Effects of tunicamycin and various monosaccharides on phytohaemagglutinin-induced autorosette formation

The structural characteristics of autologous red blood cell (ARBC) receptors on human peripheral blood lymphocytes (PBL) induced by phytohaemagglutinin (PHA) were examined. Tunicamycin, which is known to be a blocker of the protein glycosylation of N-glycosidic type glycoprotein, significantly inhibited the production of ARBC receptors. When trypsinized PBL were cultured in the presence of tunicamycin, the inhibitory effect was enhanced. When several monosaccharides were added to the mixture of ARBC and PBL pre-treated with PHA for 24 h, D-mannose caused the most inhibition, and galactose and D-fucose also caused significant inhibition. These data suggest that N-glycosidic type glycoproteins play an important role in binding sites to ARBC on PBL surface membranes and that more than one type of glycose may be involved.     

Effect of dexamethasone on RNA synthesis in human peripheral blood lymphocytes

The action of dexamethasone on lymphoid tissue in a culture of partially purified peripheral blood lymphocytes was biphasic in character. After incubation of lymphocytes in vitro with the hormone for 6 h stimulation of RNA synthesis was found. Sedimentation analysis of labeled RNA fractionated on a column containing poly-V-sepharose indicated an increase in the mRNA content and enrichment of cytoplasmic RNA with polyA sequences. Meanwhile Mn⁺⁺-dependent RNA-polymerase, sensitive to -amanitine, was activated. After cultivation of the lymphocytes with the hormone for 24 h, RNA synthesis was inhibited. The biphasic character of the action of the steroid also was observed in the rosette-formation test.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 811–814, July, 1976.     

Sex hormones enhance immunoglobulin synthesis by human peripheral blood lymphocytes

Using a protein A haemolytic plaque assay to detect immunoglobulin synthesis by human peripheral blood lymphocytes cultured with pokeweed mitogen, we have shown a significant increase in IgG plaque-forming cells in the presence of physiological concentrations of testosterone and oestradiol. Higher concentrations of these steroids inhibit plaque-forming cells. There was no difference in the response to testosterone and oestradiol of lymphocytes derived from male subjects or menstruating or post-menopausal females.     

Rosette formation by peripheral lymphocytes. II. Inhibition of the phenomenon

In a previous paper it had been shown that up to 40% of human peripheral lymphocytes will make rosettes with sheep red cells. In this study the inhibitory effect of antilymphocyte serum (ALS) and of certain other preparations on this rosette phenomenon is described. ALS was prepared in horses by injection of human peripheral lymphocytes and rosette-inhibiting, cytotoxic and agglutinating titres were determined during immunization. The ALS had been tested in vivo by regional administration to vervet monkeys bearing skin allografts. It was found that the peak of rosette-inhibiting titre appeared to provide a good index of immunosuppressive activity. These peaks, and the accompanying periods of immunosuppressive activity, were of short duration, in contrast to the levels of cytotoxic and agglutinating titres. Antihuman lymphocyte sera had considerable rosette-inhibiting titres against the lymphocytes of the baboon (Papio papio) but little against those of the vervet monkey (Cercopithecus aethiops).

Inhibition by ALS of the phenomenon described by us is unaffected by complement. Antihuman globulin serum and phytohaemagglutinin had no inhibitory effect. Digestion of ALS with pepsin to obtain the F (ab')₂ fragment had only a slight effect on rosette-inhibiting titres, while it reduced the cytotoxic activity very considerably.

The phenomenon described by us is probably different from that reported by other workers, in which only a small proportion of lymphocytes form rosettes.

     

Induction of T cell growth factor synthesis in human peripheral blood lymphocytes by staphylococcal protein A

Staphylococcal Protein A (SpA) induces T cell growth factor (TCGF) production in cultures of human peripheral blood mononuclear cells (PBMC) with titers equivalent to those induced by PHA. The optimal time and cell concentration for TCGF production were found to be the same for SpA and PHA. TCGF induction by SpA was blocked by addition of human AB-serum or human IgG, as was the mitogenic effect of SpA. An easy and inexpensive procedure is described for the quantitative removal of SpA from TCGF without loss of TCGF activity.     

Phorbol myristate acetate inhibits immunoglobulin synthesis by human peripheral blood lymphocytes

Phorbol myristate acetate (PMA), a known lymphocyte mitogen, has been shown to inhibit immunoglobulin synthesis by human peripheral blood lymphocytes. This effect occurs with low concentrations of the agent and following only 12 h contact with the lymphocytes. PMA was also capable of preventing pokeweed mitogen stimulation of lymphocytes. These experiments suggest that PMA prevents the complete differentiation of B-cells into plasma cells capable of secreting immunoglobulin.     

T-cell independence of immunoglobulin synthesis by human peripheral blood lymphocytes stimulated with SpA-containing staphylococci.

Unfractionated and T-cell depleted human peripheral blood lymphocytes (PBL) were cultured in vitro in the presence of pokeweed mitogen (PWM) and Staphylococcus aureus strain Cowan I (StaCw). After 7 days of culture, the cells were assayed for cytoplasmic immunoglobulins (Cyto-Ig) by direct staining using fluorescein-labelled F(ab'')2 fragments prepared from specific antisera against human IgG F(ab'')2. The amount of immunoglobulin of the IgM and IgG class released into the cell-free supernatants was also measured by radioimmunoassay. In unfractionated PBL StaCw, like PWM, was able to induce a significant increase of either the number of Cyto-Ig containing cells for the amount of IgM and IgG secreted into the supernatant. In contrast, the amount of IgM and IgG immunoglobulin released into the supernatant of T-cell depleted suspensions stimulated with PWM was significantly reduced in comparison with that of unfractionated populations, whereas it was unchanged in T-cell depleted vs unfractionated suspensions stimulated with StaCw. The addition of a few T lymphocytes restored the ability of T-cell depleted suspensions to produce Ig in the presence of PWM, whereas despite addition of high numbers of T cells no further augmentation of the Ig production induced by StaCw on T-cell depleted suspensions was observed. Cultures of umbilical cord blood lymphocytes (UCBL) stimulated with PWM did not generate Ig-producing cells, whereas UCBL stimulated with StaCw showed significant production of Ig of both IgM and IgG classes. The results indicate that T lymphocytes are probably not involved either with stimulation or with the suppression of Ig production induced by StaCw.     

Binding of C-reactive protein (CRP) by human peripheral blood lymphocytes in vivo.

Antigenicity of C-reactive protein (CRP) on the surface of human lymphocytes was investigated by use of indirect immunofluorescence technique with anti-CRP antibodies. CRP on the lymphocyte surface (sd-CRP) belongs to two different categories: i) CRP produced by lymphocytes and inserted into cell membrane (s-CRP), ii) CRP produced primarily by the liver and bound by the lymphocytes (sb-CRP) in calcium-dependent manner. In human peripheral blood of healthy donors approximately 2.5% of lymphocytes expressed membrane CRP (s-CRP) and 1.5% of lymphocytes bound CRP in calcium-dependent manner (sb-CRP). Percentage of s-CRP lymphocytes increased in patients with rheumatoid arthritis, while population of sb-CRP lymphocytes did not change significantly, except cases where serum CRP concentration reached more than 50 micrograms/ml. Thus, it can be concluded that CRP is bound to the distinct population of lymphocytes, bearing specific membrane receptors.     

Differentiation of human peripheral blood B lymphocytes.

Human B-lymphocyte differentiation was studied by measuring the capacity of such cells, isolated from peripheral blood, to synthesize and secrete Ig after pokeweed stimulation. Results show that a maximum incorporation of [3H]-thymidine took place 2 days before the appearance of detectable Ig-secreting cells. On the 7th day after pokeweed stimulation, when Ig synthesis and secretion are at a maximum, [3H]-thymidine uptake was low. Since inhibition of DNA synthesis 3 days after pokeweed stimulation completely prevents the generation of Ig-secreting plasma cells, initial DNA synthesis is apparently essential before Ig-secreting plasma cells can develop in response to pokeweed stimulation.     

Stimulation of human peripheral blood lymphocytes by Tamm-Horsfall urinary glycoprotein.

Tamm-Horsfall urinary glycoprotein (THP), prepared by salt precipitation of pooled urine from normal individuals, stimulated purified human peripheral blood lymphocytes (PBL) to undergo blastoid transformation. the response was measured by tritiated thymidine uptake into DNA after 6 days in culture. Several batches of THP stimulated, in varying degrees, all samples of PBL tested and the response approached that seen with the mitogens phytohaemagglutinin (PHA), Concanavalin A (Con A) and pokeweed mitogen (PWM) after 4 days in culture. The response usually exceeded that seen after 6 days with tuberculin purified protein derivative (PPD) in Mantoux positive lymphocyte donors.     

Suppression of generation of human cytotoxic effectors by lectins or lectin-activated peripheral blood lymphocytes

The lectins phytohemagglutinin, pokeweed mitogen and concanavalin A used at their optimal mitogenic concentration, or human lymphocytes activated by the same mitogens, were found to suppress the in vitro generation of cytotoxic effectors when added to a cell-mediated lympholysis (CML) mixture during the first 48 h of culture. The data suggest that the suppressive mechanism is mediated to a greater extent by an allogeneic interaction between lectin-activated cells and the allogeneic cells present in the CML mixture than by suppressor cells induced by the lectin. Since partial suppression was observed with supernatants of activated lymphocytes cultured for 18 h with allogeneic stimulating cells (but not activated lymphocytes alone), a soluble mediator may be involved in the suppressive mechanism. The mechanism of suppression therefore may be identical to the preemption phenomenon recently described in primary and secondary CML.     

Regulation of p26-bcl-2 protein levels in human peripheral blood lymphocytes.

BACKGROUND: The protein encoded by the bcl-2 (B cell lymphoma/leukemia-2) proto-oncogene has been implicated in the regulation of lymphocyte cell survival and has been shown to interfere with programmed cell death (apoptosis). Recently, controversy has emerged regarding the expression of bcl-2 in circulating peripheral blood lymphocytes (PBL) and its correlation with lymphocyte proliferation. Using immunohistochemical methods, Pezzella et al. (Pezzella F, Tse G, Cordell J, Pulford K, Gatter K, Mason D. Am J Pathol 1990; 137:225-32) detected abundant amounts of Bcl-2 protein in resting PBLs and observed an inverse correlation between bcl-2 expression and cellular proliferation in lymphoid tissues. In contrast, previous in vitro studies of bcl-2 mRNAs showed that expression of this gene is very low in unstimulated PBLs but can be markedly induced when lymphocytes are stimulated to proliferate in culture (Reed JC, Tsujimoto Y, Alpers JD, Croce CM, Nowell PC. Science 1987; 236:1295-9; Granniger W, Seto M, Boutain B, Goldman P, Korsmeyer S. J Clin Invest 1987; 80:1512-5). EXPERIMENTAL DESIGN: To resolve this issue, the regulation of p26-Bcl-2 protein levels was examined in freshly isolated and in cultured human PBLs by immunoblotting using two different polyclonal antisera specific for the human Bcl-2 protein. RESULTS: When freshly isolated from whole blood, resting PBLs contained easily detectable amounts of p26-Bcl-2 protein that did not significantly change in culture when cellular proliferation was stimulated with mitogenic lectins and lymphokines. If processing of the blood was delayed, however, p26-Bcl-2 protein was low or undetectable in resting PBLs and underwent marked increases in its relative levels after in vitro stimulation of PBLs by mitogenic lectins and lymphokines. CONCLUSIONS: The findings indicate that bcl-2 is normally expressed in quiescent circulating lymphocytes, consistent with a role for this gene in the maintenance of lymphocyte survival, and illustrate the importance of correlating in vitro and in vivo expression data.     

Polyclonal activation of human peripheral blood B lymphocytes by Fusobacterium nucleatum.

The present study examined in vitro polyclonal human B-lymphocyte (B-cell) activation (PBA) by Fusobacterium nucleatum. Pokeweed mitogen, a well-studied PBA activator, was included in some experiments for comparison. PBA was determined by the total immunoglobulin A, G, and M concentrations in the culture supernatants as measured by micro-enzyme-linked immunosorbent assay. F. nucleatum, at concentrations between 1 and 10 micrograms/ml, stimulated optimal PBA in monocyte-depleted cultures, whereas the pokeweed mitogen response was optimal in unfractionated, monocyte-containing cultures. Immunoglobulin synthesis occurred primarily between days 6 and 8 after stimulation with F. nucleatum. T lymphocytes enhanced the PBA response to F. nucleatum, particularly at a T- to B-cell ratio of 1:1. Immunoglobulin production was greater in round-bottomed wells than in flat-bottomed wells at lymphocyte concentrations of 200,000 cells per well. The PBA response, however, increased dramatically in flat-bottomed wells containing higher lymphocyte concentrations, suggesting that PBA is enhanced by cell-to-cell contact. A delay in stimulation of the lymphocytes with F. nucleatum resulted in diminished immunoglobulin production. The results provide information on the regulation of in vitro PBA induced by F. nucleatum. The data also suggest that there may be differences in the mechanisms by which F. nucleatum and pokeweed mitogen stimulate PBA.     

Elevated autorosette formation by lymphocytes of dogs affected with cyclic neutropenia (CN)

Cyclic neutropenia (CN) is an inherited disorder characterized by regularly recurring episodes of neutropenia in humans and Gray Collie dogs. Early thymic hypotrophy and lymphoid exhaustion in the CN dogs suggests there may be a lymphoid cell differentiation defect. Later manifestations of CN in dogs include arthritis, anaemia, glomerulone-phritis, and amyloidosis which are often associated with autoimmune diseases. Several Coombs' antiglobulin tests were performed and did not detect autoantibodies, however, peripheral blood lymphocytes from CN dogs formed rosettes with their own erythrocytes while in normal dogs such rosettes were extremely rare. Furthermore, when lymphocytes from CN dogs were rosetted with erythrocytes from normal dogs, the numbers of allogeneic rosettes were comparable to those formed with autologous erythrocytes. These results suggest strongly that the rosetting lymphocytes are specific for common erythrocyte surface components. Although the physiological importance of the autorosetting phenomenon is not known, the frequency of autorosette formation in CN dogs, as reported here, suggests that it may be an early indication of developing autoimmune activity.     

Inhibition of T-lymphocyte colony formation by inhibitors of mitochondrial protein synthesis.

A double layer technique which requires an underlayer of peripheral blood leucocytes, in addition to phytohaemmagglutinin (PHA) in the overlayer, to obtain good T-lymphocyte colony formation, was used to assess the effect of two inhibitors of mitochondrial protein synthesis, chloramphenicol and ethidium bromide. A significant inhibition of the colony growth of peripheral blood T lymphocytes was observed when either of the drugs was incorporated in the underlayer. The inhibitory effect was always smaller (c. 50%) when the drugs were added to the overlayer. These findings point to the existence of a T-lymphocyte colony stimulating factor(s), released mainly by the leucocyte-rich underlayer and essential for T-colony formation, the production of which is inhibited by these mitochonrial inhibitors.     

Rosette formation by peripheral lymphocytes

In preparations of human peripheral lymphocytes suspended in serum absorbed with sheep red cells, up to 30% of the lymphocytes may make rosettes with sheep erythrocytes.

Washed lymphocytes suspended in Hanks' solution make many rosettes if tested without delay. Such lymphocytes rapidly lose their capacity to make rosettes, but it can be restored by adding the serum of man or of the horse, rabbit or guinea-pig. The lymphocytes of three newborn babies, and of one adult who had no detectable antisheep agglutinin in the serum, made rosettes with sheep cells.

Rosette formation is uncorrelated with serum agglutinin levels. Many normal adults have far higher titres of agglutinins against the red cells of other animals than against sheep cells; yet their lymphocytes do not make rosettes with the cells of these other animals.

Sodium cyanide (0·01 M) abolished rosette formation, and horse antihuman lymphocyte globulin inhibits it.

It is concluded that sheep cell rosette formation by human peripheral lymphocytes is not due to humoral antibody or delayed hypersensitivity, because of the great proportion of lymphocytes that are capable of it. Its nature is obscure, but it is suggested that it may be due to a substance, not primarily an antibody, that is elaborated by a large proportion of circulating lymphocytes and cross-reacts with some red cell antigens as plant lectins do.

Caution is advised in using the system to test antihuman lymphocyte serum until more is known about it.

     

Rosette formation of pig peripheral blood lymphocytes with sheep red blood cells.

Peripheral blood lymphocytes of normal young pigs formed spontaneous and EAC rosettes with sheep red blood cells. Pretreatment of the SRBC with a sulphydryl reagent increased the percentage of spontaneous rosettes and the number of SRBC bound by the lymphocytes. The influence of an incubation period prior to centrifugation and the duration and temperature of the following incubation were tested for spontaneous and EAC rosettes. The importance is stressed to define the number of bound SRBC to call a lymphocyte a "rosette-forming cell". Rosette formation is interpreted as a quantitative rather than a qualitative marker.     

Expression of a novel protein by regenerating hepatocytes and peripheral blood lymphocytes.

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.