Tricyclic antidepressants prevent the differentiation of monocytes into macrophage-like cells in vitro.

The investigation was designed to determine whether the two tricyclic antidepressant agents (TCAs) clomipramine and imipramine and the selective reuptake inhibitor citalopram affect differentiation of human monocytes to macrophage-like cells (MAC-LCs). We established primary adherent cultures of peripheral blood monocytes and monitored their morphology, capacity for phagocytosis and antigen expression during transformation to MAC-LCs. As expected, maturation of monocytes to MACs is accompanied by changes in morphology, elevated expression of the antigens CD16 and CD51 and an increase in the percentage of phagocytic cells. Treatment of cells with the TCAs clomipramine (40 micromol/L) or imipramine (100 micromol/L) and with citalopram (100 micromol/L), for 11 days resulted in the following observations: (1) monocytes treated with TCAs never developed the morphology characteristic of the MAC-LCs; (2) TCAs reduced the percentage of phagocytic cells; (3) TCAs had little influence on the expression of CD14, CD16, CD51, and HLA-DR. However, when added after monocyte differentiation into MAC-LCs, citalopram and clomipramine no longer reduced the percentage of phagocytic cells and these effects were not simply due to irreversible cytotoxicity. Thus clomipramine, imipramine, and citalopram inhibit differentiation of human monocytes into MAC-LCs in vitro, but in a reversible manner.

Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK)

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 are diagnostic of Wegener's granulomatosis, but ANCA occur also in patients with other inflammatory disorders, such as ulcerative colitis, primary sclerosing cholangitis (PSC) and autoimmune hepatitis. As their predictive value for autoimmune liver disease remains unknown, we analysed the prevalence and antigen specificity of ANCA in patients with various chronic liver diseases (CLD). METHODS: We studied sera from 100 patients with primary biliary cirrhosis (PBC), from 76 with PSC and from 279 with various CLD, consecutively drawn during a 5-year period at the time of liver biopsy. The ANCA were detected by indirect immunofluorescence (IIF) while the antigen specificity was characterized by ELISA by using lactoferrin, neutrophil elastase, cathepsin G and BPI (bactericidal/permeability increasing protein) as antigens. RESULTS: In PBC, ANCA were detected by IIF in 39 patients (39%). The antigen reactivity by ELISA was lactoferrin in seven, elastase in 15, BPI in 20 and cathepsin G in four patients. Four patients had reactivity against more than one antigen. In PSC, IIF demonstrated ANCA in 49 patients (65%). The antigen reactivity was lactoferrin in 17, elastase in 14, BPI in 20 and cathepsin G in four patients. Twelve patients showed reactivity against more than one antigen. In CLD, ANCA were observed in sera from 55 patients (20%). Nineteen of 45 patients (42%) with autoimmune liver disease were ANCA positive versus 36/234 (15%) with non-autoimmune liver disease (P = 0.0002). Among IIF-positive patients, antibody reactivity against lactoferrin was noted in 14, elastase in 28, BPI in 25 and cathepsin G in five patients. Twenty-one patients had reactivity against more than one antigen. Elastase and BPI antibodies occurred more frequently in patients with autoimmune compared to non-autoimmune liver disease (P < 0.01). CONCLUSIONS: Anti-neutrophil cytoplasmic antibodies are prevalent in patients with chronic liver diseases, but although they occur more frequently in patients with autoimmune liver disease their specificity and sensitivity for autoimmune liver disease is low. The predominant antigens are lactoferrin, elastase and BPI, but the correlation between IIF findings and ELISA reactivity against these antigens is weak.

Antidepressant-induced lipidosis with special reference to tricyclic compounds.

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipid's suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

Biocompatibility of microplates for culturing epithelial renal cells evaluated by a microcalorimetric technique.

In the present study we have developed a microcalorimetric procedure which allows convenient investigation of biocompatibility in a microsystem. We examined the biocompatibility of a porcine renal epithelial tubule cell line LLC-PK1 and a human primary renal epithelial tubule cell (RPTEC) with microplates composed of three different materials, i.e. Thermanox, transparent film and titanium. All three materials showed equal biocompatibility with LLC-PK1 cells, judging from the attainment of steady-state power curves and the same rate of heat production per cell (2.5 microW / microg DNA). The human renal cells were poorly biocompatible with the Thermanox and transparent film. However, on titanium the RPTEC cell did adhere, as demonstrated by a steady-state power curve. The human cells also showed a higher metabolic activity (3.0 microW / microg DNA), than did LLC-PK1 cells cultured on the same type of microplates. In research on biocompatibility there is a need for alternatives to experimental animal investigations. The present technique allows studies of cellular interactions with different biomaterials in a rapid and standardized manner and may therefore prove to be a useful screening procedure.

The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis.

Our aim was to determine the prevalence of the PiZ allele for alpha 1-antitrypsin (AAT) deficiency and some relevant antineutrophil cytoplasmic antibody (ANCA) specificities in patients with ulcerative colitis (UC), and explore a possible association between these markers. In addition, we studied the relation to disease extension and activity. Sera from 141 patients with UC (72 women) were analyzed while 50 blood donors and 54 patients with acute myocardial infarction served as controls. Serum samples were screened for PiZ with ELISA and phenotyped by isoelectric focusing. BPI-ANCA and PR3-ANCA were detected by ELISA. Results were that 8.5% (12/141) of the patients with UC were PiZ carriers, higher than expected in the general Swedish population (4.7%) (p = 0.03). There was a significant difference between PiZ-carriers and non-PiZ-carriers in the extension and severity of colitis (odds ratio = 4.1, confidence interval = 1.1, 14.9; p = 0.028, and odds ratio = 9.0, confidence interval = 1.1, 73.3; p = 0.015; respectively). BPI-ANCA and PR3-ANCA were detected in 20.5% (29/141) and 12% (17/141) (p < 0.05 compared with controls for all parameters). Occurrence of BPI-ANCA and PR3-ANCA was not related to extension or severity of colitis (p > 0.05 for both variables). We observed no association between PiZ-carrier status and occurrence of BPI-ANCA or PR3-ANCA. The increased frequency of heterozygosity for the PiZ variant of AAT deficiency among patients with UC might imply a role played by protease inhibitors for regulation of inflammation and immunologic response in UC.

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.

Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and Bcl-X(L).

In order to investigate the molecular mechanism of the antineoplastic effects exerted by the antidepressive agents imipramine, clomipramine, and citalopram, we examined the effects of these compounds on cell viability, generation of reactive oxygen species (ROS), and mitochondrial membrane potential (DeltaPsi(m)) in human acute myeloid leukemia HL-60 cells. Our results indicate that exposure to these compounds causes a loss in cell viability by activating the apoptotic process, as identified by electron microscopy, DNA gel electrophoresis, and flow cytometry. The increased generation of ROS induced by these drugs was a relatively early event and preceded the loss of DeltaPsi(m). Overexpression of the antiapoptotic protein Bcl-2 or Bcl-X(L) prevents antidepressant-induced apoptosis, as well as loss of DeltaPsi(m), but does not affect the generation of ROS.

Modulation of apoptosis induced by tricyclic antidepressants in human peripheral lymphocytes.

We have recently reported that the tricyclic antidepressants (TCAs) imipramine, clomipramine, and citalopram induce apoptosis in human peripheral lymphocytes. This system is well suited for studies on the pathophysiology/physiology of apoptosis regulation. Apoptosis was determined using both DNA gel electrophoresis and flow cytometric analysis. TCA-induced apoptosis in lymphocytes was monitored in the presence of the protein synthesis inhibitor cycloheximide (CHX), the RNA synthesis inhibitor actinomycin D (Act D), the antioxidant reduced glutathione (GSH), the nuclease inhibitor aurintricarboxylic acid (ATA), the cytokine interleukin-2 (IL-2), and the immunostimulator linomide. CHX and Act D failed to prevent and actually enhanced TCA-induced apoptosis in lymphocytes, indicating that protein and RNA syntheses are not required for this process. Exogenous IL-2, GSH, and ATA protected the lymphocytes from apoptosis induced by TCAs in a dose-dependent manner, whereas linomide had no effect on TCA-induced apoptosis under our in vitro conditions. Our data demonstrate that TCA-induced apoptosis in human lymphocytes shares many common features with other stimuli-induced apoptotic processes.

Application of microcalorimetry for recording basal metabolic and Na+, K+-ATPase activity in LLC-PK1 cells, a model for the renal tubular epithelial cell.

In the present study we have employed a microcalorimetric procedure to measure the heat generated by a porcine renal tubule cell line (LLC-PK1) and its Na+, K+-ATPase. Microplates with an area of 2.2 cm2 were found to be optimal in terms of producing sufficient heat and a steady-state power curve. We compared the rate of heat production by cells in suspension and on monolayers and found a much lower value in suspension, that is, 1.42+/-0.2 versus 2.54+/-0.19 microW/microg DNA. Ouabain, the specific Na+, K+-ATPase inhibitor, caused a reduction in this heat output. The maximal inhibition in cell suspensions was 40% and remained unchanged with as much as 100 microM ouabain, the highest concentration tested. With cells cultured on microplates, ouabain in the concentration interval 0.1-3 microM caused a 25% inhibition of heat output. With 25-100 microM ouabain, a 50% inhibition was observed and at higher concentrations, no further inhibition occurred. Furthermore, upon removal of ouabain, full recovery of the Na+, K+-ATPase was observed, a process that could easily be monitored by using cell monolayers cultured on microplates.

Induction of apoptosis in proliferating lymphocytes by tricyclic antidepressants.

We have previously found that tricyclic antidepressants (TCAs) induce apoptosis in quiescent human lymphocytes. The aim of the present study was to evaluate if TCAs induce apoptosis in proliferating human lymphocytes and in established blastoid lymphocytes also. The development of conA-induced lymphoblast populations was followed by measuring the CD25 membrane expression. Three TCA compounds were run with the following concentrations: imipramine (10, 20, 30, 40, 60 microM), clomipramine (1, 10, 20, 30, 40 microM) and citalopram (40, 60, 80, 100, 180 microM). They all induced a dose-dependent apoptosis both in continuously transformed, as well as in established lymphoblasts. Preincubation of the TCA up to 48 h did not significantly increase induction of apoptosis. The three drugs tested were found to be potent inducers of apoptosis in proliferating lymphocytes. Furthermore, we found that the apoptotic populations in proliferating and in established blastoid lymphocytes were of fairly the same magnitude than in the corresponding population in TCA-incubated resting lymphocytes. In conclusion, we demonstrate that TCAs induce apoptosis in proliferating lymphocytes, as they do in quiescent lymphocytes. Furthermore, the extent of apoptosis was even more pronounced in TCA-incubated lymphoblasts compared to TCA-treated resting lymphocytes.

FK506 suppresses the mitogen-induced increase in lymphocyte adhesiveness to endothelial cells, but does not affect endothelial cell activation in response to inflammatory stimuli.

BACKGROUND: In vivo studies indicate that FK506 may affect endothelial cell activation. FK506 has also been reported to affect leukocyte adhesiveness and transendothelial migration. The purpose of the present study was to investigate the direct effects of FK506 on endothelial activation and on the increase in lymphocyte adhesiveness associated with mitogen stimulation. METHODS: Using flow cytometry and enzyme-linked immunosorbent assays, we studied the effects of FK506 on expression of E-selectin and intercellular adhesion molecule 1 and on the release of interleukin (IL) 6 and IL-8 from endothelial cells in response to inflammatory mediators. Expression of lymphocyte adhesion molecules and adhesion between lymphocytes and endothelial cells were also quantitated using flow cytometry. RESULTS: Endothelial activation in response to lipopolysaccharide, IL-1beta, or tumor necrosis factor-alpha was found to be unaffected by FK506. The increase in lymphocyte adhesiveness to endothelium seen after mitogen stimulation, on the other hand, was significantly depressed by FK506. The associated changes in the expression of CD11c, CD29, and CD31 were also significantly altered by FK506. CONCLUSION: In addition to its inhibitory effects on T-cell activation, FK506 may also interfere with processes leading to increased lymphocyte adhesiveness. This is thus a possible additional mechanism for its beneficial effects in connection with organ rejection and autoimmune diseases.

Tricyclic antidepressant-induced lipidosis in human peripheral monocytes in vitro, as well as in a monocyte-derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red.

Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.

Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro.

By employing microcalorimetry to assess overall metabolic activity in combination with other assays for specific metabolic events, we have investigated the influence of cyclosporin A and FK506 on the metabolic status of resting and mitogen-stimulated human peripheral lymphocytes. Both cyclosporin A and FK506 significantly reduced heat output from resting lymphocytes. This reduction could not be correlated with effects on DNA synthesis, lactate production, ATP levels or mitochondrial uptake of Rhodamine 123. These two drugs also potently reduced the increase in heat output seen during mitogen stimulation of lymphocytes. Both cyclosporin A and FK506 also prevented the increase in DNA synthesis, lactate production and ATP levels seen in response to mitogen stimulation. The increase in mitochondrial uptake of Rhodamine 123 during blastoid transformation was significantly reduced only by cyclosporin A. We ascribe the major part of the effects of these compounds to inhibition of the glycolytic pathway in both resting and mitogen-stimulated lymphocytes. These results indicate that the immunosuppressants cyclosporin A and FK506 exert other effects on lymphocytes than their well-established inhibitory action on calcineurin.

Inflammatory response to aorto-bifemoral graft surgery.

OBJECTIVE: To evaluate the systemic release of major cytokines and complemental activation during elective aorto-bifemoral bypass surgery and the influence on the postoperative course. DESIGN: Prospective randomised study. SETTING: University hospital, Sweden. PATIENTS: Fourteen consecutive patients with aorto-iliac occlusive disease were randomised to receive either a bifurcated e PTFE graft with stretch properties or a collagen coated Dacron graft. MAIN OUTCOME MEASURES: Immunologic parameters were assessed and included the cytokines TNF alpha (tumor necrosis factor), IL-6, IL-8, S-IL-2R and complemental factor C5a. Furthermore, acute plasma proteins, including C-reactive protein, and the different white blood cell fractions were determined. Sampling was performed frequently during surgery and postoperatively up to one month. RESULTS: An increase of serum-TNF alpha levels was observed early after declamping. This response preceded an increase of IL-6 levels and of C-reactive protein. No release of IL-8 was identified. A significant correlation between TNF alpha, IL-6 and C-reactive protein was observed (p < 0.001). A positive correlation was also observed for the degree of surgical trauma (blood loss). No significant differences between the two graft materials were encountered. The complemental system was also involved in the acute reactions and a marked increase of C5a levels was noted. A decrease of S-IL-2R levels as well as lymphocyte concentrations was also observed postoperatively and was interpreted as a downregulation of the immune-system in the immediate postoperative course. CONCLUSIONS: The results demonstrate an early and generalized inflammatory response during and after aortic surgery with involvement of different cytokines as well as the complemental system. TNF alpha appears to play a central role in the release of other cytokines, but IL-6 seemed to correlate best with later development of nonvascular complications. Few differences were found between the different grafts and the response seems to be influenced by other factors such as the surgical procedure and ischaemia/reperfusion injury.

Tricyclic antidepressants induce apoptosis in human T lymphocytes.

Apoptosis is a form of programmed cell death that is involved in cell turnover. In the present study we show that the tricyclic antidepressants (TCAS) imipramine, clomipramine and citalopram induce apoptosis in human peripheral lymphocytes. Lymphocytes were incubated with these three drugs for up to 48 h. Apoptosis was characterized by typical nucleosomal DNA fragmentation on agarose gel, as well as quantitated using 4'-6-diamidino-2-phenylindole (DAPI) staining and 3'-OH end-labeling of fragmented DNA at the single cell level. Apoptosis induced by TCAs was shown to be dose-dependent and could be detected after a 24 h incubation. The optimal concentrations of the three TCAs found to induce apoptosis were 50 microM imipramine, 20 microM clomipramine and 180 microM citalopram. Furthermore, immunofluorescence and three-color flow cytometry were used to identify the phenotype of apoptotic cells. TCA-induced apoptosis was shown to involve exclusively T-lymphocytes. Cytotoxic T-lymphocytes were more prone to undergo apoptosis than were T-helper cells. In conclusion, the present investigation clearly demonstrates that TCAs exert cell biological effects upon human T-lymphocytes. Further studies are required to determine the possible clinical relevance of these findings.

Reduced cell survival and morphological alterations induced by three tricyclic antidepressants in human peripheral monocytes and lymphocytes and in cell lines derived from these cell types.

Tricyclic antidepressant drugs are widely used for the treatment of manic-depressive disorders. As such compounds have been reported to give rise to myelin figures in lymphocytes in experimental animals, the effects of clomipramine (2.5-50 muM), imipramine (2.5-100 muM) and citalopram (5-50 muM) on human peripheral lymphocytes, granulocytes and monocytes in culture were investigated. No cytoplasmic alterations were detected in T or B lymphocytes, but large myelin figures could be seen by fluorescence and electron microscopy in monocytes. Optimal concentrations for the formation of these structures were 20 muM for clomipramine and 40 muM for imipramine. A strongly dose-dependent inhibition of the growth of Molt-4 and U937 cells was also observed with clomipramine, which was 2.5-fold as potent as imipramine in this respect. Treated U937, but not Molt-4, cells showed an increased content of fluorescent granules compared with untreated cells. Furthermore, these tricyclic antidepressants appeared to induce apoptosis in lymphocytes, as judged from the disorganization of the nucleus and the appearance of a typical DNA ladder pattern in treated cells.

Tricyclic antidepressants inhibit IL-6, IL-1 beta and TNF-alpha release in human blood monocytes and IL-2 and interferon-gamma in T cells.

Tricyclic antidepressants (TCAs) are widely used in treating depressive disorders. It has been demonstrated that, for instance, IL-1 beta and IL-6 inhibit the HPA axis, which plays a role in the development of depressions. Therefore. we were interested in investigating how TCAs influence cytokine release by T lymphocytes and monocytes respectively. Cells were incubated with either 5 microM clomipramine, 15 microM imipramine or 20 microM citalopram. IL-2 release was suppressed to 60% of the control values by clomipramine and imipramine (p = 0.001; p = 0.000), but citalopram was found to cause a much weaker inhibition (only 18%) (p = 0.16). INF-gamma release was affected to a lower degree than IL-2 release, and imipramine (34%) (p = 0.054) was more potent than clomipramine (24%) (p = 0.16) and citalopram (12%) (p = 0.059) in this case. Monocytes incubated with TCA for 4 h exhibited only limited inhibition of IL-1 beta and IL-6 release, i.e., 6-25% for all three compounds. The corresponding value for TNF-alpha release was 20-45% inhibition, with citalopram being the weakest inhibitor. After 10 h of monocytes to LPS exposure, all three compounds exerted a strong inhibition of IL-1 beta and TNF-alpha release, i.e., 60-70% with p-values below 0.012 for all of them. However the inhibition of IL-6 release was less than 35%. Citalopram was equality as potent as imipramine and clomipramine in inhibiting IL-6 release after long-term exposure of monocytes to LPS. All three TCAs elevated intracellular cAMP concentrations significantly in T lymphocytes and monocytes (p < 0.001).

A calorimetric method for continuous recording of lymphocyte proliferation.

We have developed a calorimetric technique as an alternative non-radioactive method for measuring mitogen-induced lymphocyte proliferation. Power-time curves can be recorded from as few as 2X10(5) cells suspended in 4 ml medium. This density is, however, too low for the detection of proliferation during a 72 h incubation. Cell numbers exceeding 4X10(6) suspended in 4 ml medium cause pronounced crowding and are therefore too high for use during proliferation studies. We found the optimal initial cell concentration to be 2X10(6) cells suspended in 4 ml of medium. In a four-channel bioactivity monitor, up to four separate incubations can be run in parallel. Furthermore, this method offers the advantage of one-line monitoring of cell proliferation throughout the incubation.