Down-modulation of a novel Bax-associated protein during apoptosis in normal mature B lymphocytes

A simple method for testing the assumption of independent censoring is developed using a Cox proportional hazards regression model with a time-dependent covariate. This method involves further follow-up of a subset of lost-to-follow-up censored subjects. An adjusted estimator of the survivor function is obtained for the dependent censoring model under a proportional hazards alternative. The proposed procedure is applied to an example of a clinical trial for lung cancer and a simulation study is given for investigating the power of the proposed test.     

Leukocyte-endothelium interaction during the early stages of hypercholesterolemia in the rabbit: role of P-selectin, ICAM-1, and VCAM-1

The early effects of hypercholesterolemia on leukocyte-endothelium interaction were studied in vivo in the rabbit mesenteric microcirculation. Rabbits fed a 0.5% high-cholesterol (HC) diet showed elevated plasma cholesterol levels during the 1 to 2 weeks of HC feeding (P<0.001 versus control diet-fed rabbits). Intravital microscopy of mesenteric venules revealed that leukocyte rolling had increased 10-fold (P<0.001 versus control-fed group) at the end of the first week of the HC diet, which was sustained after 2 weeks of HC feeding (P<0.001 versus control-fed rabbits). Firm adherence of leukocytes to the endothelium was moderately increased after a 1-week period of hypercholesterolemia (P<0.05) but increased by 12-fold at 2 weeks (P<0.001 versus control diet-fed and P<0.01 versus 1-week HC-fed rabbits). Upregulation of the endothelial cell adhesion molecules P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was observed immunohistochemically on the intestinal microvascular endothelium of HC-fed rabbits. P-selectin was maximally expressed within the first week of the HC diet and remained elevated during the second week of cholesterol feeding (P<0.01 versus control). In contrast, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were moderately upregulated at 1 week but were highly expressed after 2 weeks of the HC diet (P<0.05 and P<0.001 versus control, respectively). Basal release of NO from both mesenteric microvascular and aortic endothelium in cholesterol-fed rabbits was progressively reduced after 1 (P<0.05) and 2 (P<0.01) weeks. Our data suggest that enhanced leukocyte-endothelium interaction occurs in vivo in the rabbit microcirculation during the first 2 weeks of hypercholesterolemia. This phenomenon is associated with impaired basal NO release and progressive endothelial surface expression of endothelial cell adhesion molecules (ie, P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1) in the microvasculature.     

Expression of ICAM-1, VCAM-1, and LFA-1 in adenocarcinoma of the lung with observations on the expression of these adhesion molecules in non-neoplastic lung tissue

Intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and the lymphocyte function-associated antigen (LFA-1) are cell adhesion molecules thought to play an important role in the complex process of airway inflammation and tumor cell growth. The aim of this study was to examine the distribution of ICAM-1, VCAM-1, and LFA-1 in adenocarcinoma of lung and in major cellular compartments of non-neoplastic lung tissue. We examined cellular compartments in tissue from five bronchoalveolar carcinomas, three acinar adenocarcinomas, and one colon cancer metastatic to the lung. The compartments in neoplasms included the tumor cells proper, endothelial cells within the tumor vasculature, tumor stromal cells, and tumor-infiltrating lymphocytes. The compartments in non-neoplastic lung tissue included lung endothelial cells, pulmonary lymphocytes, interstitial fibroblasts, Type II alveolar pneumocytes, and bronchial epithelial cells. ICAM-1 was expressed in tumor cells from all of the nine adenocarcinomas. In contrast, VCAM-1 expression was not identified in tumor cells from any of the nine adenocarcinomas. ICAM-1 was expressed in all cellular compartments of the non-neoplastic lung tissue, whereas VCAM-1 was expressed only in pulmonary lymphocytes and interstitial fibroblastic cells. LFA-1 was uniformly expressed in tumor-infiltrating lymphocytes from each of the nine tumors and all of the lymphocytes in non-neoplastic lung tissue. This study showed major differences in the expression of ICAM-1 and VCAM-1 in tumor cells from pulmonary adenocarcinoma and also provided evidence for a wider distribution of ICAM-1, compared with VCAM-1, in non-neoplastic cellular compartments of the lung. ICAM-1 expression was particularly noticeable in bronchial and alveolar epithelial cells. Upregulation of ICAM-1 in pulmonary adenocarcinoma might foster binding by LFA-1-bearing lymphocytes, with a possible impact on the vulnerability of tumor cells to host defense mechanisms.     

Intracellular K+ suppresses the activation of apoptosis in lymphocytes

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.     

Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation

We have used the 3T3-L1 and 3T3-F442A preadipocyte cell lines to examine the expression and regulation of neuronal apoptosis inhibitory protein (NAIP) during adipocyte differentiation. When 3T3-L1 preadipocytes differentiated into adipocytes, they developed resistance to apoptosis induced by growth factor deprivation, as assessed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling. Protein expression of NAIP was markedly elevated in 3T3-L1 and 3T3-F442A adipocytes compared with that in their fibroblast-like precursors. NAIP was also present in rat white adipocytes. In 3T3-L1 cells, the increase in NAIP occurred by day 4 of the 8-day differentiation protocol, which includes exposure of confluent preadipocytes to insulin, dexamethasone, and isobutylmethylxanthine. Incubation of confluent 3T3-L1 preadipocytes with any of these components alone had no effect on NAIP expression. When 3T3-C2 cells, a control cell line that does not differentiate, were subjected to the differentiation protocol, the low NAIP levels remained unaltered. Addition of rapamycin, a p70 S6 kinase inhibitor that blocks adipocyte differentiation, to the 3T3-L1 differentiation medium prevented the rise in NAIP expression. These data demonstrate for the first time that NAIP is expressed in adipocyte cell lines and primary adipocytes. The differentiation-dependent augmentation of NAIP protein levels in 3T3-L1 adipocytes is closely correlated with the development of resistance to apoptosis induced by growth factor deprivation, suggesting a potential role for NAIP in these cells.     

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.     

Respiratory infection in lipid-fed rabbits enhances sudanophilia and the expression of VCAM-1

The pathogenesis of atherosclerosis has been related to infection of the arterial wall, but it is not clear whether this occurs before or after the development of lipid-containing lesions. Respiratory bacterial infection increases the expression of vascular cell adhesion molecule-1 (VCAM-1). We therefore examined whether a similar infection would enhance atherosclerosis in New Zealand White rabbits fed chow supplemented by 15% (w/w) egg yolk for 50 days. Rabbits with naturally acquired respiratory infection by Pasteurella multocida, pathogen-free (SPF) animals infected by P. multocida in the laboratory, and age-matched SPF rabbits maintained in a disease-free environment were used. Endothelial cells expressing VCAM-1 in the aorta between intercostal arteries 3 and 5 were identified using anti-VCAM-1 (Rb1/9) and an alkaline-phosphatase-linked secondary antibody and quantified in H?utchen preparations. The remainder of the aorta was stained with Sudan IV to show lipid deposition. The expression of VCAM-1 (mean +/- SEM per 10,000 cells) was 22 +/- 8 (n = 5) in the lipid-fed SPF rabbits, significantly different from that in the lipid-fed rabbits with naturally occurring infection (190 +/- 51 (n = 5)) or from rabbits infected in the laboratory (106 +/- 25 (n = 5)). The extent of Sudanophilia was significantly greater in the naturally infected rabbits (8.3 +/- 1.2%) or infected SPF rabbits (10.3 +/- 1.8%) than in the SPF rabbits (2.7 +/- 0.8%; P < 0.05). Antibiotic treatment in naturally infected rabbits reduced the number of cells expressing VCAM-1 and the extent of the Sudanophilia to baseline levels. Thus, Sudanophilia is enhanced by bacterial infection in rabbits fed egg yolk and is associated with a significant increase in VCAM-1.     

Expression of the alpha 5 beta 1 fibronectin receptor on T lymphocytes of patients with HIV-1 infection

AIMS: To evaluate the expression of the alpha 5 beta 1 integrin fibronectin receptor (FNR), which mediates several processes, including phagocytosis, cell motility and the immune response, on T lymphocytes of patients with HIV-1 infection. METHODS: T lymphocytes were incubated with monoclonal antibody directed against FNR and then with monoclonal antibodies, conjugated with phycoerythrin, directed against CD3, CD4 and CD8 positive cells. Expression of FNR on CD3, CD4 and CD8 positive cells was analysed using flow cytometry. RESULTS: Normal expression of FNR was observed on CD3 positive cells from asymptomatic HIV positive patients and those with AIDS. Increased expression of FNR was observed on CD8 positive cells from asymptomatic HIV positive patients and on CD4 positive cells from patients with AIDS. Increased FNR expression was observed on CD4 positive cells from patients with AIDS, particularly those with opportunistic infections caused by Pneumocystis carinii, Mycobacterium sp, Toxoplasma gondii, and Cryptococcus neoformans. CONCLUSION: Increased expression of FNR on CD8 and CD4 positive cells in asymptomatic HIV positive patients and those with AIDS, respectively, may be an epiphenomenon correlated with lymphocyte activation by HIV-1 or opportunistic infection, Further study is required to determine whether upregulation of FNR expression has a direct role in the pathogenesis of AIDS.     

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its alpha 4 beta 1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes

Lymphocyte extravasation involves a step(s) of de-adhesion to allow trans- and subendothelial migration in response to inflammatory signals. We show here that ligated VCAM-1 was rapidly internalized (t1/2 14.5 min) in ECV 304 endothelial cells and in TNF-alpha-primed human umbilical vein-derived endothelial cells (t1/2 11.2 min). The process required energy (ATP), intracellular Ca2+, an intact cytoskeletal network and active protein kinases. The internalization of VCAM-1 involved a clathrin-dependent pathway based on the observations that 1) it was inhibited in cells treated with lysosomotropic agents or with a hypertonic concentration of sucrose, and 2) internalized VCAM-1 colocalized with clathrin. In contrast, the cross-linked alpha 4 beta 1 integrin counter-receptor of VCAM-1 remained associated with the plasma membrane of purified peripheral T and Jurkat cells. Our results suggest a model where VCAM-1 would initially participate in the retention of T cells to the endothelium by binding alpha 4 beta 1 integrin. Lymphocyte de-adhesion would be facilitated as a result of the internalization of VCAM-1. The persistent cell surface expression of alpha 4 beta 1 integrin would allow the migrating T cells to interact with and receive signal(s) from its fibronectin ligand of the extracellular matrix.     

Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.     

Expression of a candidate cadherin in T lymphocytes

Cadherins are homotypic adhesion molecules that classically mediate interactions between cells of the same type in solid tissues. In addition, E-cadherin is able to support homotypic adhesion of epidermal Langerhans cells to keratinocytes (Tang, A., Amagai, M., Granger, L. G., Stanley, J. R. & Udey, M. C. (1993) Nature (London) 361, 82-85) and heterotypic adhesion of mucosal epithelial cells to E-cadherin-negative intestinal intraepithelial T lymphocytes. Thus, we hypothesized that cadherins may play a wider role in cell-to-cell adhesion events involving T lymphocytes. We searched for a cadherin or cadherins in T lymphocytes with a pan-cadherin antiserum and antisera against alpha- or beta-catenin, molecules known to associate with the cytoplasmic domain of cadherins. The anti-beta-catenin antisera coimmunoprecipitated a radiolabeled species in T-lymphocyte lines that had a molecular mass of 129 kDa and was specifically immunoblotted with the pan-cadherin antiserum. Also, the pan-cadherin antiserum directly immunoprecipitated a 129-kDa radiolabeled species from an 125I surface-labeled Jurkat human T-cell leukemic cell line. After V8 protease digestion, the peptide map of this pan-cadherin-immunoprecipitated, 129-kDa species exactly matched that of the 129-kDa species coimmunoprecipitated with the beta-catenin antiserum. These results demonstrate that T lymphocytes express a catenin-associated protein that appears to be a member of the cadherin superfamily and may contribute to T cell-mediated immune surveillance.     

Dysregulation of bcl-2, c-myc, and Fas expression during tricyclic antidepressant-induced apoptosis in human peripheral lymphocytes

Tricyclic antidepressants (TCAs) have been shown to induce apoptosis in human lymphocytes. In the present report, we investigated in parallel the regulation of the three oncogenes bcl-2, c-myc, and Fas. A reduction in c-myc and bcl-2 levels of 35-40% and 22-27%, respectively, was observed. On the other hand, Fas expression on the outer surface of the plasma membrane was increased up to 31%. In conclusion, bcl-2, c-myc, and Fas are undergoing dysregulation due to TCA-induced apoptosis.     

Intracellular Ca2+ elevation and cyclosporin A synergistically induce TGF-beta 1-mediated apoptosis in lymphocytes

Apoptosis plays an essential role in the development and homeostasis of the immune system. During lymphocyte development, potentially autoreactive cells are eliminated via the activation of a tightly regulated cell death program(s). Similar processes operate in mature lymphocytes, to control the magnitude of the normal immune response by eliminating activated lymphocytes. However, differences in susceptibility to signal-induced apoptosis between immature and mature lymphocytes are numerous. One well-characterized example occurs in response to Ca2+ elevation: peripheral T lymphocytes are resistant, while immature thymocytes are highly susceptible, to Ca2+-mediated cell death (CMCD). In this study, we show that the immunosuppressant cyclosporin A (CsA) primes splenic lymphocytes to undergo CMCD upon ionomycin stimulation. This CsA-induced CMCD affected both T and B lymphocytes. CsA-plug Ca2+-mediated apoptosis was dissected into a two-step process: first, CsA and Ca2+ synergized to induce TGF-beta 1 secretion by B cells; and then TGF-beta 1 and Ca2+ synergistically triggered T and B lymphocyte apoptosis. Together, our results suggest that lymphocyte apoptosis may play a role in CsA-induced immunosuppression via a TGF-beta-dependent mechanism.     

Age-associated modulation of apoptosis and activation in murine B lymphocytes

We examined neuronal activity in three motor cortical areas while a rhesus monkey adapted to novel visuomotor transforms. The monkey moved a joystick that controlled a cursor on a video screen. Each trial began with the joystick centered. Next, the cursor appeared in one of eight positions, arranged in a circle around a target stimulus at the center of the screen. To receive reinforcement, the monkey moved the joystick so that the cursor contacted the target continuously for Is. The video monitor provided continuous visual feedback of both cursor and target position. With those elements of the task constant, we modified the transform between joystick movement and that of the cursor at the beginning of a block of trials. Neuronal activity was studied as the monkey adapted to these novel joystick-cursor transforms. Some novel tasks included spatial transforms such as single-axis inversions, asymmetric double-axis inversions and angular deviations (also known as rotations). Other tasks involved changes in the spatiotemporal pattern and magnitude of joystick movement. As the monkey adapted to various visuomotor tasks, 209 task-related neurons (selected for stable background activity) showed significant changes in their task-related activity: 88 neurons in the primary motor cortex (M1), 32 in the supplementary motor cortex (M2), and 89 in the caudal part of the dorsal premotor cortex (PMdc). Slightly more than half of the sample in each area showed significant changes in the magnitude of activity modulation during adaptation, with the number of increases approximately equaling the number of decreases. These data support the prediction that changes in task-related neuronal activity can be observed in M1 during motor adaptation, but fail to support the hypothesis that M1 and PMdc differ in this regard. When viewed in population averages, motor cortex continued to change its activity for at least dozens of trials after performance reached a plateau. This slow, apparently continuing change in the pattern and magnitude of task-related activity may reflect the initial phases of consolidating the motor memory for preparing and executing visuomotor skills.