Surface molecules of cultured human lymphoid cells.

Cell surface molecules of cultured human lymphoid cells were selectively labeled by lactoperoxidase-catalyzed iodination and examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two major iodinated species with apparent mol. wts. of 27 000 and 35 000 daltons were detected on autoradiographs of the labeled proteins of human lymphoid cell lines believed to be of thymus-independent (B) cell origin. Neither molecule was detected on putative thymus-dependent (T) lymphoid cell lines. Metabolic labeling studies showed that both molecules are glycoproteins. Rabbit antisera against cultured B lymphoid cells made specific by absorption for B cells reacted with several labeled species from iodinated B cells including the 27 000 and 35 000 mol. wt. glycoproteins. These molecules were also detected on tonsillar lymphocytes but not on peripheral blood lymphocytes. Reciprocal absorption with B cells of rabbit antisera against cultured T cells gave antisera specifically cytotoxic for T cells. However, these sera did not precipitate iodinated proteins from Nonidet P-40 lysates of T cells.     

人胎盘间充质干细胞来源外泌体保护氧化应激损伤肺上皮细胞的机制

背景:前期研究已证实人胎盘间充质干细胞来源培养上清能够通过抑制细胞凋亡保护氧化损伤的肺上皮细胞.目的:探究人胎盘间充质干细胞来源外泌体对氧化应激损伤肺上皮细胞BEAS-2B的保护作用.方法:体外培养BEAS-2B细胞,采用含100,200,300,400,500 μmol/L过氧化氢的DME/F-12完全培养基培养细胞...     

Electrophoresis of lymphoid cells. Characterization of human B and T cells in peripheral lymphoid tissues

Small lymphocytes from human blood, tonsils, spleen and lymph nodes showed a bimodal distribution on electrophoresis, the electrophoretic mobility (EPM) of one group being slow, the other fast. The difference in mean EPM between the slow and fast groups was about 30%. The percentage of slow cells in each organ was similar to that of cells with surface-bound immunoglobulin as judged by immunofluorescence studies. It was therefore concluded that a major proportion of B cells has a relatively low net surface charge while that of the T cells is higher.     

B and T cells in lymphoid tissues of human appendix.

Human appendix lymphoid cells (HAL) react very strongly to stimulation with concanavalin A, strongly to stimulation with phytohaemagglutinin, and weakly but definitely to stimulation with lipopolysaccharide. From the results of rosette formation assay, cytotoxicity tests with anti-T cell antiserum or anti-B cell antiserum, cell surface or intracellular immunoglobulin staining with fluorescein-conjugated rabbit anti-Fab of human immunoglobulin serum, and plaque-forming cell (PFC) assay, it was concluded that human appendix lymphoid tissue is a B cell pool but includes T cells. However, both direct and indirect PFC could not be significantly demonstrated against sheep red blood cells in a 5-day HAL culture.     

Antibody-dependent tumour cytolysis by human neutrophils: effect of synthetic serine esterase inhibitors and substrates.

The requirement for serine esterase activity in antibody-dependent cellular cytotoxicity (ADCC) in human neutrophils against Raji target cells has been investigated. The lysis was prevented when the serine esterase inhibitors TPCK and TLCK (chloromethyl-ketone derivatives of tosylamino acids) were introduced into the system. Moreover, neutrophils pretreated with TPCK or TLCK and washed were inhibited as well, via a process unaffected by the presence of adequate amounts of enzymatic substrates. This suggests that the inhibition mediated by TPCK and TLCK is independent of serine esterase blockade, therefore implying the inactivation of some other step crucial to the lysis. The addition of synthetic chymotrypsin substrates (tyrosine and phenylalanine esters) impaired the Raji cell lysis in a dose-related manner without altering the constitution of neutrophil-target conjugates. Trypsin ester substrates were ineffective. These results are in agreement with the involvement of a serine esterase activity with chymotrypsin-like specificity, which should participate in the lysis at a post-binding step. We conclude that neutrophil-mediated ADCC, as developed in our model system, needs the intervention of a serine esterase or esterases, like other systems of cell-mediated cytotoxicity.     

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.     

Detachment and cytolysis of human endothelial cells by proteinase 3

Activation and degranulation of polymorphonuclear leukocytes (PMN) with release of proteolytic enzymes, such as proteinase 3 (PR3) and elastase, in the vessels of patients with Wegener's granulomatosis (WG) is thought to play an important role in the vascular endothelial cell damage. We have investigated the detachment and cytolysis of ⁵¹Cr-labeled umbilical vein endothelial cells (HUVEC) induced by highly purified, enzymatically active, PR3 and elastase. Incubation of confluent monolayers of HUVEC with 100 mU/ml of PR3 for 3 h at 37°C generally resulted in 20% detachment and 30% cytolysis. Elastase (350 mU/ml) induced approximately 40% detachment and 15% cytolysis. Both PR3-mediated and elastase-mediated detachment and cytolysis were fully inhibited by alpha-1-proteinase inhibitor (α₁PI), while anti-leukoprotease (ALP) only inhibited elastase-mediated endothelial damage. By selective inhibition of an azurophilic granule extract with either α₁PI or ALP we calculated that PR3 is responsible for 23% of the total detachment and cytolysis induced by the extract. Elastase was responsible for 60% of the detachment and 19% of the cytolysis. Detachment induced by PR3 was inhibited by three out of five IgG preparations purified from c-ANCA-positive sera of WG patients. PR3-mediated cytolysis was inhibited by each of the c-ANCA + IgG preparations and also to a limited extent by control IgG, suggesting a partial nonspecific stabilization of the endothelial cells. These studies provide evidence that besides elastase, PR3 also plays an important role in the PMN-mediated endothelial cell damage.     

Assay of immune cytolysis of lymphocytes and tumour cells by automatic determination of cell volume distribution.

Immune cytolysis (lysis) of cells due to the action of antibody in the presence of complement is usually substantiated by the uptake of vital dye by the cells, or by the escape of radiolabel from the cells. Immune cytolysis has now been assayed by determination of cell volume distribution with a Coulter multi-channel particle size analyser used in conjunction with a Coulter counter. For Ehrlich ascites and sarcoma-180 cells, volume degradation corresponding to vital staining was obtained only if trypsin (final concentration 625 microgram/ml) was added immediately after the usual 1 h incubation period for cells, antibody and complement. For L1210 leukaemia cells, trypsin was added at 0 degrees just 1 min before Coulter evaluation, to avoid potentiation of antibody-mediated cell lysis by trypsin. Immune cytolysis of mouse thymic, splenic and lymph node lymphocytes required addition of pronase (final concentration 625 microgram/ml) at 0 degrees for further disruption of antibody-damaged cells, prior to determination of cell volume distribution in the Coulter equipment. Scanning electron micrographs of L1210 cells undergoing immune cytolysis illustrated the changes in cell volume recorded by the Coulter apparatus. This new method for determination of immune cytolysis provides detailed information about the volume distribution of target cells, which permits detection of subtle changes and gives insight into the process of cytolysis. It is not intended to displace other procedures in routine use, except that complete automation of the present method is possible in future.     

CD40 associates with the MHC class II molecules on human B cells

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.     

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. II. The mechanism of null cell participation in immunoglobulin synthesis and secretion by B cells.

Immunoglobulins were synthesized and secreted by human B cells cultured with T cells with receptors for FcM (TM) helper cells, monocytes, null cells and PWM for 7 days. Immunoglobulin synthesis did not take place if the null cells were omitted from the cultures irrespective of the duration of the culture period. Null cells incorporated into the cultures at only 25% of their optimal concentration did not affect immunoglobulin synthesis markedly by the cultured B cells. However, the number of B cells in the culture could not be diluted without an accompanying marked reduction in immunoglobulin synthesis. The B cells synthesized and secreted significant quantities of immunoglobulin even when the null cells were added as late as day 6 of the 7-day culture whereas no or very little immunoglobulin was synthesized if the B cells were not present from the beginning of the 7-day culture. It was demonstrated that cultured null cells do not transform into B cells and do not attain their immunoglobulin-synthesizing function. Furthermore, cultured B cells do not transform into null cells and do not attain their helper function. The null cells can also be distinguished from the B cells on the basis of cell-surface markers, receptors, and blastogenic responsiveness to phytomitogens. It is concluded that (i) the human circulating B cells require the null cells, in addition to the TM cells, monocytes and PWM, in culture in order to synthesize and secrete immunoglobulin; (ii) the null cell signal that stimulates immunoglobulin synthesis and secretion by the B cells is probably the last signal following the TM helper cell, monocyte and PWM signals received by the B cells; and (iii) the null cells and the B cells constitute distinct lineages of cells.     

Activation of human T cells by toxic shock syndrome toxin-1: the toxin-binding structures expressed on human lymphoid cells acting as accessory cells are HLA class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding assay using 125I-labeled TSST-1 showed the presence of specific TSST-1 binding in a B cell fraction of human peripheral blood mononuclear cells and L cells transfected with DR2 genes or DR4 genes but not in a T cell fraction and control L cells. Fixation with paraformaldehyde, an inhibitor of antigen processing, did not remove TSST-1-binding activity of the transfectants. Binding of 125I-labeled TSST-1 to the transfectants was reduced by an anti-DR monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single band with TSST-1-binding activity and the same migration pattern as DR heterodimers. TSST-1-induced T cell responses, proliferation and interleukin 2 (IL2) production were observed in the presence of the transfectants but not in the presence of control L cells, while concanavalin A-induced IL2 production was observed in the presence of either the transfectants or control L cells. Presence of an anti-DR monoclonal antibody inhibited the TSST-1-induced responses. Paraformaldehyde-fixed Daudi cells were effective in supporting TSST-1-induced IL2 production by T cells. These results indicate that HLA class II molecules directly bind intact TSST-1 and perform an essential role as the TSST-1-binding structures on accessory cells in T cell activation by the toxin.     

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. I. Immunoglobulin expression on human lymphoblastoid cells established from various lymphoid cells

Forty-seven lymphoblastoid cell lines were established from human fetal lymphoid tissues, cord blood lymphocytes (CBL) and adult peripheral blood lymphocytes (PBL) by Epstein-Barr virus (EBV) infection. Their surface immunoglobulin (sIg), intracytoplasmic immunoglobulin (cIg) expression, and immunoglobulin (Ig) content in the culture supernatant were tested. Expression of sIgM, sIgG and sIgA were predominant on fetus-derived cell lines, while sIgD was the most prominent on CBL-derived cells. Though cIg expression did not vary between cell lines of different origin, Ig content in the culture supernatant differed greatly. Fetus- and CBL-derived cells secreted IgM exclusively, but PBL-derived cells secreted not only IgM, but also IgG and IgA abundantly. These results indicate that the lymphoblastoid cells established by EBV infection reflect the Ig phenotype of the cell from which they originated.     

Cross-reaction of a monoclonal antibody to human MHC class II molecules with rabbit B cells

Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells. Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype. HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules. Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules. The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells. The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes. Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells. Mesenteric lymph node and spleen cell suspensions, depleted of sIg+ cells, are devoid of 44H10+ cells, while similarly-treated appendix B cells still contain a subset of B cells which are sIg-, RABELA+ and 44H10+. These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.     

Production and regulation of interleukin 6 in human B lymphoid cells

Human B cell lines were screened for production of interleukin 6 (IL 6) by the B9 hybridoma cell bioassay. Some long-established lines such as RPMI 1788, CESS and recently established early-passage Epstein-Barr virus (EBV)-transformed lymphoblastoid lines were constitutive IL 6 producers. Other long-established lymphoblastoid lines such as RPMI 8866 and SKW6.4 and all Burkitt lymphoma lines tested were nonproducers of IL 6. Constitutive production of IL 6 in early passage lines could be enhanced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and recombinant (r)IL 4 but not by rIL 1 alpha or rIL 1 beta. Nonproducing EBV-transformed lymphoblastoid cell lines could be induced to IL 6 production by PMA, rIL 1 alpha, IL 1 beta and IL 4. Among the nonproducing lines SKW6.4 was induced to IL 6 production by PMA, rIL 1 alpha, rIL 1 beta and rIL 4, whereas RPMI 8866 was induced only by PMA, to a limited extent by rIL 1 alpha and rIL 1 beta but not at all by rIL 4. There was no induction of IL 6 by any recombinant cytokine in the two Burkitt lymphoma lines but measurable production of IL 6 was induced by PMA in one of them (EB4). Cytokines which were neither enhancers nor inducers of cell lines on their own included rIL 2, rIL 5, interferon (rIFN)-gamma, native purified IFN-alpha, tumor necrosis factor (rTNF)-alpha, rTNF-beta and purified platelet-derived transforming growth factor-beta. rIL 4 synergized with either rIL 1 alpha or rIL 1 beta in the induction of IL 6 in the nonproducing line SKW6.4. Similar effects were also seen in this line with combinations of (a) rIFN-gamma and rIL 4 and (b) IFN-alpha and both rIL 1 alpha and rIL 1 beta. rIL 4, with or without rIL 1, was more effective than rIL 6 in the induction of IgM synthesis in SKW6.4 and the effect was only partially inhibited by anti-IL 6 antiserum at a dose which totally inhibited IL 6-induced IgM production. Normal peripheral blood lymphocyte populations pre-activated by anti-immunoglobulin rosetting exhibited enhanced production of IL 6 in the presence of rIL 4 and PMA but not in the presence of rIL 1, in contrast to the behavior of adherent mononuclear blood cells which showed IL 4-induced down-regulation of IL 6 production.(ABSTRACT TRUNCATED AT 400 WORDS)     

The induction of human peripheral blood lymphoid colonies by conditioned media from human tumour cell lines.

Conditioned medium (CM) from 29 human tumour cell lines and 3 malignant pleural fluids were tested for their ability to stimulate lymphoid colony formation in semi-solid agar; 9 of 14 malignant melanomas, 3 of 6 colonic carcinomas, 2 of 5 ovarian carcinomas, 3 of 4 breast carcinomas and 1 of 3 pleural fluids from breast cancer patients contained colony-stimulating activity (CSA) for human peripheral blood lymphoid cells (PBL) in semi-solid agar. Conditioned media also stimulated PBL proliferation in liquid medium; these effects were dose dependent. With the exception of one pleural fluid, extensive dialysis of CM did not significantly increase colony formation; CM from two tumour cell lines demonstrated a significant decrease in the induction of colony formation after dialysis.     

Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. II. Separation by velocity sedimentation.

Velocity sedimentation in a zonal rotor using gradients of uniform osmolarity was used to separate leukocyte subpopulations from human blood and bone marrow. The separations were performed at high sedimentation rates having the advantage of rapidity over conventional unit gravity separations. Myeloid stem cells (CFU-C) and cells reactive with phytohemagglutinin (PHA), Concanavalin A (Con A), and in mixed leukocyte culture (MLC) were separated and their sedimentation profiles obtained. CFU-C sedimented ahead of lymphoid cells and behind mature myeloid elements. Two distinct marrow subpopulations separated by velocity sedimentation were consistently stimulated by Con A, and variably stimulated by PHA and MLC reactions. Both large cells (predominantly myeloid) and small cells (predominantly lymphoid) from bone marrow were stimulated by Con A in [3H]thymidine incorporation assays. When separated subpopulations showing stimulation by Con A were mixed, inhibition of [3H]thymidine incorporation resulted.     

Phytol induces programmed cell death in human lymphoid leukemia Molt 4B cells.

The exposure of human lymphoid leukemia Molt 4B cells to phytol which was isolated from Lolium multiflorum Lam and identified by MS, and 1H- and 13C-NMR, led to both growth inhibition and the induction of programmed cell death (apoptosis). Morphological change showing apoptotic bodies was observed in the cells treated with phytol. The fragmentation by phytol of DNA to oligonucleosomal-sized fragments that are characteristics of apoptosis was observed to be concentration- and time-dependent. These findings suggest that growth inhibition by phytol of Molt 4B cells results from the induction of apoptosis in the cells.     

Regulatory B cells protect against chronic hypoxia-induced pulmonary hypertension by modulating the Tfh/Tfr immune balance

Hypoxia-induced pulmonary hypertension (HPH) is a progressive and lethal disease characterized by the uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) and obstructive vascular remodelling. Previous research demonstrated that Breg cells were involved in the pathogenesis of pulmonary hypertension. This work aimed to evaluate the regulatory function of Breg cells in HPH. HPH mice model were established and induced by exposing to chronic hypoxia for 21 days. Mice with HPH were treated with anti-CD22 or adoptive transferred of Breg cells. The coculture systems of Breg cells with CD4⁺ T cells and Breg cells with PASMCs in vitro were constructed. Lung pathology was evaluated by HE staining and immunofluorescence staining. The frequencies of Breg cells, Tfh cells and Tfr cells were analysed by flow cytometry. Serum IL-21 and IL-10 levels were determined by ELISA. Protein levels of Blimp-1, Bcl-6 and CTLA-4 were determined by western blot and RT-PCR. Proliferation rate of PASMCs was measured by EdU. Compared to the control group, mean PAP, RV/(LV + S) ratio, WA% and WT% were significantly increased in the model group. Anti-CD22 exacerbated abnormal hemodynamics, pulmonary vascular remodelling and right ventricle hypertrophy in HPH, which ameliorated by adoptive transfer of Breg cells into HPH mice. The proportion of Breg cells on day 7 induced by chronic hypoxia was significantly higher than control group, which significantly decreased on day 14 and day 21. The percentage of Tfh cells was significantly increased, while percentage of Tfr cells was significantly decreased in HPH than those of control group. Anti-CD22 treatment increased the percentage of Tfh cells and decreased the percentage of Tfr cells in HPH mice. However, Breg cells restrained the Tfh cells differentiation and expanded Tfr cells differentiation in vivo and in vitro. Additionally, Breg cells inhibited the proliferation of PASMCs under hypoxic condition in vitro. Collectively, these findings suggested that Breg cells may be a new therapeutic target for modulating the Tfh/Tfr immune balance in HPH.