Human allogeneic CD2+ lymphocytes activate airway-derived epithelial cells to produce interleukin-6 and interleukin-8. Possible role for the epithelium in chronic allograft rejection.

BACKGROUND: The adhesion of lymphocytes to the epithelium and the release of proinflammatory cytokines are important features observed during acute and chronic allograft rejection. Development of chronic rejection in lung-transplantation patients is preceded by high levels of interleukin (IL)-6 and IL-8 protein in the bronchoalveolar lavage. Therefore, we studied the expression of IL-6 and IL-8 in cocultures of epithelial cells and allogeneic lymphocytes. METHODS: IL-6 and IL-8 protein levels were determined in supernatants of the airway-derived epithelial cell line A549 and in primary epithelial cells obtained from lung-brushings after coculturing with autologous and allogeneic lymphocytes. Transcriptional mechanisms were detected by transient transfections. RESULTS: Coculture-supernatants of epithelial cells and allogeneic CD2+ lymphocytes show high levels of IL-6 and IL-8 protein due to transcriptional activation of the respective genes in epithelial cells. Highest productions were measured when the epithelial-cell:lymphocyte ratio was 1:10. Highly purified CD4+ and/or CD8+ cells were unable to induce the same response as observed with the total lymphocyte-population. Depletion of CD4+ and/or CD8+ had no effect on the IL-6 and IL-8 production induced by the total CD2+ lymphocyte-population. However, depletion of CD56+ cells diminished the lymphocyte-induced IL-6 and IL-8 production by > 75%. CONCLUSION: These data show that allogeneic CD2+ lymphocytes are able to activate lung-derived epithelial cells, resulting in the release of proinflammatory cytokines, which have a prominent role in chronic allograft rejection observed in lung-transplantation patients.

Eosinophilic granulocytes and interleukin-6 level in bronchoalveolar lavage fluid are associated with the development of obliterative bronchiolitis after lung transplantation.

In a prospective cohort study, we assessed whether changes in total cell counts and differentiation and interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) concentrations in bronchoalveolar lavage fluid (BALF) are associated with a higher risk to develop obliterative bronchiolitis (OB). We investigated 60 lung transplant patients (follow-up of 2 to 8 yr) with either histologic evidence of OB within 1 yr after lung transplantation (n = 19) or no pathology, good outcome (GO) for at least 24 mo and well-preserved lung function, i.e., FEV > or = 80% of baseline (n = 41). Median time between lung transplantation and the first BAL was 42 d for the GO group and 41 d for the OB group (p > 0.05). In the bronchial fraction, median total cell counts (0.06 x 10(3)/ml versus 0.04 x 10(3)/ml), lymphocyte (9 x 10(3)/ml versus 2 x 10(3)/ml), and eosinophilic granulocyte counts (1 x 10(3)/ml versus 0) were significantly higher in the OB group than in the GO group (p < 0.05). In the alveolar fraction, this was the case for the median value of neutrophilic granulocyte counts (19 x 10(3)/ml versus 4 x 10(3)/ml), respectively. Median values of IL-6 and IL-8 concentrations in both bronchial (IL-6: 23 versus 6 pg/ml, IL-8: 744 versus 102 pg/ml) and alveolar fractions (IL-6: 13 versus 3 pg/ml, IL-8: 110 versus 30 pg/ml) of the BALF were significantly higher in the OB group than in the GO group. By means of logistic regression, we showed that higher total cell, neutrophilic granulocyte, and lymphocyte counts, the presence of eosinophilic granulocytes, and higher concentrations of IL-6 and IL-8 were significantly associated with an increased risk to develop OB. We conclude that monitoring cell counts, neutrophilic and eosinophilic granulocytes, IL-6, and IL-8 in BALF within 2 mo after lung transplantation in addition to the transbronchial lung biopsy (TBB) pathology will contribute to a better identification and management of the group of patients at risk for developing OB within a year.

Cyclosporine, FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells.

BACKGROUND: The immunosuppressive effects of cyclosporine (CsA), tacrolimus (FK506), mycophenolate mofetil (MMF), and prednisolone in cells from the immunological compartment are well documented. In contrast, limited information is available with respect to the effects of these immunosuppressive drugs on airway-epithelial cells, although these cells may contribute to the development of obliterative bronchiolitis (OB) through the production of interleukin (IL)-6 and IL-8. METHODS: We studied the production of IL-6 and IL-8 proteins by airway-derived epithelial cell lines and primary epithelial cell cultures obtained from lung brushings. Transcriptional mechanisms were detected by transient transfections. RESULTS: We demonstrate that CsA dose dependently induces the production of the proinflammatory cytokines IL-6 and IL-8 in both cell lines and primary epithelial cells. FK506 and MMF were also able to upregulate IL-8, although the effect was less dramatic than observed for CsA. Low concentrations of prednisolone (0.01 and 0.001 microg/ml) enhanced IL-6 and IL-8 secretion, whereas concentrations > or =0.01 microg/ml significantly diminished IL-6 secretion. Furthermore, we showed that CsA and prednisolone mediate their effects at the transcriptional level. CONCLUSIONS: The data provide evidence that relevant concentrations of CsA and MMF in vivo may enhance the inflammatory processes in the lower airways of patients after lung transplantation.

Waiting for coronary revascularization: a comparison between New York State, The Netherlands and Sweden.

OBJECTIVE: To compare waiting times for percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft (CABG) surgery in New York State, the Netherlands and Sweden and to determine whether queuing adversely affects patients' health. METHODS: We reviewed the medical records of 4487 chronic stable angina patients who underwent PTCA or CABG in one of 15 New York State hospitals (n = 1021) or were referred for PTCA or CABG to one of ten hospitals in the Netherlands (n = 1980) or to one of seven hospitals in Sweden (n = 1486). We measured the median waiting time between coronary angiography and PTCA or CABG. RESULTS: The median waiting time for PTCA in New York was 13 days compared with 35 and 42 days, respectively, in the Netherlands and Sweden (P < 0.001). For CABG, New York patients waited 17 days, while Dutch and Swedish patients waited 72 and 59 days, respectively (P < 0.001). The Swedish and Dutch waiting list mortality rate was 0.8% for CABG candidates and 0.15% for PTCA candidates. CONCLUSIONS: There were large variations in waiting time for coronary revascularization among these three sites. Patients waiting for CABG were at greatest risk of experiencing an adverse event. In both the Netherlands and Sweden, the capacity to perform coronary revascularization has been expanded since this study began. Further international cooperation may identify other areas where quality of care can be improved.

Indications for coronary revascularisation: a Dutch perspective.

OBJECTIVE: To assess the appropriateness of indications for coronary artery bypass graft (CABG) surgery and percutaneous transluminal coronary angioplasty (PTCA). METHODS: A modified Delphi group judgement process with input from a panel of six interventional cardiologists and six cardiopulmonary surgeons. There was one clinician from each of the 12 tertiary referral heart centres in The Netherlands. MAIN OUTCOME MEASURE: Ratings by panel members, on a 1 to 9 scale, of indications presented as a choice between two treatments (CABG v medical treatment, PTCA v medical treatment, and CABG v PTCA) for 1182 model cases. Each case represented a unique combination of clinical features in terms of symptoms, medical history, and results of tests. Ratings were analysed with respect to degree of agreement among panelists, degree of appropriateness of indications, and panel's preference for invasive or medical treatment. RESULTS: The panel agreed on 58.6% and disagreed on 3.2% of the indications. The panel opted for invasive treatment in 48.2% and medical treatment in 22.8%, and had no clear preference for either method in 29.0% of the cases. When compared with medical treatment, CABG was more often rated appropriate than PTCA: 35.4% v 21.6% (P < 0.001). Panel scores depended on severity of anatomical disease. For instance, for 51.5% of the model cases with one-vessel disease not including the proximal left anterior descending artery, the panel preferred medical treatment to invasive treatment, while the latter was preferred in 18% of the cases. In cases with type C lesions, the panel frequently rated PTCA as inappropriate. Panel scores were also affected by nonclinical factors. Cardiologists and surgeons rated the procedure of their own specialty higher than the alternative invasive intervention. CONCLUSIONS: The panel method yields logically consistent scores of the appropriateness of indications for carrying out medical procedures. It may be an aid in formulating clinical practice guidelines.

The appropriateness of intention to treat decisions for invasive therapy in coronary artery disease in The Netherlands.

OBJECTIVE: To determine the appropriateness of intention to treat decisions concerning coronary artery bypass grafting (CABG) and percutaneous transluminal coronary angioplasty (PTCA) for patients with coronary artery disease in The Netherlands. DESIGN: Prospective study of intention to treat decisions using a computerised expert system. SETTING: "Presentation" sessions in 10 tertiary referral heart centres in 1992. PATIENTS: 3207 consecutive patients: 1618 CABG and 1589 PTCA candidates. MAIN OUTCOME MEASURE: Percentage of invasive treatment decisions rated appropriate, uncertain, or inappropriate by the expert system. RESULTS: PTCA decisions were common for patients with one-vessel disease and CABG decisions for patients with three-vessel and left main disease. PTCA decisions outnumbered CABG decisions in acute myocardial infarction. Of CABG decisions, 84% were rated appropriate, 12% uncertain, and 4% inappropriate. The proportions for PTCA decisions were 39% appropriate, 31% uncertain, and 29% inappropriate. Type C lesion was the main determinant of inappropriateness of PTCA decisions. If type C lesions were downgraded to type A/B lesions the rate of inappropriate PTCA decisions dropped to 6%. CONCLUSIONS: Clinicians in tertiary referral centres in The Netherlands favoured CABG if vessel disease was extensive or involved the left main artery, and PTCA for patients with less extensive disease and with acute myocardial infarction. Few CABG decisions were inappropriate. The main determinant of inappropriateness of PTCA decisions was its intended use in patients with type C lesions.

Adhesion properties, intermediate filaments and malignant behaviour of head and neck squamous cell carcinoma cells in vitro.

This study compared phenotype and behaviour of seven head and neck squamous cell carcinoma (HNSCC) cell lines and normal epithelial cells. Indications were found that in HNSCC cells: 1) loss of the cell-cell adhesion molecule E-cadherin was correlated with loss of epithelioid cell morphology but not with loss of cell-cell cohesion; 2) reduced expression of the cell-cell adhesion molecule desmoglein was unrelated to cell shape or motility; 3) high expression of the cell-substrate adhesion molecule alpha 6 beta 4, a receptor for laminin, corresponded with epithelioid colony formation while, in our adhesion assay, low expression of alpha 6 beta 4 paradoxically coincided with an increased adhesion to laminin; 4) presence of vimentin intermediate filaments coincided with loss of anchorage dependency. We propose that by simultaneous downregulation of E-cadherin, replacement of alpha 6 beta 4 by an aberrant laminin receptor and co-expression of vimentin a malignant phenotype arises of spindle-shaped, motile, anchorage-independent HNSCC cells with enhanced laminin-binding capacity.

Characterization of a rat oral squamous cell carcinoma cell line UHG-RaC '93 induced by 4-nitroquinoline-1-oxide in vivo.

This study describes several characteristics of a cell line, UHG-RaC '93 derived from rat oral squamous cell carcinoma induced by the carcinogen 4-nitroquinoline-1-oxide (4NQO). The cell line was established from explant cultures without support of fibroblast feeder cells and continued for > 30 passages. UHG-RaC '93 had a high mitotic rate with a population doubling time of 25 h and a high rate of squame production. The first passage had a low colony-forming efficiency in agarose gel, whereas later passages did not grow at all in semi-solid medium. Phenotype selection was furthermore apparent from a gradual increase of the trypsin-detachment time. Cytogenetic analysis showed that UHG-RaC '93 was hypotetraploid with an average of 74 chromosomes. Abnormalities compared to the normal karyotype were assessed and consisted mainly of breakpoints at (1)(q5?3), (3)(p1), (3)(q11q23), (11)(p?11), (13)(p13) and a derivative (12)t(12;13)(q10;q10). The karyotype remained stable for at least 26 passages. The expression of typical epithelioid markers like cytokeratins and desmoglein corresponded with normal rat oral keratinocytes. However expression of alpha 6 beta 4-integrin was altered. Squame production, immunophenotype and anchorage dependency indicated that UHG-RaC '93 had the same features of a well-differentiated carcinoma with a low degree of agressiveness as the original tumour. The stable karyotype of this cell line provides a basis for further analysis of the effect of 4NQO on the genotype, phenotype and behaviour of rat oral keratinocytes.

Characterization of human larynx carcinoma cell lines HLaC'79 and HLaC'82: a common origin but diverged malignancies.

As part of a study on the relationship of tumour phenotype and behaviour, we have characterized two head and neck squamous cell carcinoma cell lines, derived from human laryngeal carcinomas and designated HLaC'79 and HLaC'82. Cytogenetic analysis revealed that HLaC'79 and HLaC'82 shared 10 major chromosome rearrangements indicating that the cell lines had a common origin. In the extremely complex chromosomal patterns, abnormalities were found in chromosomes 1, 3 (surplus 3q) and 5 (i(5p) x 2). Both cell lines displayed constitutive expression of vimentin and were capable of anchorage-independent growth in agarose gels. However, in spite of their common origin specific differences were found. Cells of HLaC'79 were spindle shaped and formed tumours in athymic mice. In contrast, cells of HLaC'82 had a compact morphology, contained less vimentin, were more contact inhibited and were not tumorigenic. These results indicate that malignant transformation in HLaC'82 was partially reversed.

Use of cultured mucosal grafts to cover defects caused by vestibuloplasty: an in vivo study.

PURPOSE: In oral and maxillofacial surgery palatal mucosal grafts are routinely used to cover mucosal defects caused by vestibuloplasty. However, the quantity of palatal mucosa is a limiting factor in more extensive operations. This study investigated whether autologous cultured sheets of mucosa can serve as a dressing for these wounds. MATERIALS AND METHODS: Punch biopsies (diameter, 4 mm) were taken from the hard palate of eight patients (five men, three women; mean age 43 years). Epithelial cells were enzymatically dissociated from these tissue specimens and grown in vitro in the presence of a fibroblast feeder layer. Within 3 weeks, a transplantable epithelial sheet of about 20 cm2 was obtained. The sheet was detached from the culture flask by enzyme treatment and fixed to a carrier of Vaseline (Cheeseborough Ponds Inc, Greenwich, CT) gauze. Using a split-mouth technique, the sheet was placed on half of a mucosal defect created by vestibuloplasty, while the other half of the defect was covered by a conventional split-thickness palatal graft. Both the cultured and conventional graft were held in place by the patient's relined denture fixed with perimandibular sutures. One week postsurgery, the denture and Vaseline gauze were removed. Three months after vestibuloplasty, biopsy specimens of each grafted site were taken and processed for light and transmission electron microscopy (LM, TEM). RESULTS: Three months postsurgery, the grafted mucosa of both sites bore close resemblance to palatal mucosa. Both the cultured and split-thickness grafts were vascularized, did not evoke a homograft reaction, and showed a smooth graft/lip mucosal junction and minimal wound contraction. LM and TEM revealed that both types of grafts formed a fully differentiated keratinizing mucosa with a well-developed basement membrane and rete ridges, comparable with the histology and ultrastructure of palatal mucosa in situ. CONCLUSION: It was concluded from this study that cultured mucosa can serve as a proper dressing for mucosal defects after vestibuloplasty.

The distribution of peripheral vascular disease in a Dutch Caucasian population: comparison of type II diabetic and non-diabetic subjects.

OBJECTIVES: To study the distribution of peripheral vascular disease and the relationship to diabetes. DESIGN: Prospective population based study of Dutch caucasian inhabitants. METHODS: From a total of 10,500 subjects aged between 50 and 75 years, 2484 Caucasian inhabitants were screened with respect to their glucose tolerance. Subjects using oral antidiabetic agents or insulin were classified directly as having diabetes mellitus. The other participants were screened using two oral glucose tolerance tests and classified using WHO criteria. A group of 173 people with diabetes and a representative age/sex stratified sample of 288 non-diabetic subjects were seen in the vascular laboratory. Carotid artery disease was investigated with Duplex scanning, arm and leg artery obstructions with real time frequency analysis of continuous wave Doppler signals and indirect blood pressure measurements. RESULTS: Comparing diabetic with non-diabetic subjects, we found significantly more obstructions of the carotid arteries (8.7 vs 2.8%), arm arteries (2.3 vs 0%), as well as leg arteries (31.8 vs. 18.4%). The same holds if only the crural artery obstructions were compared (23.7 vs 16.0%). Two of the four diabetic subjects with arm artery obstructions had retrograde vertebral flow, three of them had carotid artery obstructions as well, and three also had leg artery obstructions. More than half of the subjects with a carotid artery obstruction, also had leg artery obstructions. CONCLUSIONS: Peripheral vascular disease is common in diabetes, but most are asymptomatic.

The effect of detachment of cultured epithelial sheets on cellular ultrastructure and organisation.

In this morphological study the (ultra)structural changes that lead to contraction of detached cultured epithelium were investigated. Keratinocytes, isolated from human skin and oral mucosa, were grown to form stratified cell sheets. The multilayers were examined with light and electron microscopy before, during and after detachment from the culture vessel. Attached epithelium had a stretched morphology with flattened cells and nuclei. Evidence is provided that after enzymatical detachment with dispase (1) basal cells became columnar by contraction of actin bundles in the basal cortex, which was accompanied by blebbing of the basal cell membrane; (2) in all cell layers cytokeratin bundles contracted resulting in displacement of desmosomes and a spherical shape of the cells and nuclei. By slow dispase-detachment at 4 degrees C or by quick mechanical detachment, shrinkage of the sheet was partly suppressed but contraction of cytokeratin and related events occurred indicating that these were the result of the spontaneous reassembly of the intermediate filament system. The results suggested that the shape and ultrastructure of all cells in an epithelial multilayer are dependent on the interaction of the basal cells with the underlying extracellular matrix.

Ca(2+)-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients.

The influence of a transmembrane pH gradient on the Ca(2+)-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.

Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching.

The fusion of influenza virus with cultured cells has been investigated. The virus was labelled with the fluorescent probe octadecyl rhodamine B and fusion was monitored as fluorescence dequenching due to dilution of the probe from the viral into a cellular target membrane. Fusion with the plasma membrane does not occur, unless the extracellular pH is temporarily lowered. At neutral pH fusion occurs only after a lag phase of 10-15 min, the time required for virus internalization, and the reaction is inhibited by NH4Cl, indicating that it takes place in an intracellular acidic compartment, most likely the endosome. This suggests that influenza virus infects cells via the endocytic pathway.

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles.

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing.

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion.

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes.

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.