Synthesis and secretion of alpha 1-microglobulin by human lymphocytes.

alpha 1-Microglobulin (alpha 1-m) was purified by column chromatography from a supernatant fluid of cultured T and B lymphocytes stimulated with mitogens. This protein had a molecular weight of 33,000 as determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, migrated in the alpha 1-region on immunoelectrophoresis and proved immunologically identical to alpha 1-m which had been purified from the urine of patients with renal tubular disorders. Using indirect immunofluorescence, alpha 1-m was detected on the surface of both T and B lymphocytes, displaying the same intensity on each cell type. These findings indicate that alpha 1-m is actively produced and secreted by T and B lymphocytes.     

Human recombinant interleukin-2 is mitogenic to human lymphocytes

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.     

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.     

Corticosteroids decrease the expression of beta 2-microglobulin and histocompatibility antigens on human peripheral blood lymphocytes in vitro.

The in vitro effect of two different glucocorticoids (prednisolone and dexamethasone) on the expression of beta 2-microglobulin and HLA-A-A, -B and -C-antigens on the surface of cultured lymphocytes was measured by quantitative immunofluorescence (flow cytofluorometry) and by a radioimmunoassay. Both antigens were found to be decreased, dexamethasone typically in a concentration of 10-6 mol/l causing a decrease in surface beta 2-microglobulin of 15% after an incubation period of 24 hr. The expression of two other lymphocyte surface antigens, Igm and Thy antigens, measured in parallel with beta 2-microglobulin and HLA antigens, was not affected by the same culture conditions. The steroid effect was not due to masking of the affected antigens, but was completely abolished by inhibition of protein synthesis.     

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.     

Genetic differences in the mitogenic response of peripheral blood lymphocytes in the chicken.

The mitogenic responses of chicken peripheral blood lymphocytes were compared in two partially inbred chicken lines and in their hybrids. The lymphocytes of line P chickens showed a higher phytohaemagglutinin (PHA) response than those of line V chickens and (P X V)F1 hybrids. The Con A responses were of the same magnitude in all chicken lines. The higher PHA response in line P chickens was not sex-linked, apparently was not associated with the major histocompatibility complex and it did not correlate with immunoglobulin G allotypes.     

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.     

Limulin (Limulus polyphemus lectin): mitogenic effect on human peripheral lymphocytes.

The lectin from hemolymph of Limulus polyphemus was purified by affinity chromatography on insolubilized bovine submaxillary mucin. The purity of the protein was checked by crossed immunoelectrophoresis. Agglutination of human red blood cells was completely abolished after neuraminidase treatment, while other cells were still agglutinable after the same treatment but required a higher concentration of lectin. Limulin was able to simulate about 50% of human peripheral lymphocytes. This mitogenic effect could be inhibited by bovine submaxillary mucin but not by the disialylated mucin. Related to the known oligosaccharide-binding specificity of limulin and of the other nonspecific activators of lymphocytes, the authors suggest that lymphocyte stimulation is triggered by binding to a glyco-conjugate bearing the following carbohydrate chains: NANA leads to GalNac leads to or NANA leads to Gal leads to GlcNAc leads to Man.     

Suppression of in vitro antibody response of human peripheral blood lymphocytes by a heat-labile factor in normal human serum.

When fresh autologous serum was added to normal human peripheral blood lymphocytes (PBL), it suppressed greater than 90% of the in vitro anti-SRBC response of these cells. Heating the serum for 30 min at 56 degrees C reversed this suppression, Serum from a patient with rheumatoid arthritis and circulating immune complexes had no suppressive effect on the anti-SRBC response of normal human PBL, but serum from patients having the same disease, without circulating immune complexes, did suppress over 90% of the plaque-forming cell response. Serum from an agammaglobulinaemic patient was also suppressive. Addition of serum from patients with congenital deficiencies of C2, C3, C5 and C8 also has a suppressive effect. Absorption of normal serum with immune complexes markedly decreased levels of C1 and C4, and also reversed the suppressive effect of this serum. These data suggest that a heat-labile factor in normal human serum which can be absorbed by immune complexes suppresses the antibody response to a T-dependent antigen. Other immune suppressors found in normal human serum are heat-stable or do not suppress in the presence of normal serum proteins. Thus the suppressive protein described in these studies may be unique. It is possible that either C1 or C4 or both may play a role in the suppression noted here.     

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.     

Suppressed peripheral blood lymphocyte blastogenesis in pre- and postpartal sheep by chronic heat-stress, and suppressive property of heat-stressed sheep serum on lymphocytes

Phytohemagglutinin (PHA) and concanavalin A (Con A)-induced blastogenesis of peripheral blood lymphocytes was examined in heat-stressed pre- and postpartal sheep. The peak responses of lymphocytes to PHA and Con A in heat-stressed sheep revealed significant reduction before and after parturition compared with those in the corresponding control animals kept under thermoneutral conditions. Furthermore, the effect of serum from control or heat-stressed sheep on PHA-induced lymphocyte blastogenesis was examined. Supplementation of serum from heat-stressed sheep significantly suppressed the blastogenesis of lymphocytes obtained from healthy sheep, bovine, and human donors. Unlike dexamethasone, heat-stressed sheep serum did not inhibit IL-2 production by PHA-stimulated human peripheral blood lymphocytes. These results indicate that the immunosuppression of heat-stressed sheep is in part mediated by serum factor(s) that can modulate T-cell function in a species nonspecific manner.     

Spontaneous thyroglobulin autoantibody production by peripheral blood lymphocytes is regulated by T cells and is augmented by soluble T cell factors

In this study we report the spontaneous production of autoimmune anti-thyroglobulin antibodies (Tg Ab) by peripheral blood lymphocytes from patients with autoimmune thyroid disease and the use of these cells to study the regulation of this production in vitro. Using a mitogen free microculture system we have found a significant correlation between an individuals' serum titre and the amount of Tg Ab produced in vitro. At low B:T cell ratios Tg Ab production decreased. Specific depletion of suppressor T cells and helper T cells was accomplished using the monoclonal antibodies OKT 8 and OKT 4 respectively. Suppressor cell depletions resulted in increases in Tg Ab production while depletion of helper cells had no consistent effect. The addition of pokeweed mitogen had no stimulatory effect on spontaneous thyroglobulin antibody production. In contrast, when T cell conditioned media was added to the cells, Tg Ab production increased substantially in all patient cultures tested but not in normal control cultures. The results of this study are consistent with the existence of functional thyroid-antigen specific suppressor cells in the peripheral blood which are actively involved in the regulation of autoantibody producing B cells. The results obtained with the cultures maintained in conditioned media show that autoimmune lymphocytes are extremely sensitive to one or more stimulatory factors present in the media and suggests an important role for the production of soluble lymphocyte factors and the expression of receptors for these factors in the aetiology of autoimmune thyroid disease.     

Characterization of lymphocytes which inhibit proliferation of human T cells exposed to lymphocyte-derived mitogenic factors.

Culture supernatants of PHA-activated human lymphocytes (active SUPs) contain factors which are strongly mitogenic for fractionated peripheral T cells. This stimulation is suppressed by certain non-T lymphocytes. It is shown that these suppressor cells can inhibit an ongoing response of T cells to active SUP and that this inhibition is reversible. Using various rosette sedimentation techniques for fractionating subpopulations of lymphocytes it is concluded that the suppressor cells lack membrane-associated receptors for C3 but possess receptors for the Fc part of IgG. This subset of lymphocytes may be an important regulator of lymphocyte proliferation during immune responses.     

Formation of auto-rosettes by peripheral blood lymphocytes.

A mean of 3-4% (0-5-19-5%) of the peripheral blood lymphocytes of normal adults were shown to bind three or more autologous erythrocytes in vitro to form auto-rosettes. Marked individual fluctuations were observed. Increased percentages were observed in patients with cancer, but not in other selected groups, including a group who had undergone thymectomy for myasthenia gravis. Auto-rosette formation was shown to be a property of T cells by the demonstration of (a) simultaneous binding of autologous and sheep erythrocytes, (b) non-inhibition of auto-rosette formation by anti-immunoglobulin, and (c) formation of auto-rosettes by mitogen-stimulated T blasts. Auto rosette formation is a property of high percentages of human thymocytes, and of lymphocytes treated with neuraminidase or stimulated to blast-cell trans-formation by phytomitogens. It is suggested that auto-rosette formation by these cells is related to their relatively low content of cell-coat sialic acid, as compared with untreated T lymphocytes. The possible influence of the cell coat on lymphocyte function is discussed briefly.     

Avidin-binding to peripheral blood B lymphocytes and monocytes.

Avidin-coated magnetic beads bind peripheral blood B lymphocytes and monocytes. This unwanted reactivity is not due to the membrane expression of avidin target molecules since beads coated with a biotin-binding analogue are non-reactive and binding occurs even when cellular carbohydrate-binding sites are not active, in the absence of Mg2+ and Ca2+ cations, or when they are blocked by a alpha-D-glucose or alpha-D-mannose in presence of Ca2+ and Mg2+. The non-polar residues of avidin appear not to be engaged in a hydrophobic bond with the membrane molecule since suroptimal quantities of serum albumin do not prevent the avidin binding. It is suggested that ionic interactions explain the binding of avidin-coated beads to B lymphocytes and monocytes and that these can be inhibited with high molecular weight serum molecules or with 0.4 M NaCl.     

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.     

Activation of human peripheral blood lymphocytes by concanavalin A dependence of monocytes.

The activation of human peripheral blood lymphocytes or isolated T lymphocytes by concanavalin A (Con A) is hightly potentiated by the presence of autologous, mitomycin C-treated monocytes. The optimal lymphocyte: monocyte ratio within a broad dose range is 1:1 when the incorporation of [14C]thymidine is expressed as total incorporation per culture tube and 1:10 when expressed per lymphocyte. A five-to-ten-fold increase of total DNA synthesis is noted in the presence of 10-90% monocytes. The data may help to explain the wide variations in Con A responsiveness of human peripheral lymphocytes which may be partly related to differences in purification which give rise to cell preparations containing varying amounts of monocytes.     

Cell-mediated immune response to bacterial products in human tonsils and peripheral blood lymphocytes.

Lymphoproliferative responses of tonsillar tissue lymphocytes and peripheral blood lymphocytes to phytohemagglutinin and specific bacterial product antigens were studied in children undergoing tonsillectomy and adenoidectomy. Tonsillar tissue lymphocytes responded to optimal concentrations of phytohemagglutinin. Varidase, and streptolysin-O in a manner similar to peripheral blood lymphocytes. Higher base-line mitogenic activity in tonsillar lymphocytes was frequently associated with the presence of Staphylococcus aureus in the tonsils. Tonsillar tissue lymphocytes from 23% of the subjects with the highest base-line mitogenic activity manifested a decreased response to in vitro stimulation with mitogens or antigens. In subjects with such preactivated tonsillar lymphocytes, the proliferative responsiveness of blood lymphocytes to mitogen and antigens was markedly increased after tonsillectomy and adenoidectomy. These observations suggest the existence of in vitro correlates of cellular immunity to bacterial products in the mucosal surfaces. In addition, it is proposed that tonsils may possess immunosuppressive activity for peripheral blood lymphocytes, which may be related to local tonsillar infections.