Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance.

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.

Dendritic cells improve the generation of Epstein-Barr virus-specific cytotoxic T lymphocytes for the treatment of posttransplantation lymphoma.

BACKGROUND: The use of immunosuppressive therapies after solid organ transplantation has been shown to increase a patient's risk for Epstein-Barr virus (EBV)-associated lymphoma. A potential therapy for this disorder is the adoptive transfer of EBV-specific cytotoxic T lymphocytes (CTLs). We proposed that dendritic cells (DCs) could be loaded with EBV antigens and be used to improve the in vitro generation of EBV-specific CTLs. METHODS: Autologous EBV-transformed B-lymphoblastoid cell lines (BLCLs) were generated from normal donors, and CTLs were initiated by culturing peripheral blood mononuclear cells with DCs alone, disrupted BLCLs alone, intact, irradiated BLCLs alone, and DCs loaded with disrupted BLCLs. Lytic activities were determined with a 4-hour chromium-release assay against autologous BLCLs, and statistical calculations were performed by a Student t test assuming equal variance. RESULTS: The lytic activity of CTLs generated with DCs loaded with disrupted BLCLs reached 78% and was statistically significant (P < .01) at all effector/target ratios compared with CTLs generated with DCs alone, disrupted BLCLs alone, or intact BLCLs alone. Total numbers of CTLs were also greater than those of control groups for DCs loaded with disrupted BLCLs. CONCLUSIONS: DCs improved the in vitro generation of EBV-specific CTLs as evidenced by this group's significantly increased lytic activity over that of the control group. The improved lytic activity of DC-generated EBV-CTLs suggests that adoptive transfer of these cells could lead to a more effective immunotherapeutic response against posttransplantation EBV-associated lymphoma.

[In vitro exposure of a human bronchial epithelial cell line with nitrogen dioxide induces enhanced transcription and liberation of pro-inflammatory cytokines]. / In-vitro-Expositionen einer humanen bronchialen Epithelzelllinie mit Stickstoffdioxid induzieren eine vermehrte Transkription und Freisetzung proinflammatorischer Zytokine.

Studies of in vivo inhalation of nitrogen dioxide (NO2) have demonstrated a transient pulmonary inflammation. This study was done to determine the contribution of airway epithelial cells to the release of inflammatory mediators following NO2 exposure. Confluent cultures of the human bronchial epithelial cell line BEAS-2B on Transwell-Col filters were exposed for 1 h to air or NO2 up to 1.5 ppm with the apical fluids removed with 5% CO2 at 37 degrees C. The cells were hydrated with Hanks' Balanced Salt Solution (HBSS) in the basolateral compartment. Sequential reverse transcription and quantitative cDNA amplification (RT-PCR) was used to measure inflammatory mediator mRNA abundance in BEAS-2B cultures. When compared to air-exposed cells, NO2 induced increases in IL-6 (23.4-fold) and IL-8 (30.9-fold) mRNA abundance. The NO2-dependent increases in mRNA expression reached a maximum between 0 and 1 h post exposure and returned to baseline levels within 24 h. IL-6 and IL-8 proteins as measured by enzyme-linked immunosorbent assays (ELISA) were also elevated in supernatants recovered from NO2-exposed BEAS-2B cells. These studies suggest that exposure to NO2 induces the synthesis and release of inflammatory mediators from airway epithelial cells that may participate in the pathogenesis of airway disease.

Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells.

Acute exposure of animals and humans to ozone results in decrements in lung function, development of airway hyperreactivity, inflammation, edema, damage to pulmonary cells, and production of several compounds with tissue damaging, fibrinogenic or fibrotic potential. The contribution of airway epithelial cells and alveolar macrophages to these processes is unclear. In this study we have directly exposed human alveolar macrophages and human airway epithelial cells to ozone in vitro and measured the cytotoxic effects of ozone, as well as the production of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and fibronectin, all of which are substantially elevated in the bronchoalveolar lavage fluid of humans exposed to ozone. Cells were grown on rigid, collagen-impregnated filter supports, and the interaction of cells with ozone facilitated by exposing them to the gas with medium below the support but no medium on top of the cells. The results show that, although macrophages are much more sensitive to ozone than epithelial cells, they do not produce increased amounts of IL-6, IL-8, or fibronectin following ozone exposure. In contrast, epithelial cells produce substantially more of all three proteins following ozone exposure, and both IL-6 and fibronectin are secreted vectorially. An immortalized human airway epithelial cell line (BEAS 2B) was used in these experiments since human airway epithelial cells are infrequently available for in vitro studies. Data from this study extend previous findings which suggest that the BEAS cell line is a useful model to study the interaction between airway epithelial cells and environmental toxicants.

The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators.

Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. We therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of our data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

Solubility of vesicular stomatitis virus M protein in the cytosol of infected cells or isolated from virions.

The peripheral membrane M protein of vesicular stomatitis virus purified by detergent extraction of virions and ion-exchange chromatography was determined to be a monomer in the absence of detergent at high salt concentrations. Reduction of the ionic strength below 0.2 M resulted in a rapid aggregation of M protein. This self-association was reversible by the detergent Triton X-100 even in low salt. However, aggregation was not reversible by high salt concentration alone. M protein is initially synthesized as a soluble protein in the cytosol of infected cells, thus raising the question of how the solubility of M protein is maintained at physiological ionic strength. Addition of radiolabeled M protein purified from virions to unlabeled cytosol from either infected or uninfected cells inhibited the self-association reaction. Cytosolic fractions from infected or uninfected cells were equally effective at preventing the self-association of M protein. Self-association could also be prevented by an irrelevant protein such as bovine serum albumin. Sedimentation velocity analysis indicated that most of the newly synthesized M protein is monomeric, suggesting that the solubility of M protein in the cytosol is maintained by either low-affinity interaction with macromolecules in the cytosol or interaction of a small population of M-protein molecules with cytosolic components.

Cell-mediated cytotoxicity and the reorientation of effector cell granules towards the target cell are inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone.

Cell-mediated cytotoxicity involves a localized secretory process in which lytic agents stored in specialized granules of the effector cells are released upon contact with the appropriate target cell membrane and cause membrane damage. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibits cytotoxicity of natural killer cells, cytotoxic T-lymphocytes and lymphokine-activated killer cells. This inhibition is due to an effect of CCCP on the cytolytic cells, rather than on their targets, and is reversible. Treatment with CCCP does not inhibit the formation of effector-target conjugates, but seems to affect the programming of the effector cells for lysis. CCCP only inhibits lysis if added during a certain period of the lytic cycle: it has an effect only if added before, or within 5 minutes of the initiation of killing by a pulse of Ca++. Effector cells treated with CCCP retain their characteristic beta-glucuronidase-positive granules, but in the presence of the drug, these are no longer oriented to face the contact area with the target cell membrane.

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity.

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.

Cytolysis of actinomycin D-treated target cells by cell-free supernatants from human monocytes.

The lytic mechanism of human peripheral blood monocytes was studied by using as targets actinomycin D-treated WEHI-164, an NK-insensitive murine fibrosarcoma cell line. Monocytes, but not lymphocytes, lysed WEHI-164 target cells pre-treated with actinomycin D within 6 h in 51Cr-release assays. Because cytolysis could not be inhibited competitively by unlabeled WEHI/D target cells, contact-independent mechanisms of cytolysis were investigated. Cell-free supernatants collected from monocytes cultured for 4-6 h at 37 degrees C lysed target cells as effectively as effector cell preparations of monocytes. Supernatants from lymphocytes cultured in parallel were not cytolytic. Cytolytic activity was not detected in supernatants from preparations of monocytes that were held on ice. However, monocytes produced cytolytic activity whether they were isolated by adherence or remained unseparated in suspensions of mononuclear cells. The cytolytic activity in cell-free supernatants (CFS) from monocytes was unaffected by incubation with protease inhibitors. CFS activity was destroyed by heat. Storage of CFS at 37 degrees, 22 degrees, 4 degrees, or -20 degrees C for 24 h decreased cytolytic activity; however, loss of cytotoxicity was minimized by storage at 4 degrees C. The cytolytic substance detected in 4-h CFS from monocytes appeared to be a protein(s) based on the sensitivity of the cytolytic activity to proteases. Cytolytic activity of CFS eluted from Sephacryl 200 in a single peak with an apparent molecular weight between 25,000 and 45,000 daltons.

Lipopolysaccharide (LPS) stimulates fresh human monocytes to lyse actinomycin D-treated WEHI-164 target cells via increased secretion of a monokine similar to tumor necrosis factor.

We have studied the effects of lipopolysaccharide (LPS) on tumoricidal activity of human monocytes freshly isolated from peripheral blood. Actinomycin D-treated WEHI-164 cells were used as targets because they are NK insensitive and are lysed rapidly by monocytes in 6-hr 51Cr-release assays. Monocytes exhibited significant spontaneous activity without endotoxin. Monocytes either pretreated for 1 hr with LPS or assayed in the presence of LPS exhibited 100- to 1000-fold increased cytolytic activity. A half-maximal response was observed with 100 pg/ml LPS. Lipid A was as effective as intact LPS but required slightly higher doses. Monophosphoryl lipid A had no effect. Supernatants of monocytes cultured 5 hr contained sufficient cytolytic activity to account for levels of cytolysis mediated by monocytes directly. Doses of LPS from 10 pg/ml to 10 micrograms/ml produced parallel increases in cell-mediated and supernatant-mediated lysis. Lymphocytes did not produce cytolytic supernatants. Cytolytic activity appeared in monocyte supernatants after 30 min and peaked after 4 to 7 hr regardless of the LPS concentration; longer incubation led to a loss of activity. Cytolytic activity was heat labile and trypsin sensitive, and was recovered from Sepharose S-200 columns in a single peak with an apparent m.w. between 25,000 and 40,000. Actinomycin D or cycloheximide treatment of monocytes before the addition of LPS inhibited cytolytic monokine production. Cytolytic monokine activity was partially neutralized by specific rabbit antisera to human tumor necrosis factor (TNF). We conclude that, although fresh human monocytes exhibit spontaneous tumoricidal activity, LPS is a potent activating agent. Its stimulatory effects depend on new transcription and translation and are mediated by enhanced secretion of a cytolytic monokine similar to TNF.

Elicitation of natural killer cells in beige mice by infection with vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) infection of beige (bg/bg) mice induced levels of natural killer cytolytic activity comparable to that of uninfected normal bg/+ controls, but considerably less than natural killer activity in VSV-infected bg/+ mice. In contrast, VSV-infected bg/bg and bg/+ mice had essentially equivalent amounts of anti-VSV cytotoxic T-lymphocyte and antibody activity. VSV infection induced comparable levels of interferon in both bg/bg and bg/+ mice. Therefore, the decreased natural killer activity in bg/bg mice could not be attributed to an inability to produce interferon.