Peripheral Blood Mononuclear Cell Preparations Research Articles

Induction of thyroid autoantibody production: synergistic effect of B cell mitogen combined with T cell mitogen.

In vitro production of thyroglobulin autoantibodies (TgAb) and thyroid microsomal autoantibodies (McAb) by peripheral blood mononuclear cells (PBMC) stimulated with the B cell mitogen Staphylococcus aureus Cowan I (SAC) and the T cell mitogen pokeweed mitogen (PWM) was examined in 35 normal subjects (NC) and 64 patients with autoimmune thyroid disease (AITD) using an enzyme-linked immunosorbent assay technique. Low concentrations of SAC plus PWM resulted in a synergistic effect on thyroid autoantibody production as well as nonspecific immunoglobulin G production. With such maximal stimulation, TgAb production was detected in all PBMC preparations from serum TgAb-positive patients with AITD; TgAb production was also detected in some NC (46%) and serum TgAb-negative patients with AITD (39%), but the levels of TgAb production were low. Similarly, McAb production was marked in PBMC preparations from serum TgAb-negative but McAb-positive patients. TgAb-secreting cells were also detected in NC by the plaque-forming cell (PFC) assay. The response patterns of PBMC to mitogen (Nil, PWM, and SAC plus PWM) in terms of TgAb production varied among serum TgAb-positive patients with AITD, but not among NC and serum TgAb-negative patients with AITD. Serum TgAb titers were significantly correlated with the in vitro production of TgAb by PBMC with no stimulation (r = 0.64; n = 99; P less than 0.001), with stimulation by PWM (r = 0.75), and with stimulation by SAC plus PWM (r = 0.87); the correlation coefficient increased with the efficiency of stimulation of B cell differentiation. Similar results were found for McAb production. These data suggest that 1) optimal in vitro thyroid autoantibody production occurs with B cell mitogen (SAC) acting synergistically with T cell mitogen (PWM); 2) sufficient numbers of resting B lymphocytes specific for Tg or microsomal antigens are present in some NC PBMC; 3) stages of thyroid-specific B cell differentiation in PBMC vary among serum thyroid autoantibody-positive patients with AITD; and 4) the potential of PBMC to produce thyroid autoantibodies may correlate with the capacity of thyroid-derived lymphocytes. Thus, the circulating lymphocytes may provide a useful vehicle by which sequential changes occurring at the tissue level may be examined.

Rat Monocytes in a Model of Combined Injury Express the OX8 Antigen

We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) [whole-body irradiation (500 cGy 60Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)] for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.

Studies on the mechanism of cortisol inhibition of human natural killer cell activity: Effects of calcium entry blockers and calmodulin antagonists

The role of Ca 2+ in mediating the inhibition by glucocorticoids of human natural killer (NK) activity was investigated using Ca 2+ entry blockers (verapamil and its desmethoxy-derivatives LU46973 and LU47093) and calmodulin antagonists (pimozide and two naphthalenesulfonamide derivatives, W-7 and W-13). Peripheral blood mononuclear (PBM) cell preparations were incubated for 20 h with 1×10 −6 M Cortisol and these agents in various combinations (concentration range: 1×10 −7 − 1×10 −5 M) and then assayed in a direct 4-h cytolytic assay using 51Cr-labeled K 562 target cells. Exposure to cortisol led to a significant reduction of NK cell activity (about 50% with respect to the spontaneous activity). Ca 2+ entry blockers displayed per se a dose-dependent depressive effect on cytotoxicity and gave significant enhancement of cortisol-dependent inhibition. Calmodulin antagonists were per se minimally effective but clearly amplified the cortisol-mediated inhibition. Raising extracellular Ca 2+ by CaCl 2 or intracellular Ca 2+ by the ionophore A23187 yelded an appreciable reduction of these effects. Our data are compatible with the view that extracellular and intracellular Ca 2+ play a role in the control of human NK cell activity. Moreover, it is conceivable that the mechanisms involved in glucocorticoid inhibition of NK cell activity involve Ca 2+-dependent pathways.

Antigen-induced and polyclonal B-cell responses in human peripheral blood lymphocyte cultures

This work was designed to delineate the anti-hapten antibody (Ab) response induced by trinitrophenol-polyacrylamide (TNP-PAA) beads from the nonspecific B-cell response which concomitantly occurs in human peripheral blood mononuclear cell (PBMC) cultures. Indeed human PBMC produce consistent amounts of immunoglobulins when cultured at high cell density in the presence of fetal bovine serum, regardless of the presence of antigen. In contrast, the stimulation of such cultures by TNP-PAA leads to an Ab response characterized by the following: cells secreting anti-hapten Ab at a high rate (detected by a plaque-forming cel (PFC) assay); a 10–30 times enhancement in the number of hapten-specific binding cells (detected by a rosetteforming cell (RFC) assay); the production of anti-TNP IgM Ab (detected by an ELISA assay). The anti-TNP response is specifically triggered by the particulate antigen, as shown by the following: (i) The TNP-PAA antigen induces a clear-cut increase in the amount of anti-TNP Ab whereas it only marginally increases that of total IgM. (ii) The anti-TNP Ab response is specifically abolished when anti-TNP RFC are depleted from the PBMC preparation before the initiation of the cultures. (iii) The anti-TNP Ab response is specifically abolished when PBMC are triggered by TNP-PAA in the concomitant presence of a soluble TNP-protein conjugate. These results demonstrate the ability of polymeric antigens to specifically activate human peripheral blood B cells.

The effects of adherent cells on measurement of the hyper-responsiveness of rheumatoid B lymphocytes to Epstein-Barr virus

Rheumatoid peripheral blood mononuclear cells show an increased responsiveness to superinfection with Epstein-Barr virus (EBV). We have investigated the role of adherent cells in this hyperresponsiveness using two different methods of adherent cell depletion. Depletion of adherent cells from both rheumatoid and normal mononuclear cells, using either dextran bead columns or plastic petri dishes, produced inconsistent changes in the response of autologous non-adherent cells to EBV. The addition of supernatants of cultured rheumatoid adherent cells also produced an inconsistent change in response although normal adherent cell supernatants increased the responsiveness of autologous non-adherent cells to EBV. The inconsistencies observed are discussed with respect to adherent cell subpopulations present in rheumatoid and normal peripheral blood mononuclear cell preparations when using recognized methods of adherent cell depletion.

Increased natural killer cell activity in sarcoidosis.

We have studied NK cell activity in 32 patients with sarcoidosis and in 29 control subjects. Cytotoxicity was increased in both active and inactive sarcoids, the lytic activity of sarcoid peripheral blood mononuclear cells (PBMC) being approximately 50% higher than that of control subjects. The number of lymphocytes bearing Fc receptors for IgG (FcR-IgG) was correlated with NK cell activity, both parameters being increased in the sarcoid group. Sarcoid NK cell activity was reduced to control levels after depletion of plastic adherent cells, whilst inhibition of cytotoxicity by PGE2 was increased using non-adherent sarcoid PBMC. Plastic depletion had no effect on NK cell activity or its inhibition by PGE2 using PBMC from control subjects. The results demonstrate the presence of a weakly adherent, PGE2 insensitive NK cell in sarcoid PBMC preparations.

The formation of surface membrane vesicles from schistosomula of Schistosoma mansoni.

Intact surface membrane vesicles were obtained and purified from the schistosomula of Schistosoma mansoni. The method of preparation of the vesicles involved treating the schistosomula in a buffered high salt medium for 1 h at 4 degrees C (Step 1), and then for 1 h at 25-37 degrees C (Step 2). Vesicles were formed from mechanically- and skin-transformed schistosomula, and their size and number depended on the temperature of Step 2. The majority of the vesicles expressed surface membrane concanavalin A receptors and parasite antigens on their outermost surfaces. When incubated with sensitized peripheral blood mononuclear cell preparations from humans exposed to schistosomal preparations, the vesicles stimulated lymphocyte transformation as effectively as soluble egg and adult worm antigens. Although both preparations contained identical proteins, small vesicles were less effective than large vesicles in stimulating lymphocyte transformation. Hence, vesicles prepared under a variety of conditions might be used to study those factors which influence the presentation of membrane-bound surface antigens to the immune system of the host.

Changes in purine salvage pathway enzyme activities during human lymphocyte differentiation induced by thymosin fraction 5.

Adenosine deaminase (ADA) and purine nucleoside Phosphorylase(PNP), two purine salvage pathway enzymes, are essential to normal immune function. These two sequential enzymes have separate roles in the functioning of the immune system since deficiencies of each result in distinct clinical and laboratory immunodeficient states (Giblett et al., 1972; Giblett et al., 1975). Also, these two enzymes have been studied in different malignant cell lines as well as in peripheral blood mononuclear cell preparations (PBMC) from individuals with different types of lymphoid malignancies(Tritsch and Minowada, 1978; Blatt et al., 1980). Results from these studies have suggested that ADA and PNP may serve as markers for cell differentiation.

T cell enrichment and depletion of human peripheral blood mononuclear cell preparations. Unexpected findings in the study of the functional activities of the separated populations

In the present communication we present the results of a study undertaken to assess the effectiveness of several methods of depleting human peripheral blood of thymus-derived (T) cells, and to assess the purity of the T cell-enriched populations obtained. The methods of T cell depletion employed included rosetting with unmodified sheep red blood cells (SRBC) in the cold at high erythrocyte to lymphocyte ratios; rosetting with S-2-aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC; and cytotoxic depletion using anti-Leu-1, a monoclonal antibody directed toward a pan-T cell antigen, and rabbit complement. The methods used to enrich for T cells included rosetting with unmodified or AET-treated SRBC, and passage through a nylon wool column. • The purity of the T-enriched and T-depleted populations obtained was assessed by (a) repeat rosetting with unmodified SRBC, (b) staining for cell surface immunoglobulin (sIg), (c) quantitating the number of Ig secreting cells (ISC) in a reverse hemolytic plaque assay, (d) quantitating the proliferative responses to the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) and to the B cell mitogen Staphylococcus aureus Cowan (SAC), and (e) quantitating the development of ISC following culture with PWM, with or without the addition of an irradiated T cell-enriched population. • By all parameters investigated, the depletion of T cells from peripheral blood by rosetting with AET-treated SRBC or by treatment with anti-Leu-1 and complement was far more effective than by rosetting with unmodified SRBC under optimal conditions. Of particular note was the finding that functional assays (ISC at time zero (T 0), proliferative response to SAC, the development of ISC on culture with PWM) revealed a much higher degree of B cell contamination in all of the T cell-enriched populations than would have been suspected from the extent of the depletion of sIg staining cells.

Interaction of wheat germ agglutinin with human peripheral blood mononuclear cells. Binding kinetics and flow microfluorometric analysis

We have used binding experiments with radiolabeled lectin and flow microfluorometry to characterize the interaction of wheat germ agglutinin (WGA) with human peripheral blood mononuclear cell preparations. Results indicated that WGA bound to these cells in a complex fashion described by a curvilinear (upwardly concave) Scatchard plot and a nonlinear dissociation curve. Binding was rapid and reversible and was inhibited 80–90% by 0.1 M N-acetylglucosamine, a specific haptenic inhibitor of WGA binding. Approximately 1 5 of the total binding was attributable to monocytes, (but phagocytic uptake was excluded), and the remainder was due to lymphoid cell variants. Kinetics of binding were only modestly altered by formalin fixation of the cells mitigating against a role for site-site interactions in the generation of the non-linear Scatchard plot. Flow microfluorometric analysis revealed that the capability to bind WGA was a characteristic of only 35% of blood mononuclear cells and further allowed identification of a small cell subclass constituting less than 10% of the total that nonetheless accounted for fully one half of the entire WGA binding potential of these cells.

Suppressor Cell Function in Sarcoidosis

We investigated the role of suppressor cells in the depressed cellular immunity of patients with sarcoidosis. The mean response in 16 patients with active sarcoidosis to three concentrations of phytohemagglutinin was significantly (P less than 0.01) less than control values. Passage of the cells over glass wool resulted in a 116% increase in response to phytohemagglutinin in patients and a 39% decrease in control subjects. Addition of indomethacin to phytohemagglutinin cultures increased the response of cells in patients with sarcoidosis by 192% +/- 32% versus a 112% +/- 18%-increase for control subjects (mean +/- SEM, P less than 0.05). Patients had an increased percentage of monocytes in peripheral blood mononuclear cell preparations, and the percent monocytes correlated with the percent increase in phytohemagglutinin response after glass wool passage (r = 0.62, P less than 0.05). Thus, several factors contribute to the depressed phytohemagglutinin response in sarcoidosis patients: an increased suppression by the prostaglandin-producing suppressor cell, an increased percentage of monocytes, and an as yet undefined factor.