Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.     

Diagnostic and predictive role of cell-free midkine in malignant pleural effusions

Purpose

The detection of circulating nucleic acids has long been explored for the diagnosis and prognosis of a variety of clinical conditions. The aim of this study was to detect the cell-free mRNA expression of midkine (MK) in patients with effusions and its potential diagnostic and predictive value.

Methods

Effusions were collected prospectively from 168 patients. The cell-free RNA was extracted from effusions, and the mRNA expression of MK was detected using real-time PCR. The expression of carcinoembryonic antigen (CEA) and biochemical markers in effusions were also assayed. Primary cancer cells were isolated from the malignant effusions (n = 46). Compared with culture cell lines, the response of these cancer cells to chemotherapeutic agents was determined by CCK-8 assay.

Results

The expression of cell-free MK mRNA was significantly higher in the malignant group than in the benign group (0.13 vs 0.01, P < 0.001). The sensitivity and diagnostic accuracy of MK were 77.5 and 81.5 %, while a combination of CEA and MK reached 86.9 % sensitivity and 88.7 % accuracy. In addition, cell-free MK mRNA expression was significantly correlated with inhibitory rate of cisplatin (R = ?0.72, P < 0.01).

Conclusions

Measurement of cell-free MK mRNA levels in effusion supernatant yields a high diagnostic accuracy and a potential predictive value.     

Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation

This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation.     

Macrophage-derived chemokine in malignant and tuberculous pleural effusions

BACKGROUND AND OBJECTIVES: Macrophage-derived chemokine (MDC/CCL22) is recognized as a T-helper (Th) 2-type chemokine. Both malignant and tuberculous pleural effusions are typically lymphocytic pleural effusions. Tuberculous pleural effusions have a more polarized Th1 reaction than malignant effusions, which are predominantly Th2 in nature. The aim of this study was to compare the levels of MDC in malignant pleural effusions with those in tuberculous pleural effusions to help delineate the role of MDC in Th2 versus Th1 effusions. METHODS: Forty-three patients with pleural effusions (32 malignant, 11 tuberculous) were studied. The concentration of MDC in the pleural effusion was measured by ELISA. RESULTS: The median concentration of MDC was lower in malignant pleural effusions than in tuberculous pleural effusions (P < 0.005). CONCLUSIONS: MDC has been reported to both promote and suppress antitumour immunity. The low concentration of MDC in malignant effusions is likely to minimise its antitumour activity but the precise role of MDC in malignant and tuberculous effusions needs to be investigated further.     

Carcinoembryonic antigen levels in benign and malignant pleural effusions

One hundred ninety-one unselected fluid specimens submitted routinely for cytologic examination were assayed to determine whether the measurement of carcinoembryonic antigen (CEA) levels in pleural effusions is useful in detecting malignancy. The mean +/- SD CEA level of 103 benign effusions was 4.1 +/- 2.9 ng/ml. Only one benign effusion had a level greater than 12 ng/ml (18 ng/ml). Benign inflammatory effusions (pneumonia, empyema) had a higher mean CEA activity (6.2 +/- 3.4) than effusions caused by congestive heart failure (2.9 +/- 1.5) (p less than 0.001). Twenty-four (34%) of 70 malignant effusions had a CEA level greater than 12 ng/ml, and 28 (40%) were "positive" by cytologic study. Thirty-eight (54%) were detected by one or both methods. Ten malignant effusions were positive by CEA (greater than 12 ng/ml) alone. These data suggest that the determination of CEA activity levels, when used in conjunction with other clinical findings, may be useful in detecting malignant pleural effusions.     

Osteopontin is upregulated in malignant and inflammatory pleural effusions

Background and objective:   Osteopontin (OPN) is an important mediator of inflammation and cancer progression. In the present study, we asked whether pleural fluid (PF) and serum OPN concentrations differed between patients with pleural effusions of different aetiologies, and whether assessment of OPN levels was useful for diagnostic purposes.
Methods:   One hundred and nine consecutive patients with pleural effusions of different aetiologies were recruited prospectively during daily clinics. OPN levels were measured by ELISA.
Results:   PF OPN levels were 10-fold higher in exudates than in transudates and were significantly correlated with markers of pleural inflammation and vascular hyper-permeability, such as PF/serum LDH or protein ratios, PF protein and PF vascular endothelial growth factor levels. Patients with malignant pleural effusions had higher PF and lower serum OPN concentrations than those with benign disease. The diagnostic accuracies of PF and PF/serum OPN for malignancy were 71.5% (95% CI: 64–80) and 70.6% (95% CI: 62–80), respectively.
Conclusions:   OPN levels were elevated in exudative pleural effusions, as compared with the levels in blood or transudative pleural effusions. While PF and PF/serum OPN were higher in patients with malignancies, the diagnostic accuracy of the tests was not sufficient to permit routine use in clinical practice.     

Interleukin-16 in tuberculous and malignant pleural effusions.

The aim of this study was to explore the presence of interleukin (IL)-16 in pleural effusions, the correlation between IL-16 levels and cytological parameters, as well as the chemoattractant activity of IL-16 on CD4+ T-lymphocytes. Total nucleated cell and differential counts, and IL-16 concentrations in the pleural effusion from 32 patients with tuberculous pleurisy and 30 patients with lung cancer were determined. Three-colour flow cytometry was performed to determine T-lymphocyte subsets in cell pellets of pleural effusion. The chemoattractant activity of IL-16 for CD4+ T-lymphocytes was also analysed. The levels of IL-16 were significantly higher in tuberculous than in malignant effusions. However, IL-16 levels could not be used for diagnostic purposes due to significant overlap between the two groups. Positive correlations were found between the IL-16 levels and CD4+ T-cells, and pleural fluid was chemotactic for CD4+ T-cells in vitro. Intrapleural administration of IL-16 to patients produced a marked progressive influx of CD4+ T-cells into the pleural space. Compared with malignant pleural effusion, interleukin-16 appeared to be increased in tuberculous pleural effusion. Interleukin-16 levels were positively related to the numbers of CD4+ T-cells, and interleukin-16 could directly induce CD4+ T-cell infiltration into the pleural space.     

Soluble HLA class I, HLA class II, and Fas ligand in blood components: a possible key to explain the immunomodulatory effects of allogeneic blood transfusions

The immunomodulatory effect of allogeneic blood transfusions (ABT) has been known for many years. However, a complete understanding of the effects of ABT on the recipient's immune system has remained elusive. Soluble HLA class I (sHLA-I), HLA class II (sHLA-II), and Fas ligand (sFasL) molecules may play immunoregulatory roles. We determined by double-determinant immunoenzymatic assay (DDIA) sHLA-I, sHLA-II, and sFasL concentrations in different blood components. sHLA-I and sFasL levels in red blood cells (RBCs) stored for up to 30 days and in random-donor platelets are significantly (P <.001) higher than in other blood components and their amount is proportionate to the number of residual donor leukocytes and to the length of storage. Blood components with high sHLA-I and sFasL levels play immunoregulatory roles in vitro as in allogeneic mixed lymphocyte responses (MLR) and antigen-specific cytotoxic T-cell (CTL) activity, and induce apoptosis in Fas-positive cells. These data suggest that soluble molecules in blood components are functional. If these results are paralleled in vivo, they should be taken into account in transfusion practice. Blood components that can cause immunosuppression should be chosen to induce transplantation tolerance, whereas blood components that lack immunosuppressive effects should be preferred to reduce the risk of postoperative complications and cancer recurrence.     

sFasL检测对结核性与恶性胸腔积液鉴别诊断价值的探讨

目的探讨sFasL对恶性胸腔积液与结核性胸腔积液鉴别诊断价值。方法应用ELISA法分别检测32例恶性胸腔积液和43例结核性胸腔积液中sFasL的含量，对结果进行统计学处理。结果结核性胸腔积液组sFasL（13．56±5．38ng／m1）显著高于恶性胸腔积液组（5．72±2．59ng／m1），二者具显著性差异（P〈0．001）。以10ng／ml为临界值，胸腔积液中sFasL〉10ng／ml诊断为结核性胸腔积液的敏感性为81．4％（35／43），特异性为81．3％（26／32），临床诊断符合率为81．4％（61／75）。结论sFasL对结核性、恶性胸腔积液的鉴别诊断有临床实用价值。     

Efficacy and safety of iodopovidone pleurodesis in malignant pleural effusions

Background and objective: Pleurodesis is one of the best methods of controlling malignant pleural effusions (MPE), a distressing complication of metastatic disease. In recent studies of a wide range of pleural diseases, iodopovidone was used as a sclerosing agent for pleurodesis and demonstrated good results with low morbidity. The aim of this study was to evaluate the efficacy and safety of iodopovidone pleurodesis in MPE. Methods: A retrospective analysis was performed on patients with MPE who underwent pleurodesis at our institution between 2005 and 2008. All patients underwent instillation of 20 mL of 10% iodopovidone, 80 mL of normal saline and 2 mg/kg of lidocaine through a chest tube, which was clamped for 2 h. The tube was removed when the daily output of fluid was <200 mL. Data on the requirement for additional pleural procedures, adverse events and survival were collected. Results: Sixty‐one pleurodesis procedures were performed in 54 patients. No procedure‐related mortality was observed. Adverse events occurred after 11 (18%) pleurodesis procedures. The most frequent complication was mild thoracic pain that occurred immediately after 10 (16.4%) procedures, and one patient developed pleural empyema that was treated with drainage and antibiotics. A success rate of 98.4% was observed. Except for the patient who developed pleural empyema, none of the other patients had recurrences of pleural fluid or required additional pleural procedures during the follow‐up period (mean of 5.6 months). Conclusions: Iodopovidone pleurodesis was successful and was associated with only a few minor complications. It appears to be a good option for the management of recurrent MPE.     

sFasL检测对结核性与恶性胸腔积液鉴别诊断价值的探讨

目的探讨sFasL对恶性胸腔积液与结核性胸腔积液鉴别诊断价值。方法应用ELISA法分别检测32例恶性胸腔积液和43例结核性胸腔积液中sFasL的含量,对结果进行统计学处理。结果结核性胸腔积液组sFasL(13.56±5.38 ng/ml)显著高于恶性胸腔积液组(5.72±2.59 ng/ml),二者具显著性差异(P＜0.001)。以10 ng/ml为临界值,胸腔积液中sFasL>10 ng/ml诊断为结核性胸腔积液的敏感性为81.4%(35/43),特异性为81.3%(26/32),临床诊断符合率为81.4%(61/75)。结论sFasL对结核性、恶性胸腔积液的鉴别诊断有临床实用价值。     

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia

Objectives: Waldenstrom Macroglobulinemia (WM) is a B‐cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta‐2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. Methods: We assessed the serum levels of the total soluble HLA‐I molecules and the HLA‐Gs molecules in 105 patients with IgM‐related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. Results: We found higher levels of HLA‐Is in WM, compared to IgM MGUS and healthy donors. HLA‐Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA‐Is at the cut‐off of 1.8 μg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA‐Is level was strongly associated with high serum β2M level >3 mg/L [OR = 2, (CI 95% 1.1–5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5–4.1), P = 0.06] and haemoglobin ≤11.5 g/dL [OR = 3.3, (CI 95% 1.2–9.7); P = 0.03]. High levels of serum HLA‐Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM‐MGUS status. Conclusion: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.     

胸液P-选择素检测对结核性与恶性胸腔积液鉴别意义的探讨

目的　检测结核性及恶性胸腔积液患者胸液P-选择素水平,探讨其对鉴别良恶性疾病的意义。方法 采用酶联免疫吸附法(ELISA法)检测48例结核性胸液及50例恶性胸液P-选择素水平。结果 恶性胸液P-选择素水平(18.76±8.45μg/L)明显高于结核性胸液P-选择素(7.43±5.32μg/L)(P<0.01)。结论 测定胸液P-选择素水平有助于结核性及恶性胸液的鉴别。     

Proinflammatory cytokines and fibrinolytic enzymes in tuberculous and malignant pleural effusions

OBJECTIVES: To measure tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in pleural effusions caused by tuberculosis (TB) and malignancy and their relationship with plasminogen activator inhibitor type I (PAI-1) and tissue type plasminogen activator (tPA), and to compare the differences between tuberculous and malignant pleural effusions. In addition, the relationship between the effusion levels of these parameters and the development of residual pleural thickening was evaluated in the patients with tuberculous pleurisy. DESIGN: Prospective study. MATERIALS AND METHODS: TNF-alpha, IL-1beta, PAI-1, and tPA were measured simultaneously in blood and pleural fluid using an enzyme-linked immunosorbent assay in 33 patients with tuberculous and in 30 patients with malignant pleural effusions. Residual pleural thickening was measured and defined as a pleural thickness of >/= 10 mm found on chest radiographs at the completion of anti-TB chemotherapy in tuberculous pleurisy patients. RESULTS: In both groups, the levels of proinflammatory cytokines and fibrinolytic enzymes were significantly higher in pleural fluid than in blood. The levels of TNF-alpha and PAI-1 were significantly higher in tuberculous than in malignant effusions. In contrast, malignant pleural fluid had significantly higher values of tPA than did tuberculous pleural fluid. In tuberculous effusions, the values of PAI-1 and the PAI-1/tPA ratio correlated positively and the levels of tPA correlated negatively with those of TNF-alpha and IL-1beta. In malignant pleural fluid, positive correlations were found between the values of proinflammatory cytokines (TNF-alpha and IL-1beta) and PAI-1. Residual pleural thickening was found in 9 of 33 patients (27. 3%) with tuberculous pleurisy. The pleural fluid values of TNF-alpha, IL-1beta, and PAI-1 were significantly higher and the concentrations of tPA were significantly lower in tuberculous pleurisy patients with residual pleural thickening. CONCLUSIONS: Compared to malignant pleural effusion, fibrinolytic activity in pleural fluid was reduced in tuberculous effusion. Pleural inflammation caused by TB may enhance the release of proinflammatory cytokines, particularly TNF-alpha, which subsequently may increase PAI-1 and decrease tPA in pleural fluid. The imbalance of PAI-1 and tPA in pleural space may lead to fibrin deposition and pleural thickening.     

Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I

Natural killer (NK) cells activate quickly in response to pathogens, tumors, and allogeneic hematopoietic cell transplants. Modulating the NK cell response are clonally distributed NK cell receptors that survey cells for change in the expression of major histocompatibility complex (MHC) class I and structurally related ligands. Here the enzyme-linked immunospot (ELISPOT) assay, intracellular cytokine staining (ICS), and short-term culture were used to quantify the response of bulk NK cell populations from human donors to HLA class I-deficient 221 cells and to 221 cells transfected with single HLA class I allotypes. NK cells in cultures containing interleukin-2 (IL-2) or IL-12 exhibited specificities of HLA class I-mediated inhibition that correlated well with those previously defined using NK cell clones in long-term culture and with the frequencies of cells expressing particular inhibitory HLA class I receptors. Culture with IL-12, but not IL-2, gave an increased frequency of cells expressing CD94: NKG2A but no change in killer immunoglobulin-like receptor (KIR) expression. For some heterozygote combinations of KIR3DL1 alleles, ICS can be used to compare the functional properties of the 2 allotypes. Thus, both the low-expressing KIR3DL1*005 and the high-expressing KIR3DL1*002 gave similar inhibitory response on challenge with an HLA-B*5801 ligand. The single-cell assays developed here should facilitate future population study and clinical analysis of human NK cell regulation by MHC class I.     

Soluble HLA class I antigens in sera of patients with chronic hepatitis.

Soluble HLA Class I antigens in sera (serum-HLA Class I, s-HLA Class I) of patients with chronic hepatitis (CH) were measured with an enzyme-linked double determinant immunoassay (E-DDIA). The mean titers of s-HLA Class I antigens of patients with CPH (mean +/- standard deviation, 2.22 +/- 1.60), CAH2A (2.24 +/- 1.65) or CAH2B (2.73 +/- 1.46) were significantly higher than that of normal subjects (0.36 +/- 0.27) (P less than 0.01). The titer of s-HLA Class I correlated significantly with the level of serum glutamic pyruvic transaminase (s-GPT) (r = 0.73), and weakly with serum level of beta 2-microglobulin (r = 0.43). In patients with chronic hepatitis type B (CH-B) treated with human lymphoblastoid interferon alpha (IFN-alpha), the titer of s-HLA Class I antigens increased. The increased level of s-HLA Class I antigens in the clinical course of chronic hepatitis may be caused by their release from necrotizing hepatocytes which have acquired the expression of HLA Class I antigens on the cell-surface membrane during viral infection.     

Platelet activation by cross-linking HLA class I molecules and Fc receptor.

The effect on platelet activation of monoclonal antibodies directed against common determinants of the HLA class I heavy chain molecule was studied. Cross-linking W6/32, an anti-HLA class I of IgG2a subclass, led to platelet activation. Two other antibodies of the same subclass did not have this effect on platelets. The lack of activity of the F(ab')2 fragments suggests that the activation signal is mediated by the platelet Fc receptor (Fc gamma RII). Indeed, except for a higher sensitivity of W6/32 to aspirin and apyrase, activations by cross-linking IV-3 (an anti-Fc gamma RII) and W6/32 are similar at the level of InsP3 formation, calcium mobilization, pH modifications, and activation of protein kinase C and myosin kinase. When HLA class I molecules and Fc gamma RII are cross-linked together, platelet activation occurs. This is not observed when a control IgG2a is substituted for W6/32 or when CD9 and Fc receptor are cross-linked together. This suggests that HLA class I molecules and Fc gamma RII synergize to activate platelets.