclear leukocytes and membrane preparations was analyzed by computer methods. Whole viable cells bind the chemoattractant with a single dissociation constant (K_D) of 22.3 ± 2.4 nM and contain an average of 55,000 receptors percell. In contrast, the binding data using membrane preparations were consistent with the presence of two classes of binding sites with average K_Ds of 0.53 ± 0.01 nM and 24.4 ± 1.2 nM. The high affinity receptors accounted for ca. 25% of the binding sites. Increasing the receptor occupancy did not affect the rate of dissociation of the ligand-receptor complex thus negative cooperativity is not a likely explanation for the complex binding isotherms. On the other hand, the dissociation kinetics did agree with the two affinity receptor model.     

Bilirubin inhibits the chemotactic activity of human polymorphonuclear leukocytesin vitro

Using the migration of human polymorphonuclear leukocytes in agarosein vitro, it was established that bilirubin inhibits migration (chemotaxis) of these cells upon stimulation with both complement-derived (zymosan-activated serum) and bacteria-derived (abacterial filtrate ofE. coli) chemotactic agents.     

The release of eosinophil chemotactic activity and eosinophil chemokinesis inhibitory activity by mononuclear cells from atopic asthmatic and non-atopic subjects

The goal of our study was to assess the chemotactic activity for eosinophils (ECA) and neutrophils (NCA) and histamine releasing activity (HRA) in crude supernatants of mononuclear cells in monosensitized atopic asthmatics and healthy controls. Chemotactic activity for ECA and neutrophils was measured in supernatants of cultured mononuclear cells with modified Boyden's chamber and HRA was assessed on healthy donor basophils. With respect to ECA generation two distinct subgroups of subjects were distinguished: releasers [ECA (+)] and non-releasers [ECA (-)]. In atopic and non-atopic ECA (+) the mean ECA index was 3.78 +/- 0.49 and 2.47 +/- 0.27 respectively (P > 0.05). Supernatants from the remaining subjects (seven of 22 atopic and five of 11 non-atopic) did not express ECA, but revealed significant inhibitory activity for chemokinesis of eosinophils (mean chemotactic index 0.25 +/- 0.16 and 0.48 +/- 0.22 for atopic and non-atopic non-releasers respectively). Stimulation with antigen of MNC from atopic and with PHA from non-atopic ECA (-) restored cells ability to release ECA. Sephadex gel chromatography revealed that supernatants of MNC contained chemotactic and chemokinesis inhibitory activity in different fractions. The spontaneous productions of NCA and HRA by mononuclear cells was similar in ECA releasers and non-releasers, although the HRA was higher following stimulation with PHA in the non-atopic ECA (+) subgroup. Our study demonstrated, for the first time, that MNC are capable of generating not only chemotactic activity but also chemokinesis inhibitory activity for eosinophils.     

Chemotactic response of splenic mononuclear cells to angiotensin II in murine schistosomiasis

Murine Schistosomiasis mansoni is a parasitic infection associated with a delayed-type hypersensitivity granulomatous reaction to the schistosome eggs. This reaction is characterized by the accumulation of mononuclear cells and other inflammatory cell types around the eggs. Granuloma macrophages produce angiotensin II (AII), which appears to have immunoregulatory function. By using an in vitro chemotaxis assay, this study demonstrated that AII is a chemotactic factor for splenic mononuclear cells derived from infected mice. The response was bimodal, with peak activities occurring at 10(-10) and 10(-6) M. AII was chemotactic for T lymphocytes, B lymphocytes, and a large population of unidentified mononuclear cells at the optimal chemotactic concentrations of the peptide. At high concentrations, AII was also chemotactic for phagocytic mononuclear cells. Sar1, ala8-AII, an analog of AII with antagonist activity, completely blocked AII-induced chemotaxis. A [3H]-AII binding assay revealed high-affinity specific binding on spleen cells. The binding was rapid, was dependent on radioligand concentration, and was reversible. These latter observations suggest that the chemotactic activity of AII for subpopulations of splenic mononuclear cells is mediated via AII receptors.     

Conformation and activity in angiotensin II peptides

Angiotensin II and its competitive inhibitor [Sar¹, Ile⁸]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d₆-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.     

The assimilation of tri- and tetrapeptides by human erythrocytes

Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F. and Kuchel, P.W. (1985) Biochem. J. 227, 833-842) but that the transmembrane flux rates for tetrapeptides are considerably less. 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo. Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave Km = 2.11 +/- 0.08 and 23.4 +/- 0.9 mmol/l and Vmax = 307 +/- 3 and 905 +/- 22 mmol/h per 1 packed cells, respectively. In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide. Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport; the values were Km = 11.6 +/- 1.1 and 56 +/- 18 mmol/l and Vmax = 12.9 +/- 3.0 and 36.4 +/- 3.2 mmol/h per 1 packed cells, respectively, for the previously mentioned peptides. The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides. The above mentioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions; these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo.     

Consequences of chemosensory phenomena for leukocyte chemotactic orientation

The stochastic nature of cell surface receptor-ligand binding is known to limit the accuracy of detection of chemoattractant gradients by leukocytes, thus limiting the orientation ability that is crucial to the chemotactic response in host defense. The probabilistic cell orientation model of Lauffenburger is extended here to assess the consequences of recently discovered receptor phenomena: "down-regulation" of total surface receptor number, spatial asymmetry of surface receptors, and existence of a higher-affinity receptor subpopulation. In general, a reduction in orientation accuracy is predicted by inclusion of these phenomena. An orientation signal based on a simple model of chemosensory adaptation (i.e., a spatial difference in relative receptor occupancy) is found to be functionally different from the signal suggested by an experimental correlation (i.e., a spatial difference in absolute receptor occupancy). However, in the context of receptor "signal noise," the signal based on adaptation yields predictions in better qualitative agreement with the experimental orientation data of Zigmond. From this cell orientation model we can estimate the effective time-averaging period required for noise diminution to a level allowing orientation predictions to match observed levels. This time-averaging period presumably reflects the time constant for receptor signal transduction and locomotory response.