Cytokines modulate endothelial cell intracellular signal transduction required for VCAM-1-dependent lymphocyte transendothelial migration.

Vascular cell adhesion molecule-1 (VCAM-1) activates endothelial cell NADPH oxidase which catalyzes production of reactive oxygen species (ROS). This activity is required for VCAM-1-dependent lymphocyte migration. The focus of our study was to determine whether these VCAM-1-dependent functions are modulated by cytokines. TGF-beta1 or IFN-gamma pretreatment of mouse endothelial cell lines inhibited VCAM-1-dependent B and T cell transendothelial migration without affecting initial lymphocyte adhesion. Neutralizing anti-TGF-beta1 blocked the effects of TGF-beta1 pretreatment of endothelial cells, whereas addition of anti-TGF-beta1 after TGF-beta1 pretreatment of the endothelial cells did not block TGF-beta1-mediated inhibition. Neutralizing anti-IFN-gamma also blocked the inhibitory effects of IFN-gamma. TGF-beta1 and IFN-gamma blocked migration by inhibiting the VCAM-1-stimulated production of low levels of ROS (0.1-0.9 microM H2O2). These results demonstrate that both TGF-beta1 and IFN-gamma directly affect the endothelial cells' ability to promote lymphocyte migration. IL-4 had differing effects on T and B cells during transmigration. IL-4 augmented T cell migration across the endothelial cell lines but did not affect T cell adhesion. Conversely, IL-4 increased B cell adhesion to the endothelial cell lines without affecting migration. In summary, cytokines can directly modulate microvascular endothelial cell intracellular signaling, demonstrating a new level of cytokine regulation of lymphocyte diapedesis.

Different orders for acquisition of apoptotic characteristics by leukocytes.

Apoptotic leukocytes undergo cellular changes that are used as markers for "early" versus "late" stages of apoptosis. To ascertain if the order for acquisition of these changes is unique to specific hematopoietic cell types, we compared four leukocyte cell types and the following five apoptotic characteristics: MC540 incorporation, annexin V-FITC binding, propidium iodide (PI) labeling of hypodiploid nuclei, DNA fragmentation by a colorimetric assay, and cell membrane permeability to PI. The order for acquisition of these apoptotic characteristics was significantly different for each of the leukocyte cell types and for the mode of induction of apoptosis. It is interesting that the nuclear changes but not the membrane changes studied in mouse spleen cells required caspase activity. In summary, the acquisition of these apoptotic characteristics occurs through caspase-dependent and caspase-independent mechanisms, and importantly, the order for acquisition of the characteristics is specific for the cell type and for the mode of induction of apoptosis.

Novel alpha 4-integrin ligands on an endothelial cell line.

The unique combination of adhesion molecules expressed on endothelial cells is thought to mediate the specificity of leukocyte-endothelial cell interactions. In this study, murine endothelial cell lines were used as a model to identify novel adhesion molecules that participate in these cellular interactions. Lymphocyte adhesion to the continuous endothelial cell lines mHEVa and mHEVc required alpha 4-integrin. Interestingly, lymphocyte alpha 4-integrin bound to VCAM-1 as well as an unknown ligand on the mHEVa cell line. We have demonstrated that this VCAM-1-independent adhesion to the mHEVa cells was not mediated by other known alpha 4-integrin ligands (fibronectin, alpha 4-integrin itself, or MAdCAM-1). Two novel alpha 4-integrin ligands (p50 and p10) were isolated from the mHEVa cell line but not the mHEVc cell line by B cell alpha 4-integrin-specific ligand binding of radiolabeled mHEV cell membrane proteins. These results provide the first direct evidence that novel ligands for alpha 4-integrin exist on membranes from endothelial cells.

Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase.

Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0-24 h. This migration was inhibited by anti-VCAM-1 or anti-alpha4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.

Human and murine high endothelial venule cells phagocytose apoptotic leukocytes.

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0-6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosis in vivo. Mice were treated with a natural corticosterone (4-pregnene-11 beta,21-diol-3,20-dione) at levels detected during stress or malnutrition (93-180 micrograms serum cortisol/dl). At 4-12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.

A novel flow cytometric method for quantifying phagocytosis of apoptotic cells.

Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin. An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, nonbound lymphocytes were removed and the mono-layers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothellal cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA+ CD45+); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA+ CD45-); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems.

Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes.

Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells. Morphologically the cells were 20-30 microns in diameter and exhibited contact inhibition. The cells were not myeloid in origin because they lacked sodium fluoride-inhibitable nonspecific esterase activity, myeloperoxidase activity, and F4/80 antigen. The cell line phenotypes were compared to high endothelial venule (HEV) cells in tissue sections. HEV cells in lymph node tissue sections expressed endoglin, PECAM-1, ICAM-1, VCAM-1, laminin, fibronectin, collagen IV, H2Kd, MECA 79, MECA 325, and vWF. The cell lines expressed endoglin, VCAM-1, fibronectin, and H2Kd. The cell line derived from cervical lymph nodes also expressed laminin and H2Dd. Neither cell line expressed collagen IV, IAd, ICAM-1, ICAM-2, dendritic cell antigen, or PECAM-1. They also did not express MECA antigens or intracellular vWF, consistent with reports of many cultured endothelial cells. To further substantiate cell ine identification, antiserum generated against the cell lines bound specifically to HEV cells in frozen lymph node tissue sections and to both of the lymph node-derived cell lines but not control cell lines. Thus, the lymph node derived-cell lines expressed molecules found on HEV cells in vivo and most importantly retained the functions of tubule formation, lymphocyte adhesion, and promotion of lymphocyte migration.

Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action.

The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta-adrenergic antagonists propranolol and nadalol did not affect the catecholamine-mediated inhibition of lymphocyte activation. Although lymphocytes contain beta-adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors.

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard (L-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was "mimicked" by the membrane penetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The beta-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal or L-PAM TuB spleen cells. Since the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8+ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.

Functional capacity of the residual lymphocytes from zinc-deficient adult mice.

Zn deficiency has been shown to reduce host defence drastically. It was of interest to determine the capacity of the residual lymphocytes from Zn-deficient mice to proliferate and produce lymphokines in response to stimulation since there are many Zn-dependent metalloenzymes that might be altered by the deficiency. To address this question, young adult A/J mice were provided Zn-deficient or Zn-adequate diets or restricted amounts of a Zn-adequate diet for 30 d. Splenocytes from moderately or severely Zn-deficient adult A/J mice gave normal proliferative responses and generated adequate interleukin II (IL-2) activity when stimulated with the mitogen Concanavalin A. However, splenocytes from deficient mice exhibited a higher degree of proliferation (about 150%) and production of IL-2 in response to foreign target cells compared with T-cells prepared from mice provided a Zn-adequate diet. B-cells from deficient mice stimulated in vivo with sheep erythrocytes produced fewer total numbers of plaque-forming cells (PFC) per spleen. Nevertheless, the proportion or number of PFC/10(6) viable splenocytes and the amounts of IgM and IgG antibody produced per PFC were equivalent to those of adequately-fed and restricted-fed controls. The previously described responses were not significantly affected by whether the level of Zn in the culture medium was adequate or limiting. Based on these tests it appeared that the residual splenic lymphocytes of Zn-deficient mice were able to carry out many fundamental immune processes.

Optimization of peroxide production by resident macrophages.

Previous reports indicated that resident macrophages produced low or nondetectable quantities of H2O2 (1-10 nmol H2O2/mg resident macrophage protein) when measured by the phenol red assay described by Pick and Mizel [5]. However, modifications of the assay that included addition of calcium to assay solutions, alterations in cell-harvesting methods, increasing the concentration of cells analyzed, incubating the cells in ambient air, and enhancing the interactions between macrophages and particulate stimulants by centrifugation resulted in increased production of peroxide. Each variable was instrumental in improving the assay conditions and the combination of changes dramatically increased the amount of peroxide (30-60 nmol H2O2/mg resident macrophage protein) obtained from murine resident macrophages, making it possible to better assess the role of these cells in microbicidal killing.

Mouse hepatitis virus infection suppresses modulation of mouse spleen T-cell activation.

Natural infection by mouse hepatitis virus (MHV) can affect interpretation of immunological studies in mice. MHV, a collective term describing a group of corona viruses, is found in natural infections in over 70% of laboratory mouse populations in the U.S.A. and Canada. Natural outbreaks of MHV in our animal colony afforded us the opportunity to study MHV-induced immunosuppression as well as the effects of MHV infection on neurotransmitter immunomodulation. Concanavalin A (Con A)-stimulated DNA synthesis by spleen T lymphocytes from MHV-infected mice was 20-50% that of non-infected mice. The MHV infection also altered neurotransmitter modulation of spleen T-lymphocyte activation. In contrast to noradrenaline ablation of Con A-activated DNA synthesis by spleen lymphocytes from non-infected mice, DNA synthesis by the infected group was not inhibited by noradrenaline or dibutyryl-cAMP. These effects of MHV infection were specific for spleen T lymphocytes since MHV infection did not alter Con A stimulation of thymocytes, lipopolysaccharide stimulation of spleen B lymphocytes, or noradrenaline inhibition of thymocyte and B-cell DNA synthesis. MHV infection also did not alter spleen T-lymphocyte subset proportions. Thus, MHV infection inhibits spleen T-lymphocyte activation and blocks in vitro catecholamine and cAMP regulation of spleen T-cell activation but does not affect activation of thymic cells or spleen B cells.

Possible roles for zinc in destruction of Trypanosoma cruzi by toxic oxygen metabolites produced by mononuclear phagocytes.

The effects of a single nutrient deficiency on immune function is now most extensively characterized using the dietary zinc deficient murine model. Deficiencies in zinc have rapid adverse effects on host defenses of humans and rodents. This impaired defense seems to be, in part, the result of a reduction in number of lymphocytes available for surveillance since residual lymphocytes are able to carry out many normal functions. In vitro, the lymphocytes were able to proliferate at a normal rate as well as produce antibodies or interleukin 2 in response to mitogens or antigens even when cultured in autologous serum to reduce the possibility of restoration of zinc deficient functions. Conversely, mononuclear phagocytes (MNP) from deficient mice had a significantly reduced capacity to associate with and kill the parasite Trypanosoma cruzi (T. cruzi) which causes Chaga's disease. Moreover, indicating the specificity of the deficient function, a short incubation of ZnCl2 but not other metals completely restored the capacity of MNP from deficient mice to take up and kill T. cruzi. Dependency on H2O2 production by the MNP's oxygen burst for killing of T. cruzi.suggested that MNP from zinc deficient mice might produce smaller amounts of H2O2. The possibility that zinc might play an integral role in the oxygen burst seemed evident from the ability of zinc to quickly restore the killing capacity of MNP from the zinc deficient mice. Further, the renewed interest in the role of metals in the production of highly reactive oxidants in biological systems prompted a literature search to identify enzymes and/or reactions known to be involved in the generation of oxygen radicals or toxic oxygen metabolites that might be zinc dependent. The literature review provided herein indicates many possible roles for zinc in the generation of toxic oxygen species. The data indicated that normal levels of H2O2 are produced by MNP from zinc deficient mice. The amount of H2O2/mg macrophage protein is normal in response to phorbol or opsonized zymosan but reduced in response to direct stimulation by T. cruzi. However, the reduced H2O2 production by T. cruzi-stimulated zinc deficient MNP was due to reduced stimulation as a result of fewer T. cruzi associated with the MNP. Thus, H2O2 levels/parasite were the same as zinc adequate controls. Yet, this does not preclude the possibility that reduced killing of T. cruzi by MNP from zinc deficient mice may be due to a function for zinc in the actual killing process or in the production of some other agent important in the killing of T. cruzi.