Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line.

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [gamma-(32)P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19-36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line.

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

Expression of a functional Fas death receptor by human foetal motoneurons.

The expression of the apoptosis inducer Fas (CD95/APO-1) surface receptor by human foetal neurons was investigated in vitro and ex vivo. Immunofluorescence studies of brain and spinal cord cells in primary cultures and of cryosections obtained from 9- and 10-week-old human foetuses, respectively, showed that all Fas-expressing cells were motoneurons (5.3 and 4.2% of the neurons in brain or spinal cord cultures, respectively) on the basis of morphology, reactivity with the monoclonal antibody SMI-32, a mostly motoneuronal marker and acetylcholine esterase expression. Fas was undetectable on the other cell types in culture. The ability of Fas to induce apoptosis of cultured cells from both tissues was determined by using the terminal transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) method combined with the same double-staining procedure. Under basal culture conditions, about 9% of cells, all glial fibrillary acidic protein-expressing astrocytes, were apoptotic. After a 48-h incubation with Fas ligand, mean 28.5% of brain motoneurons and 29.4% of spinal motoneurons underwent apoptosis, with an inhibition by Z-IETD-FMK, a caspase-8 inhibitor. Hence, Fas appears to be functional through a caspase-8-dependent pathway in a subpopulation of human foetal motoneurons.

Expression of interleukin 13 receptor in glioma and renal cell carcinoma: IL13Ralpha2 as a decoy receptor for IL13.

Glioma and renal cell carcinoma (RCC) cells express high affinity interleukin 13 (IL13) binding sites, but only RCC cell proliferation was inhibited by IL13. Both of these two cell types are IL2-receptor (gamma)c chain-negative. We thus used these cell models to investigate the patterns of expression of IL13Ralpha1, IL13Ralpha2, and IL4Ralpha chains and the role of IL13Ralpha2 in the response to IL13. Using new specific antibodies and flow cytometry, we observed a similar surface expression of IL4Ralpha and IL13Ralpha1 chains in most RCC and glioma cells, whereas IL13Ralpha2 was only present on five of six glioma cell lines. In all glioma cell lines, the amount of IL13Ralpha2 expression was 10 to 30 times higher than that of the two other chains. Although there was no surface or intracellular expression of IL13Ralpha2, its mRNA was detected in three of seven RCC cell lines. The expression on RCC cells of IL13Ralpha2 mRNA and/or that of high-affinity IL13 binding sites is not sufficient to predict IL13Ralpha2 protein expression. Blocking experiments showed that IL4 and IL13 strongly inhibited RCC cell proliferation through a unique receptor composed of IL4Ralpha and IL13Ralpha1 chains. Using RCC cells stably transfected with IL13Ralpha2 cDNA, we showed that the overexpression of IL13Ralpha2 decreased the response to IL13 but not that to IL4. Our results demonstrate that IL13Ralpha2 acts as a decoy receptor for IL13 and that it may exert a tight regulation of IL13 activity without impairing the IL4 response of the same cell target.

In vitro induction of neuronal apoptosis by anti-Fas antibody-containing sera from amyotrophic lateral sclerosis patients.

Sera from 26% of patients with sporadic amyotrophic lateral sclerosis (ALS) induced in vitro apoptosis of a human neuroblastoma cell line, as detected by two methods, and most contained anti-Fas autoantibodies. In contrast, Alzheimer sera (studied as controls) very rarely induced apoptosis and did not contain detectable anti-Fas antibodies. Soluble Fas-ligand levels in ALS sera were not different from those in normal sera, except for slightly higher levels in a single case. In mixed cultures of rat embryonic brain and spinal cord cells, ALS sera (and agonistic anti-Fas monoclonal antibodies and soluble Fas-ligand) induced the apoptosis of a subpopulation of neurons. These neurons were motoneurons on the basis of staining with the monoclonal antibody SMI 32 and Fas expression was restricted to these SMI 32-positive neurons. These data are compatible with the hypothesis of the participation of an autoimmune mechanism possibly related to anti-Fas autoantibodies in certain ALS patients.

Antibodies to human adhesion molecules and von Willebrand factor: in vitro cross-species reactivity in the xenotransplantation setting.

Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD3 1, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

Circulating cell adhesion molecules in HIV1-infected patients as indicator markers for AIDS progression.

Cell adhesion molecules play an important role during immune responses. Circulating (c) forms of these molecules have been used as monitors of disease progression. In this study, we have investigated serum levels of ICAM1, ICAM2, ICAM3 and VCAM1 in HIV-infected patients. Our results showed that levels of cICAMs and cVCAM1 are increased during HIV infection. Among an HIV-infected population, the cICAM2 level was higher in the asymptomatic group compared to the AIDS group. In contrast, the cICAM1 level was higher in the AIDS group compared to the asymptomatic group. No difference between the two groups was observed in cICAM3 and cVCAM1 levels. A significant correlation was found between cICAM1, cICAM2 and cVCAM1 in both populations. We also showed that the cICAM1/cICAM2 ratio was correlated with the increase in the c beta 2 microglobulin level and the decrease in CD4 T-cell counts in the AIDS group. These results indicate that serum cCAM1 and cICAM2 in HIV infection could be additional markers to discriminate between asymptomatic and progressor patients.

Heterogeneity of antigen expression among human umbilical cord vascular endothelial cells: identification of cell subsets by co-expression of haemopoietic antigens.

First described as an interface between blood and tissues, the endothelial cells are now known to be also involved in haemostasis, inflammatory and immune responses. Recent studies have reported that the endothelial cells represent a heterogeneous population of cells with organ-specific properties. This report describes the phenotype analyzed by flow cytometry of human umbilical vascular endothelial cells (HUVEC). For this purpose, we use monoclonal antibodies (Mabs) recognizing B cell antigens (CD10, 19, 20, 21, 22, 23, 24, 38, 40), T cell antigens (CD1, 2, 3, 4, 5, 6, 7, 8), myeloid antigens (CD13, 14, 15, 34, 35, 64), platelet antigens (CD9, 31, 51, 62P), NK and non-lineage cell antigens (CD16, 45, 56, 57), activation antigens (CD25, 30, 69, 71) and adhesion molecules (CD11a, CD11b, 29, 44, 54, 62E, 102, 106). Mabs recognizing MHC ClI or ClII molecules are also tested. Firstly, we show that HUVEC co-express some haemopoietic antigens with different levels of expression. Secondly, this study reveals that the HUVEC population does not represent a homogeneous cell population. Different endothelial cell subsets are identified. These phenotypical differences could reflect specialization of HUVEC performing different functions. The significant of haemopoietic antigen expression on the HUVEC surface will be discussed.

Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD11b/CD18 through the A domain.

The interactions between the leukocyte-specific beta 2-integrins cluster of differentiation (CD) Ag CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs), play important roles in many adhesion-dependent leukocyte functions. ICAM-1 is known to be a ligand for both CD11a/CD18 and CD11b/CD18. ICAM-2, whose two extracellular Ig domains show the highest homology to the two NH2-terminal domains of ICAM-1, has been previously shown to be a ligand for CD11a/CD18. We recently found that a 22-amino acid CD11a/CD18-binding peptide, P1, derived from the first domain of ICAM-2, also binds to purified CD11b/CD18. In the present study, we demonstrate that the ICAM-2 protein interacts with CD11b/CD18, and the binding is through the CD11b A domain.

A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50.

We have made a monoclonal anti-CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM-1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM-1 and ICAM-3. The binding to ICAM-2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand-specific manner by engagement of CD44.

Effects of intravenous immunoglobulins (IVIG) on peripheral blood B, NK, and T cell subpopulations in women with recurrent spontaneous abortions: specific effects on LFA-1 and CD56 molecules.

Polyspecific IgG given intravenously at high dose (IVIG) are increasingly used as an immunomodulating therapy in autoimmune diseases. However, very few studies have dealt with the action of IVIG on the expression of the leukocyte markers. During a clinical trial in which 13 young and healthy women received IVIG to prevent unexplained recurrent abortions we have evaluated by flow cytometry the action of IVIG on 17 clusters of leukocyte differentiation (CD). We found that the IVIG perfusions (0.5 g/kg) induced an increase in the number of polymorphonuclear and monocyte cells in the peripheral blood. This effect lasted 8 days. The IVIG treatment had no effect upon T cell populations stained with antibodies specific for CD2, CD3, CD4, CD8 and on CD4+CD45RA+, CD4+CD29+, CD8+CD28+, CD8+CD28- subpopulations. A weak decrease in the B cell number was observed. The most striking phenomenon was the decrease in the number of CD56+ cells, whereas CD16+ and CD57+ cells were unaltered. By the double-staining technique we showed that CD56+CD16+ cells became CD56-CD16+ cells. Moreover, IVIG decrease the expression level of the LFA-1 molecule on monocytes and lymphocytes. The other adhesion molecules studied remained steady (CD11b, CD49d, CD49e, CD29, CD28, and CD62L). This study has shown that IVIG have no effect on 15 of 17 CD used but downmodulate two adhesion molecules playing a key role in the immune system.

Selective detection of human hepatitis B virus surface and core antigens in peripheral blood mononuclear cell subsets by flow cytometry.

The presence of hepatitis B surface protein (HBs) and hepatitis B core protein (HBc) was investigated, by flow cytometry, on the surface of peripheral mononuclear cells (PBMC) from cells of the following phenotype: CD3 (T lymphocytes), CD4 (T helper/ inducer), CD8 (T cytotoxic/suppressor), CD19 (B lymphocytes) and CD56 [natural killer (NK) cells] among eight patients suffering from chronic hepatitis B and five healthy HBV-negative subjects. This study demonstrated the presence of HBsAg and HBcAg on the lymphocyte surface for most of the patients. The mean percentage of labelled cells was 17% for HBsAg and 15% for HBcAg. Among the different lymphocyte subsets only B lymphocytes and the NK cells expressed HBsAg for 57% and 26% of cells, respectively. Similarly HBcAg was also detected among CD19 and CD56 cells only. Polymerase chain reaction (PCR) was used to search for the presence of hepatitis B virus (HBV) DNA and RNA in PBMC, using primers located in the S gene. HBV DNA was detected with variable intensity in the CD3, CD4, CD19 and CD56 subsets following their separation with a cell sorter. For HBV RNA the signal obtained after PCR and Southern blotting was higher for CD56 and CD19 cells than for CD3 cells and undetectable for CD4 cells. This study demonstrates that replication and transcription of the HBV can occur in CD19- and CD56-positive cells. Positive signals in CD3 cells may be due to contamination of this subpopulation by NK cells.

Monitoring of early events of experimental woodchuck hepatitis infection: studies of peripheral blood mononuclear cells by cytofluorometry and PCR.

The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37% +/- 25 and 17% +/- 15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.

Immunosuppressive property of a very high purity antihaemophilic preparation: a low molecular weight component inhibits an early step of PHA induced cell activation.

Immune deficiency has been reported in haemophiliac patients receiving antihaemophilic factor VIII preparations, but the mechanisms involved in the immunosuppression are not fully understood. By using the proliferative response of peripheral blood mononuclear cells to phytohaemagglutinin (PHA) as a test system, we investigated the inhibitory influence of a very high purity antihaemophilic factor (AHF) preparation on T cell proliferation and on T lymphocyte activation molecules. We observed that this preparation reduced significantly the PHA-induced mononuclear cell proliferation, independently of the monocyte concentration. The AHF preparation did not act through a cytotoxic mechanism or a steric hindrance of PHA. The AHF preparation had no effect on the immediate expression of T lymphocyte activation molecules such as CD54 (ICAM-1). In contrast, the very high purity AHF reduced the induced expression of two early T cell activation molecules: CD25 (interleukin-2 receptor) and CD71 (transferrin receptor). The very high purity AHF also had the capacity to inhibit the up-regulation of two late activation antigens, CD38 and CD11a/CD18, and to inhibit the induced expression of HLA-DR molecule, defined also as a late T cell activation molecule. The CD45R expression level, used as a control marker, was not changed after AHF exposure. The very high purity AHF therefore influenced an early step of cell proliferation. We have also shown that the immunoregulatory properties of the preparation were not restricted to the factor VIII itself, but resulted from the presence of dialysable and low molecular weight components in the preparation.

Demonstration of woodchuck hepatitis virus infection of peripheral blood mononuclear cells by flow cytometry and polymerase chain reaction.

Peripheral blood mononuclear cells (PBMCs) from 10 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of WHV surface (WHs) and core (WHc) antigens (WHsAg and WHcAg) by cytofluorometry using fluorescein isothiocyanate-conjugated anti-WHs and anti-HBc-purified immunoglobulins from woodchuck and human sera. The presence of viral DNA and RNA was detected in the serum and PBMCs from the same blood samples by polymerase chain reaction (PCR) with two primer sets located in the S and C genes of the WHV genome. Seven animals were found positive for both WHsAg and WHcAg on the surface of PBMCs: four WHV-chronic carriers, two WHsAg-positive animals with acute WHV infection, and one woodchuck which was bled during the incubation phase of WHV infection and which became WHsAg-positive only 1 month later. Sixteen to 71% of the studied leukocyte population expressed WHsAg with a low density of expression whereas 7 to 72% expressed WHcAg with a high density of expression. Only two cases were positive for WHsAg without WHcAg on PBMCs, one WHV chronic carrier and one anti-WHs-positive animal. All woodchucks positive for WHcAg and/or WHsAg by cytofluorometry were positive also for WHV DNA and RNA in PBMCs by PCR. The tenth animal was found negative for both viral antigens as well as for WHV DNA and RNA in PBMCs despite the presence of persistent viral DNA in the serum as detected by PCR. Five healthy woodchucks devoid of WHV serological markers served as negative controls. These results obtained with a novel approach further confirm, in the woodchuck model, that a significant proportion of PBMCs are probably permissive for WHV replication. The possible immunopathogenic implications of the phenomenon are discussed.

Selective detection of human hepatitis B virus surface and core antigens in some peripheral blood mononuclear cell subsets by flow cytometry.

The presence of HBs and HBc antigens was investigated, by flow cytometry, on the surface of peripheral mononuclear cells (PBMC) from the following phenotype: CD3 (T lymphocytes), CD4 (T helper/inducer), CD8 (T cytotoxic/suppressor), CD19 (B lymphocytes) and CD56 (NK cells) among 8 patients suffering from chronic hepatitis B and 5 healthy HBV-negative subjects. This study demonstrated the presence of HBsAg and HBcAg on the lymphocyte surface for most of the patients. The mean percentage of labelled cells was 17% for HBsAg and 15% for HBcAg. Among the different lymphocyte subsets only B lymphocytes and the NK cells expressed HBsAg for 57% and 26% of cells, respectively. Similarly HBcAg was also detected among CD19 and CD56 cells only. PCR was used to search for the presence of HBV DNA and RNA in PBMC, using primers located in the S gene. HBV DNA was detected with variable intensity in the CD3, CD4, CD19 and CD56 subsets following their separation with a cell sorter. For HBV RNA the signal obtained after PCR and Southern blotting was higher for CD56 and CD19 cells than for CD3 cells and undetectable for CD4 cells. This study demonstrates that replication and transcription can occur in CD19 and CD56 cells. Positive signals in CD3 cells may possibly be due to contamination of this subpopulation by NK cells.