Effects of adrenoreceptor stimulation on in vitro spontaneous lymphocyte adhesion

The influence of adrenergic agents: alpha, beta-agonists adrenalin and noradrenaline, alpha-agonist mesaton (phenylephrine) and beta-agonist isadrin on spontaneous adhesion of lymphocytes of healthy donors in vitro was studied. It was established, that the influence on lymphocyte alpha-adrenoreceptors caused stimulation, and on beta-adrenoreceptors--inhibition of the lymphocyte spontaneous adhesion. With the help of alpha-adrenoblocking agent phentolamine and beta-adrenoblocking agent propranolol it was demonstrated, that the adrenergic effects revealed were pharmacologically specific.     

Effect of stimulation of dopamine and histamine receptors on spontaneous lymphocyte adhesion in vitro

With the help of spontaneous lymphocyte adhesion test the influence of dopamine and histamine on the adhesion of peripheral blood lymphocytes has been studied in healthy donors. It was shown that dopamine enhanced spontaneous lymphocyte adhesion, with the optimal concentrations of the substance for the realization of a stimulating effect being 10(-4)-10(-6) M. The effect of the enhancement was pharmacologically specific, as it was completely blocked by dopamine receptor-blocker--haloperidol. It was demonstrated that low concentrations of histamine (10(-7)-10(-8) M) enhanced, while higher concentrations (10(-6)-10(-3) M) inhibited lymphocyte adhesion. Dimedrol, but not cimetidine abolished the enhancement of spontaneous lymphocyte adhesion, while the adhesion inhibition was blocked by cimetidine, but not dimedrol. Thus, the revealed histaminergic enhancement and inhibition of spontaneous lymphocyte adhesion is mediated through the influence of histamine on H1- and H2-receptors, respectively.     

Study of the participation of the alpha-chain of the LFA-1 antigen in the phenomenon of lymphocyte adherence inhibition using ICO-11 monoclonal antibodies in vitro

The influence of monoclonal antibodies (Mabs) ICO-11 on the ability of lymphocytes from breast cancer patients to react in lymphocyte adherence inhibition (LAI) test has been studied in vitro. It has been demonstrated that Mabs ICO-11 in the dilutions 1/4 and higher blocked the reaction to tumor extracts in LAI-test, without affecting the reaction to the extracts of normal tissues and a spontaneous adhesion of lymphocytes of healthy donors as well. The addition of the control supernatants of myeloma cells X63.Ag8. 653 to the test-system in the same dilutions caused no influence upon LAI-reaction and spontaneous adhesion of lymphocytes from healthy donors. A possible participation of alpha-chains of function-associated antigens in the binding of tumor-associated antigens to T-cells in the inductive phase of the reaction of lymphocyte adherence inhibition in vitro has been suggested.     

The effect of Leu- and Met-enkephalins on the adenosine deaminase and 5'-nucleotidase activities of the lymphocytes under conditions of stress stimulation of metastasis

A correlation between changes of the hypothalamic mediator processes, hormone level in blood plasma, catecholamines and their catabolites in urine and changes of the activity of adenosine metabolism enzymes in the thymus and the spleen lymphocytes has been observed in C57B1 mice with Lewis lung carcinoma. Effect of leu- and met-enkephalines on neuroendocrine stress-realizing mechanisms proved to be different, that is responsible for their contrary effect on the metastatic growth--leu-enkephaline inhibits and met-enkephaline stimulates the process. It has been shown that an inhibitory effect of leu-enkephaline is greatly related to its stimulating influence on the thymus lymphocyte activity and a decrease of the functional activity of the spleen lymphocytes.     

Effect of cholinergic stimulation on spontaneous lymphocyte adhesion in vitro

With the help of spontaneous lymphocyte adhesion test it was demonstrated that acetylcholine and carbacholine enhanced the adhesion of lymphocytes. The enhancement revealed was completely abolished by a specific M-cholinergic blocker--atropine, but not N-cholinergic blocker--hexonium. Thus, the enhancement of spontaneous human lymphocyte adhesion by cholinergic stimulation is mediated by M-cholinoreceptors. It is suggested that the phenomenon revealed is mediated by the enhancement of cGMP intracellular levels after cholinergic stimulation.     

Inhibition of copulatory behavior in male rats by D-ALA2-Met-Enkephalinamide

D-Ala²-Met-Enkephalinamide (DALA), a synthetic analog of met-enkephaline resistant to enzymatic degradation, was injected intraventricularly to sexually experienced male rats paired with receptive females. A dose of DALA of 6 μg which did not influence spontaneous motor activity, completely suppressed the copulatory behavior of all animals tested. A dose of 3 μg significantly increase mounting and intromission latencies, but did not influence other measures of the copulatory behavior. The effect of DALA was prevented by naloxone (1 mg/Kg), a specific inhibitor of opioid receptors. The results suggest that enkephalins may play a role in the regulation of copulatory behavior.     

99mTc-HMPAO labelling inhibits cell motility and cell proliferation and induces apoptosis of NC-NC cells

(99m)Tc-hexamethyl-propylenamine-oxime ((99m)Tc-HMPAO)-labelled leukocytes have been used in standard diagnostic procedures for the detection of infection and inflammation. Although some investigators have already pointed out that labelling of leukocytes with (99m)Tc-HMPAO has detrimental effects on the cells, still very little is known regarding the effects of ionizing radiation on lymphocyte function. The effects of (99m)Tc-HMPAO-labelling on lymphocyte adhesion, proliferation, mitotic index, migration and apoptosis were evaluated. The lymphoblastoid cell line NC-NC was used as the lymphocyte population. (99m)Tc-HMPAO-labelling decreased cell adhesion, proliferation, mitotic index and motility, whereas it induced apoptosis and cell-cycle arrest. The rate of decrease in cell proliferation was up to 70% (P<0.001) by day 4 after labelling. (99m)Tc-HMPAO-labelling led a 35% decrease (P<0.001) in adhesion ability of the cells on fibronectin at 16h. Using the Boyden chamber motility assay, it was shown that both spontaneous and monocyte chemotactic protein (MCP-1)-induced lymphocyte motility were strongly reduced by (99m)Tc-HMPAO-labelling. The decrease in motility was approximately five-fold (P<0.05). In addition, a 12-fold increase (P<0.05) was observed in apoptosis of the (99m)Tc-HMPAO-treated cells compared with control cells. Besides, it was shown that cell-cycle arrest was induced starting from the 3rd day after treatment with (99m)Tc-HMPAO. Our observations indicate that (99m)Tc-HMPAO-labelling has damaging effects on lymphocyte function including cell adhesion, proliferation, mitotic index, motility and cell cycle under in vitro conditions.     

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

Mechanisms of release of atrial natriuretic factor. I. Effect of several agonists and steroids on its release by atrial minces

The effect that several substances may have on ANF release by atrial slices and on its tissular content was investigated. alpha- and beta-adrenergic and cholinergic agonists, vasopressin, met-enkephaline, dexamethasone and DOC, in concentration ranging from 10(-4) to 10(-8) M, were added into the incubation media and incubated 1 and 4 hours. No changes were observed in ANF concentration either in the media or in its tissular concentration as measured by a specific radioimmunoassay. When intact rats were previously treated with DEXA, DOC or DEXA + DOC and their atria incubated "in vitro", an increase in the release of ANF was observed in the Dexa-treated group only, but all treated groups had higher tissular ANF concentration. It is concluded that neither alpha- or beta-adrenergic, nor cholinergic agonists or vasopressin and met-enkephaline stimulate ANF release "in vitro". On the other hand steroids may regulate ANF release and synthesis in the intact rat. It seems likely that the ANF released into the media corresponds to a short peptide.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.     

A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.     

Endothelial PI 3-kinase activity regulates lymphocyte diapedesis

Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 +/- 6 or 34 +/- 7%, respectively, vs. control; mean +/- SE; P < 0.05). Similarly, EC knockdown of the p85alpha regulatory subunit of PI 3-kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 +/- 10% vs. control (P < 0.05). EC PI 3-kinase inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC "docking structure" after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.     

Statin-derived 1,3-oxazinan-2-ones as submicromolar inhibitors of LFA-1/ICAM-1 interaction: stabilization of the metabolically labile vanillyl side chain

Modification of the vanillyl substituent on a potent, semisynthetic lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 binding inhibitor of the statin family resulted in metabolically more stable analogues that displayed submicromolar inhibitory activity in vitro and considerable anti-inflammatory activity in vivo. The benzodioxole derivative 2b emerged with the best overall profile.     

Effect of T cell activation on lymphocyte-endothelial cell adherence and the role of VLA-4 in the rat.

Lymphocyte migration from the blood is in part controlled by lymphocyte surface adhesion molecules, such as VLA-4 and LFA-1. Small lymph node lymphocytes from rats adhere poorly to rat microvascular endothelial cells (EC), while lymphoblasts from antigen-challenged lymph nodes have an enhanced adherence to EC and preferentially migrate to inflamed tissues. This lymphoblast adherence is partially inhibited by anti-VLA-4. The effects of in vitro activation of lymph node lymphocytes on lymphocyte-EC adhesion were examined. In vitro stimulation of T cells with concanavalin A or calcium ionophore and phorbol myristate acetate (PMA) for 1-4 hr caused a marked (7- to 12-fold) increase in lymphocyte adhesion to unstimulated and IFN-gamma- or LPS-treated EC. This adhesion was partially inhibited by anti-VLA-4, was not associated with increased VLA-4 expression, was partially inhibited at 4 degrees C, and was virtually eliminated at 4 degrees C with anti-VLA-4. Anti-CD3 or IL-2 stimulation of T cells also enhanced lymphocyte adhesion but required 2-3 days of culture. This adhesion was not inhibited by anti-VLA-4 and was almost totally inhibited at 4 degrees C, suggesting a primarily LFA-1-mediated adhesion. In conclusion, stimulation of T cells with Con A or calcium ionophore plus PMA caused a rapid enhancement of lymphocyte-EC adhesion mediated in part through VLA-4, while stimulation of T cells with anti-CD3 or IL-2 enhanced lymphocyte adhesion apparently independent of VLA-4.     

Effect of somatostatin on the proliferation of mouse spleen lymphocytes in vitro

The effect of somatostatin on the spontaneous proliferation of mouse spleen lymphocytes was investigated in vitro. The rate of 3H-thymidine incorporation was used as an index of lymphocyte proliferation. Somatostatin in a concentration of 10(-7) M enhanced the lymphocyte proliferation and abolished the antiproliferative effect of rat hypothalamic extract. Lower concentrations of somatostatin slightly decreased the lymphocyte DNA synthesis.     

Pathways to chronic inflammation in rheumatoid synovitis

Postcapillary venules resembling the high endothelial venules (HEVs) of lymphoid tissues have often been observed at sites of chronic inflammation. We have therefore postulated that such venules may be an important site of lymphocyte migration into rheumatoid synovial membrane and that inflammatory cell products may act on endothelial cells (ECs) to increase lymphocyte emigration. Electron microscopic examination of rheumatoid synovial membranes showed that a strong correlation existed between the proportion of lymphocytes in perivascular tissue and the height/base ratio of the ECs in those areas. In addition, binding experiments showed that peripheral blood mononuclear cells preferentially bound to ECs in sections of rheumatoid synovial membrane that had the morphological appearance of HEVs. In vitro binding experiments, in which lymphocyte adhesion to human umbilical vein EC monolayers was measured, showed that adhesion was enhanced by preincubation of the ECs with interferon-gamma or interleukin 1 (IL 1). The central role of IL 1 in increasing lymphocyte migration into the rheumatoid synovial membrane was also supported by the findings that IL 1 is chemotactic for lymphocytes, ECs can secrete IL 1, and IL 1 activity is readily detectable in synovial fluids of rheumatoid arthritis patients.     

Molecular mechanisms of lymphocyte extravasation. I. Studies of two selective inhibitors of lymphocyte recirculation

Pertussigen, a protein toxin purified from Bordetella pertussis, and fucoidin, a high molecular weight sulfated polysaccharide, were analyzed for their ability to inhibit lymphocyte recirculation in vivo. Pertussigen treatment of lymphocytes resulted in a dosage- and time-dependent loss of their ability to localize in lymph nodes or Peyer's patches. This toxin-induced alteration did not reverse after extended lymphocyte culture in toxin-free media, and had no effect on lymphocyte viability or activation by mitogens. Furthermore, pertussigen-treated lymphocytes retained the ability to specifically adhere to high endothelial cells in an in vitro binding assay. Kinetic studies suggested that the toxin's molecular action on lymphocytes is analogous to that reported for pancreatic islets and hormone-responsive cultured cell lines. Inhibition of lymphocyte recirculation by fucoidin was also observed in vivo. Fucoidin-mediated inhibition of lymphocyte localization to peripheral lymph nodes was reversible with time, and could not be effected by pretreatment of lymphocytes with the polysaccharide. Furthermore, we confirmed the observation that fucoidin blocks lymphocyte adhesion to high endothelial cells in vitro. On the basis of these observations, we propose that the mechanism of lymphocyte extravasation involves a specific receptor-mediated binding event followed by an adenylate cyclase-dependent activation of cell motility. Fucoidin is capable of interfering with the primary adhesion event, whereas pertussigen selectively inhibits the second process to block lymphocyte recirculation in vivo.     

Mg2+-dependent doxycycline-induced inhibition of leukocyte adhesion

The method for estimating in vitro leukocyte adhesion was modified in relation to investigation of the effect of antibiotics and other pharmacological agents on cell activation. The adhesion intensity was estimated in standard 96-cavity microplates from the content of beta-hexosaminidase in the adherent cells. That made it possible to easily automatize the method by using the equipment for enzyme immunoassay. It was shown that spontaneous and induced adhesion of guinea pig leukocytes depended on temperature and bivalent ions. The level of spontaneous adhesion mainly depended on Mg ions. Doxycycline, 0.1 to 100 micrograms/ml, did not induce degranulation of leukocytes and had no cytotoxic action. Doxycycline in supratherapeutic concentrations (greater than 10 micrograms/ml) inhibited the spontaneous leukocyte adhesion. In special experiments it was found that the doxycycline's inhibitory effect was partially reversible and associated with ++chelation of Mg ions rather than Ca ones.     

Demonstration of the lymphokine mechanism of the reinforcement of lymphoid proliferation during liver regeneration in mice

Changes in the ability of mouse splenocytes to proliferate early after partial hepatectomy have been studied. Proliferative activity of mouse splenocytes in mixed lymphocyte culture and in spontaneous proliferation in vitro test increased within 4, 17 and 24 hours after operation as compared with pseudo-operated control. After 4 hours of cultivation splenocytes of partially hepatectomized mice excreted into culture medium factor(s) that enhanced proliferative activity of intact mouse lymphocytes both in vitro and in vivo. The factor is thermolabile, operates nonspecifically, and is produced by T-lymphocytes.     

Effect of staphylococcal toxin on lymphocyte receptors and function

Staphylococcal toxin at a concentration of 10(-3) inhibits in vitro the rosette-formation of lymphocytes, taken from healthy donors and patients with purulent septic diseases, with sheep and mouse red blood cells, changes the ratio of lymphocyte subpopulation, modifies the spontaneous antigen- and mitogen-dependent migration of leukocytes. The latter phenomenon is not linked with disturbances in the lymphokine-producing activity of lymphocytes, but results from changes in the migration properties of granulocytes.