IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors.

The recent understanding of plasma cell (PC) biology has been obtained mainly from murine models. The current concept is that plasmablasts home to the BM and further differentiate into long-lived PCs (LLPCs). These LLPCs survive for months in contact with a complex niche comprising stromal cells (SCs) and hematopoietic cells, both producing recruitment and survival factors. Using a multi-step culture system, we show here the possibility to differentiate human memory B cells into LLPCs surviving for at least 4 months in vitro and producing immunoglobulins continuously. A remarkable feature is that IL-6 is mandatory to generate LLPCs in vitro together with either APRIL or soluble factors produced by SCs, unrelated to APRIL/BAFF, SDF-1, or IGF-1. These LLPCs are out of the cell cycle, express highly PC transcription factors and surface markers. This model shows a remarkable robustness of human LLPCs, which can survive and produce highly immunoglobulins for months in vitro without the contact with niche cells, providing the presence of a minimal cocktail of growth factors and nutrients. This model should be useful to understand further normal PC biology and its deregulation in premalignant or malignant PC disorders.

Effects of caffeine and chlorogenic acid on propidium iodide accessibility to DNA: consequences on genome size evaluation in coffee tree.

Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo-intraspecific variation in genome size. Two important compounds present in coffee trees-caffeine and chlorogenic acid-modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes.

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems.

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

Functional interaction of the gp80 and gp130 IL-6 receptors in human B cell malignancies.

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

Modification of surface marker expression on CD14 monocytes of allergic patients after lysis or Ficoll purification.

It has been observed that peripheral blood monocytes are often in a primed or activated state in inflammatory diseases such as asthma. However, the majority of these studies have been performed using cells which have been purified by density gradient centrifugation on Percoll or Ficoll-Hypaque. Using cytofluorimetry, we compared the expression of monocyte surface markers of monocytes from untreated blood with monocytes purified by erythrocyte lysis or density centrifugation using the Ficoll technique. Monocytes from two groups of subjects were analyzed: healthy subjects and allergic patients. When compared with untreated blood, the percentage of CD16-positive cells, and the sMFI was significantly greater after monocyte purification (lysis or Ficoll). The expression of CD62L (percentage and sMFI) was modified after monocyte purification. Such modification of these two surface markers was predominantly observed on monocytes from allergic patients, and not on monocytes from healthy subjects. This study suggests that surface marker analysis should be performed on unfractionated whole blood in order to avoid modification of monocyte antigens.

Quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes.

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.

Reliable flow cytometric estimation of nuclear DNA content in coffee trees.

Flow cytometry gave high resolution of genome size in two coffee taxa (Coffea liberica dewevrei and C. pseudozanguebariae). Propidium iodide (PI) and Petunia hybrida were used as dye and internal standard, respectively. Proportionally between the DNA content and the digitized fluorescence signal was checked. Five main results were evident. First, optimal experimental conditions were established for peak location estimation (mean or mode), staining time (at least 2 minutes), high voltage (557 V) for the photomultiplier tube (PMT), and PI concentration (333 micrograms/ml). Second, a parameterization of the effects of high voltage and PI concentration were deduced from curve fitting. Third, two biases in DNA content estimation were recorded for high voltage and PI concentration, and were minimized. Fourth, the genome sizes of C. liberica dewevrei and C. pseudozanguebariae were estimated with accuracy 2C = 1.421 +/- 0.005 pg and 2C = 1.129 +/- 0.005 pg, respectively. Fifth, between-genotype variance was emphasized within each taxon.

Detection of membrane and soluble interleukin-6 receptor in lymphoid malignancies.

We studied the membrane expression of the gp80 chain of IL-6 receptor (IL-6R) by quantitative flow cytometry in chronic lymphocytic leukaemia (CLL) and leukaemic centrocytic lymphoma using a panel of seven monoclonal antibodies. IL-6R was detected in 18/26 CLL cases and 4/7 lymphoma cases, with a mean antigen density < 3000 molecules/cell. Multiple labelling experiments confirmed the IL-6R expression by neoplastic cells. Specific mRNA was found by RT-PCR in neoplastic cells. A specific ELISA test was designed using two anti-IL-6 receptor MAbs to measure the serum soluble IL-6R (sIL-6R) in CLL (n = 48). B-cell non-Hodgkin's lymphoma (NHL; n = 40), and monoclonal gammopathy (MG; n = 32). SIL-6R was higher in CLL (170 +/- 12.6 ng/ml) in NHL (160 +/- 12 ng/ml) and MG patients (183 +/- 23 ng/ml) than in age-matched controls (100 +/- 5.6 ng/ml; P < 0.001) and higher in high-grade than low-grade NHL. No correlation was noted with a previous treatment. Among CLL cases the patients classified as stage B according to the Binet's staging of the disease had the highest sIL-6R values, thus suggesting a link with tumour cell mass.

Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line.

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain incompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPIb expression. Moreover, although these agents used alone did not affect GPIIb/IIIa expression, they markedly reversed phorbol ester-induced GPIIb/IIIa expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPIb and GPIIb/IIIa expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis.

Characterization of very acidic phagosomes in breast cancer cells and their association with invasion.

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

Leukemia-associated changes identified by quantitative flow cytometry. II. CD5 over-expression and monitoring in B-CLL.

The CD5 antigen density on B cells was studied on fetal spleen, cord blood, and adult peripheral blood (after immunomagnetic bead purification) using an indirect immunofluorescence technique. In fetal spleen, there was a continuum in CD5 expression, whereas all cord blood and less than 20% adult peripheral blood B cells were CD5+. Mean CD5 antigen density on these normal cells was low (3-6 x 10(3) molecules/cell); eight to 20 times lower than on normal T lymphocytes. In adult blood, less than 10% B cells expressed more than 3 x 10(3) CD5 molecules/cell. In chronic malignancies, 34/35 cases had a CD5 antigen density lower than on residual T cells, but mean antigen density was higher (14.8 +/- 2.1 x 10(3) molecules/cell) than on normal B cells. Sixteen cases of chronic lymphocytic leukemia (50%) expressed a CD5 density above 10 x 10(3) molecules/cell. This aberrantly high CD5 expression was used to detect neoplastic cells after dilution in normal lymphocytes, with a limit of detection between 1:100 and 1:1000. Quantitation of the CD5 antigen allows better characterization of the B1 population and should be used for the monitoring of chronic malignancies.

Autocrine regulation of proliferation of cerebellar granule neurons by nerve growth factor.

Premigratory cerebellar granule neurons, which highly express nerve growth factor (NGF), low (gp75NGFR) and high (gp140trkA) affinity NGF receptors, were used as a physiological model to investigate the effects of NGF on neuronal replication. Studies in vivo and on cultures showed that NGF stimulates DNA synthesis, mitotic activity and related cell acquisition by initiating the entry of cells into the S phase and regulating their time in the G1 and S phases. The NGF-induced effects were blocked in vivo and in vitro by both monoclonal anti-blocked in vivo and in vitro by both monoclonal anti-NGF and anti-gp75NGFR antibodies. These results clearly demonstrate that NGF is essential for the crucial first step of cerebellar ontogenesis and support the idea that low affinity receptors are involved in the biological response, possibly by interacting with gp140trkA. By comparison with a number of well known mitogens, the high affinity form could be the main transducer of the mitogenic signal pathway. The early developing cerebellum appears therefore to be the first autocrine (and/or paracrine) model of NGF action on neurogenesis in the CNS.

Epitope analysis of human IL-6 receptor gp80 molecule with monoclonal antibodies.

Gp80 human IL-6R was studied using 7 murine mAb (M37, M91, M113, M139, M164, M182 and M195) obtained after fusion of splenocytes of Balb/c mice immunised with a mixture of recombinant IL-6 receptor (rIL-6R) and cells from 2 cell lines expressing IL-6R. These were U266, which is IL-6 independent and XG-1 which is IL-6-dependent. In ELISA the 7 mAb reacted against the rIL-6R and against the natural soluble form found in plasma (nIL-6R), which both lack transmembrane and cytoplasmic domains. However, M195 reacted less with the natural than with the recombinant soluble IL-6R. Using FACS analysis, the 7 mAb were shown to bind to U266 cells but not to the Namalva cell line which is deprived of IL-6R. This showed that they all recognised the membrane form of the IL-6R. Three of the anti-IL-6R mAb reacted with rIL-6R by Western blotting. Four different epitopes of the molecule were identified, either by cross-blocking experiments of mAb binding to IL6R in ELISA or by the biosensor Biacore technology. A group of 4 mAb (M37, M113, M139 and M164) and another mAb (M195) identified 2 different epitopes involved in IL-6 binding. These antibodies were able to inhibit the binding of IL-6 to IL-6R and the proliferation of the IL-6-dependent XG-1 cell line. M91 and M182 recognized 2 other epitopes that were not involved in IL-6 binding. As expected, M91 did not inhibit XG-1 proliferation; in contrast, M182 interfered with the proliferative response of the XG-1 cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of CD24 and CD45 antigens in parallel allows a precise determination of B-cell maturation stages: relevance for the study of B-cell neoplasias.

Quantitative expression, i.e. absolute number of monoclonal antibody molecules bound per cell, was evaluated for CD24 and CD45 by flow cytometry with standards of fluorescence intensity on a panel of normal and neoplastic B cells. The CD24 antigen was expressed at homogeneous high level in fetal bone marrow and liver. Its density decreased progressively in the other normal tissues in parallel with the B-cell maturation. The ratio between CD24 density measured on fetal bone marrow B cells and that seen on adult peripheral B cells was 6:1. The CD45 antigen density was lower on fetal bone marrow cells than in the more mature stages. Fetal spleen lymphocytes and all the mature B lymphocytes displayed the same CD45 density than that seen on normal adult peripheral T cells. The CD24/CD45 antigen density ratio was precisely related to the stage of B-cell maturation. The same pattern of variation of CD24 and CD45 antigen density was seen on B-cell neoplasias, with a significantly higher value of CD24 and lower value of CD45 in acute lymphoblastic leukemias than in chronic malignancies. CD24 and CD45 antigen levels were frequently out of the range observed in the corresponding normal population. Among ALL patients, a low CD24/CD45 antigen density ratio was associated with a good prognosis. These data confirm the interest of an absolute quantitative study for widely distributed antigens.

Expression of angiotensin I converting enzyme mRNA in rabbit gastric epithelial cells.

Angiotensin I converting enzyme (ACE) is a dipeptidyl carboxypeptidase synthesized by endothelial cells from many vascular beds as well as by extravascular tissues. Two forms of ACE have been characterized, a pulmonary form and a testicular form. Previously, in the gastrointestinal tract, we localized ACE in the rabbit gastric fundic tissue. In the present study, Northern blot analysis demonstrated the expression of a 5 kb ACE mRNA in fundic mucosa, identical in size to pulmonary ACE mRNA. In order to confirm the epithelial origin of this ACE, we have purified fundic epithelial cells by a flow cytometry technique by which endothelial cells were excluded and the population was enriched in intermediate and chief cells. Using reverse transcription and polymerase chain reaction with specific oligonucleotides, we have amplified from the enriched fundic epithelial cell RNA a 874 bp fragment, the restriction map of which is identical to that of rabbit lung. These findings demonstrate that in gastric mucosa ACE is expressed in fundic epithelial cells and seems to be similar to the pulmonary form.

[Study of S phase synchronization of the human gastric cancer line HGT-1]. / Etude de la synchronisation en phase S de la ligne cancéreuse gastrique humaine HGT-1.

The growth of the human gastric cancer cell line HGT-1 is regulated by a gastrin-like peptide through an autocrine process. In order to analyse the mechanism of action of this peptide, a study at different steps of the cell cycle was considered; so, a blocking of this cell line by thymidine or hydroxy-urea was studied by cytofluorimetry. In normal growth conditions in 10% FCS medium, 57% of the cells were in G0/G1 phase and 31% in S phase. A treatment with 2 mM hydroxy-urea followed by 4 hours in 10% FCS medium led to 85% of the cells in S phase. By successive treatments with thymidine and hydroxy-urea followed by 1 hour in 10% FCS medium, 2 peaks of S phase corresponding to 86% of the cells were observed; after 24 hours, cells were distributed as found for the unconfluent cell line, whatever the treatment. On the other hand, the thymidine kinase activity of unconfluent cells which was relatively elevated as compared to other cell lines (278 mU/mg protein), was increased by synchronisation with hydroxyurea followed by 1 hour in 10% SVF medium (338 mU/mg protein); after 8 hours in 10% FCS medium, this activity decreased at the value observed for cells treated with thymidine followed by 1 hour in 10% FCS medium (214 mU/mg protein). In conclusion, a synchronisation either by thymidine or by hydroxy-urea, led to a blocking of the HGT-1 cell line at different steps of the cell cycle, leading to a better knowledge of its autocrine growth regulation.

Contrasting effect of transforming growth factor type beta 1 (TGF-beta 1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3-CD4-CD8-) thymocytes.

Transforming growth factor beta (TGF-beta) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4-6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4-6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-beta 1 on the IL-2R 55kD alpha chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-beta 1 was able to increase both the percentage of CD3-DN cells expressing IL-2R alpha chains and the expression of IL-2R alpha chain in these cells. This stimulatory effect of TGF-beta 1 was distal from early transduction events. In addition, TGF-beta 1 was found to modulate CD3-DN cell proliferation. During differentiation in the thymus, CD3-DN cells transiently express the IL-2R alpha chain of the IL-2R and these IL-2R+ CD3-DN cells are preprogrammed to down-regulate the IL-2R alpha chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-beta 1 on IL-2R alpha chain expression in these in vitro differentiating CD3-DN cells. We found that TGF-beta 1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3-DN cells, the effect of TGF-beta 1 on IL-2R expression seems to be restricted to proliferating cells.

CD5 B lymphocyte antigen in monoclonal gammopathy.

Because B lymphocytes bearing the CD5 antigen have been involved in many B-cell malignancies, we have investigated the presence of the CD5 B-cell antigen on B and plasma cells in monoclonal gammopathy. Quantification of CD5 B cells was made in the peripheral blood of seven individuals with monoclonal gammopathy of undetermined significance (MGUS) and in that of 21 patients with multiple myeloma (MM). The bone marrow of ten patients with MM was also studied. Patients with progressive MM presented a significant reduction in both B and CD5 B lymphocytes (i.e., percentages and absolute numbers), when compared with individuals with MGUS and patients with stable MM. These latter individuals and patients did not differ from healthy donors. No CD5 B cells were found in the bone marrow of patients with MM. Moreover, no CD5 antigen could be detected on eight freshly established human myeloma cells lines including six totally dependent on interleukin-6. However, it was weakly expressed on two standard myeloma cell lines not requiring exogenous interleukin-6 (i.e., RPMI 8226 and U 266). In conclusion, our data show mainly an overall reduction of the polyclonal CD5 B lymphocytes similar to what is observed for the other polyclonal B lymphocytes in patients with active MM. Finally, the expression of the CD5 antigen human myeloma cell lines is not constant.

Major immunoglobulin capping deficiency in the peripheral blood B cells of patients with Sjögren's syndrome.

The capping of surface immunoglobulins (sIg) is a major characteristic of normal B lymphocytes. Thus, we have investigated sIg capping by peripheral blood (PB) B cells of patients with Sjögren's syndrome (SS) and we have found a major deficiency in these patients. In 12 healthy donors (HD), 8 +/- 2.8% of PB mononuclear cells were B cells (i.e. expressing the B-cell antigens CD19, CD20 and CD21 simultaneously) and more than 90% of these PB B cells were able to cap their sIg. In 12 experiments performed using PB lymphocytes from seven patients with SS, a major capping deficiency was noted with only 30% of PB B lymphocytes being able to cap sIg. This defect was not related to an expansion of the B-cell subpopulation expressing the CD5 antigen and was not observed in five patients with rheumatoid arthritis lacking SS. Capping of sIg via antigen binding (i.e. antigenic modulation) constitutes the initial signal for B-cell activation. This process is involved in anti-viral defence and could have a potential pathogenetic role in autoimmune diseases. This impaired B-cell function presently described represents an immune defect which could be important in the pathogenesis of SS.