Lysosome-associated membrane proteins h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells which mediate cell adhesion to vascular endothelium.

Lysosome-associated membrane proteins (LAMPs) are transmembrane lysosomal glycoproteins which are detectable at the cell surface of lymphocytes in patients with scleroderma and systemic lupus erythematosus. While these proteins have been shown to mediate adhesion of tumor cells to vascular endothelial selectins, the function of LAMPs expressed at the cell surface of peripheral blood lymphocytes has not been previously examined. In the present study, the role of lamp2 (CD107b) in lymphocyte adhesion to vascular endothelium and the factors which influence in vitro cell surface expression of both lamp1 (CD107a) and lamp2 (CD107b) are examined. Freshly isolated PBMCs and unstimulated PBMCs in the culture had low levels of cell surface lamp1 and lamp2 expression which were significantly increased following PHA stimulation (P < 0.0001). A dose-dependent response to PHA and the effect of varying concentrations of serum were defined. Kinetic analysis revealed that the majority of the increase in both lamp1 and lamp2 occurred within the first 2 hr of incubation and that a subset of PBMCs maintained expression for at least 96 hr. Incubation of cells with colchicine and cycloheximide modified the cell surface expression of these proteins. Interleukins 2, 4, 6, and 8 had only a modest effect on the degree of cell surface lamp1 and lamp2 expression, though they did significantly affect the distribution of expression among different subtypes of lymphoid cells. Under the conditions utilized in this study, cell surface LAMP expression was confined primarily to CD56+ cells and to CD3+ cells. Functional analysis utilizing a fluorescence-based adhesion assay revealed that cell surface lamp2 mediates adhesion of PBMCs to vascular endothelium, possibly by interacting with endothelial selectins. LAMPs likely contribute to the migration of activated leukocytes to sites of inflammation in vivo.

Vagal and sympathetic components of the heart rate reflex in chronic portal vein stenosis.

The effects of portal hypertension and portosystemic shunting on autonomic components of the heart rate (HR) baroreflex and on skeletal muscle blood flow changes were investigated using the chronic portal vein-stenosed rat. Phenylephrine- and sodium nitroprusside-induced changes in mean arterial pressure (MAP), HR, and skeletal muscle conductance (SMC) were assessed before and after muscarinic or beta-adrenoceptor blockade. Stenosed rats had lower MAP than sham-operated rats (90 +/- 3 vs. 81 +/- 2 mmHg, P < 0.05), and their portal pressure was higher (7.4 +/- 0.5 vs. 13.9 +/- 1.0 mmHg, P < 0.005). Phenylephrine pressor responses were reduced in stenosed animals, their associated bradycardic responses were enhanced [-1.912 +/- 0.109 vs. -1.427 +/- 0.148 beats per minute (bpm)/mmHg, P < 0.01], and their SMC responses were diminished. Methylatropine abolished bradycardic responses and enhanced pressor responses without affecting SMC. After propranolol, reflex bradycardic responses in stenosed rats were less than in shams (-0.492 +/- 0.085 vs. -0.738 +/- 0.058 bpm/mmHg, P < 0.01), and their pressor and SMC responses became indistinguishable from shams. In contrast, tachycardic responses to nitroprusside-induced hypotension before propranolol were impaired in stenosed rats (-1.492 +/- 0.114 vs. -2.225 +/- 0.347 bpm/mmHg, P < 0.05), and their SMC responses were reduced. Muscarinic blockade did not affect HR or SMC responses to hypotension in either stenosed or sham rats. beta-Adrenoceptor blockade, however, prevented hypotension-induced tachycardia, enhanced nitroprusside depressor responses, and eliminated the between-group differences in SMC responses. These studies indicate that increased gain of the parasympathetic limb of the cardiac baroreflex was responsible for attenuated pressor responses to phenylephrine in portal vein-stenosed animals and that beta-adrenoceptors contributed to skeletal muscle vascular hyporesponsiveness to phenylephrine in portal-vein stenosed animals. Altered beta-adrenoceptor function also appears to contribute to impaired chronotropic and skeletal muscle conductance responses to hypotension.

Quantitation of CD62, soluble CD62, and lysosome-associated membrane proteins 1 and 2 for evaluation of the quality of stored platelet concentrates.

BACKGROUND: Platelets become activated during storage, which results in secretion of granules, vesiculation of microparticles, secretion of protein, and a number of other biochemical and morphologic processes that decrease the utility of platelet concentrates stored for transfusion. STUDY DESIGN AND METHODS: To evaluate the quality of stored platelet concentrates, the cell surface expression of specific activation-dependent antigens (CD62 and lysosome-associated membrane proteins 1 and 2 [LAMP-1, LAMP-2]) on platelets stored in a hospital blood bank over a 7-day period was examined. Relative microparticle counts and the expression of CD62 by microparticles, as well as platelet concentrate supernatant levels of soluble CD62, were determined. RESULTS: The percentage of platelets expressing CD62 increased significantly from Day 1 to Day 5 (p < 0.05) of storage; the mean fluorescence values for CD62 did not. In contrast, the mean fluorescence values of LAMP-1 and LAMP-2 rose significantly (p < 0.01 and p < 0.05, respectively) between Days 1 and 5. Significant declines in CD62, LAMP-1, and LAMP-2 percent expression and mean fluorescence were seen on Day 6 of storage (p < 0.001). Microparticle numbers increased significantly during storage and correlated with levels of CD62 protein (free and membrane-bound) (r = 0.95 vs. Day 2, p < 0.05; r = 0.88 vs. Day 5, p < 0.05). CONCLUSION: Flow cytometric evaluations of the expression of cell surface CD62, LAMP-1, and LAMP-2 are complementary tests that, especially when used in conjunction with the quantitation of CD62 protein, provided a simple and effective means of evaluating the quality of platelet concentrates stored for transfusion.

Natural killer cells and gamma delta T cells in scleroderma: relationship to disease duration and anti-Scl-70 antibodies.

OBJECTIVES: To examine the expression of the natural killer (NK) antigen CD56, and T cell receptor delta chain antigen (TCR delta), expressed on the gamma delta T cell subset, in patients with scleroderma, and to correlate levels of expression with clinical characteristics. METHODS: Peripheral blood mononuclear cells (PBMCs) from 15 patients with scleroderma and 11 controls were obtained from heparinised blood on a ficoll/hypaque gradient, stained with monoclonal antibodies, and examined by flow cytometry for expression of CD56 and TCR delta. RESULTS: Overall, the proportion of PBMCs expressing CD56 in the patient group (14.4 (SEM 2.6)%) was not significantly different from controls (8.75 (2.6)%). The greatest levels of expression were found in patients late (more than three years) in their disease course (18.1 (3.3)%) and in patients who did not express anti-Scl-70 antibodies (17.1 (3.5)%). The proportion of gamma delta T cells was significantly lower in the patient group (1.61 (0.52)% v control 2.61 (0.46)%) (p < 0.05). Patients early in their disease or with anti-Scl-70 antibodies accounted for the reduction in gamma delta T cells (0.71 (0.29)% and 0.96 (0.41)% (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: This study emphasis that NK and gamma delta T cell numbers vary depending upon patient characteristics and may help explain prior contradictory reports. Decreased numbers of gamma delta T cells were seen in scleroderma patients, especially those with anti-Scl-70 antibodies and a disease duration of less than three years.

Alteration in lymphocyte phenotype associated with administration of adjuvant levamisole and 5-fluorouracil.

Levamisole (LMS) and 5-fluorouracil (5FU) administered adjuvantly are effective in reducing the relapse rate following surgical resection of Duke's stage C colon carcinoma. It has been postulated that LMS acts to stimulate the immune system and that this is one mechanism through which this drug exerts its antitumor effects. In this study, peripheral blood mononuclear cells (PBMC) were analyzed in nine patients with surgically resected colon carcinoma prior to initiation of adjuvant LMS/5FU and at several subsequent times while patients were on therapy. Changes in lymphocyte phenotype and soluble interleukin-2 receptor (sIL-2R) between pre-study samples and samples obtained during adjuvant LMS/5FU were evaluated. Significant increases were seen in the proportion of PBMC expressing natural killer (NK) antigen CD56 (14.7 +/- 2.4% versus 18.1 +/- 2.6%; P < 0.05) and surface IL-2R (CD25; 0% versus 0.42 +/- 0.15%; P < 0.05), in sIL-2R (314 +/- 86 U/ml versus 736 +/- 173 U/ml; P < 0.05), and in the CD4:CD8 ratio (2.34 +/- 0.93 versus 3.47 +/- 1.23; P < 0.01). A significant decrease in the proportion of CD8+ PBMC (24.7 +/- 3.8% versus 18.8 +/- 2.6%; P < 0.01) and total CD8+ PBMC (537 +/- 118 versus 324 +/- 37; P < 0.01) was seen. The increase in CD56+ cells correlated with sIL2R levels (r = 0.46; P < 0.05). No changes were noted for CD3, CD4, CD5, CD14, CD16, CD19, CDw49a, or TCR delta. The greatest increase in CD56+ cells and the smallest reduction in CD8+ cells were seen in the subgroup of patients who remained disease-free following adjuvant chemotherapy. This study suggests that adjuvant LMS/5FU has significant stimulatory effects on the immune system, which correlate with patient outcome and may account at least in part for its clinical efficacy.

Social drinking and the immune response: impairment of lymphokine-activated killer activity.

The effect of limited and intermittent alcohol ingestion on the immune response in humans has not been extensively studied. The authors, in this study, evaluate peripheral blood mononuclear cell cytotoxicity before and after alcohol ingestion in a setting designed to mimic social drinking. Eleven healthy volunteers consumed two 12 oz (355 mL) cans of beer in 30 minutes while eating pizza. Five control individuals ingested non-alcoholic beverages. Natural killer and lymphokine-activated killer activity were determined for peripheral blood mononuclear cells obtained before and 30 minutes after alcohol ingestion. Interleukin 2-induced lymphokine-activated killer activity was significantly reduced in blood samples obtained after alcohol ingestion when compared with pre-alcohol samples (p < 0.01). Natural killer activity (unstimulated) was not affected by alcohol ingestion. The authors demonstrate that ingestion of a small amount of alcohol impairs the cytotoxic capacity of peripheral blood mononuclear cells. Alcohol in the context of social drinking may have deleterious effects on the immune system's ability to clear virus-infected cells or cells that have undergone neoplastic transformation, especially for individuals with pre-existing immunosuppression.

Correlation of serum interleukin-8 and cell surface lysosome-associated membrane protein expression with clinical disease activity in systemic lupus erythematosus.

Cell surface expression of lysosome-associated membrane proteins (LAMPs) correlates with serum interleukin-8 (IL-8) levels, shorter disease duration, greater functional impairment from disease-related symptoms and soluble IL-2 receptor levels (sIL-2R) in patients with scleroderma. In this study of 46 patients with systemic lupus erythematosus (SLE), the relationship of serum IL-8 and cell surface LAMP to two clinical measures of disease activity, the SLEDAI and SLAM scales, was evaluated. IL-8 levels were determined on serum samples by the immunometric sandwich enzyme immunoassay technique. Cell surface LAMP expression was determined by flow cytometric quantitation of peripheral blood mononuclear cells with monoclonal antibodies directed against two of the major LAMP proteins, lamp1 and lamp2. The clinical disease activity scales correlated significantly with each other, with C3 levels, serum IL-8, C4, dsDNA and sIL-2R. Lamp1 and lamp2 expression correlated with the SLAM but not the SLEDAI scale. Serum IL-8 levels were elevated in 49 of 51 samples tested (44 of 46 patients) and had a stronger correlation with disease activity than C4, dsDNA and sIL-2R levels. Significantly higher levels of IL-8 were seen in patients with evidence of renal involvement. Serum IL-8 and cell surface LAMP expression may be useful indicators of disease activity in patients with SLE. The possible role of IL-8 in the pathogenesis of SLE requires further investigation.

Intestinal myoelectrical activity and transit time in chronic portal hypertension.

This study was designed to determine the effects of portal hypertension on intestinal myoelectrical activity and propulsion. In a single surgery, adult rats were implanted with a serosal electrode at each quarter of the small intestine, and portal hypertension was produced by calibrated constriction of the portal vein. To determine intestinal transit, portal vein-stenosed (PVS) and sham-operated animals were chronically implanted with a catheter in the proximal small intestine. Transit time was determined by measuring the progression of radioactive chromium along the bowel. Studies were conducted 6, 9, and 14 days after surgical preparation. Portal hypertension was associated with both transient and persistent changes in intestinal myoelectrical activity during the experimental period. Slow wave frequency was significantly reduced in the proximal small intestine on all test days and in the distal small intestine on day 14. Occurrence of the migrating myoelectric complex was reduced on days 6 and 9. Phase III amplitude was significantly reduced in the distal small intestine on all test days. Changes in intestinal myoelectrical activity in PVS animals were not associated with measurable changes in intestinal propulsion. The results suggest that both transient and persistent changes in intestinal myoelectrical activity occur during the 2-wk period after portal vein stenosis. The functional significance of the changes is unknown.