Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Role of monocytes in pokeweed mitogen-induced differentiation of human peripheral blood lymphocytes

Separate stimulation (“pulsing”) method of different cell populations with pokeweed mitogen (PWM) was used to study the regulatory role of monocytes in the PWM-induced plaque-forming cell response of human peripheral blood lymphocytes. T cells, B cells, and monocytes were separated, pulse-stimulated with PWM, extensively washed, and cocultured with unstimulated cell populations without additional PWM. Pulse-stimulated T cells helped unstimulated B cells to differentiate into immunoglobulin-secreting cells. This generation of helper T cells by PWM-pulsing was enhanced by monocytes in the presence of free PWM, as well as by PWM-pulsed monocytes in the absence of free PWM. A coculture of pulse-stimulated B cells and unstimulated T cells produced more substantial B-cell differentiation than the coculture of stimulated T cells and unstimulated B cells. Further enhancement of the latter response was obtained when B cells were pulse-stimulated in the presence of monocytes. However, pulse-stimulated B cells did not differentiate in the absence of T cells, and monocytes were unable to replace this T-cell function. It appears that there are several pathways by which PWM induces B-cell differentiation and in each, monocytes play an enhancing role.     

Regulation of lipogenesis in mitogen-stimulated human lymphocytes by thyroid hormones

The hormonal regulation of precursor incorporation into cellular lipids has been investigated in human lymphocytes stimulated with phytohemeagglutinine. Addition of thyroxine (5 micrograms/ml) for 72 h increased incorporation of [14C]acetate into the triacylglycerol fraction to 290% above the hormone-free control values. Incorporation into the cholesterol fraction was elevated up to 188% under the same conditions. Triiodothyronine was less effective than thyroxine: maximal effects were 153% of the control for triacylglycerols and 142% for cholesterol. Similar results were obtained when [14C]palmitic acid was used as a precursor for triacylglycerol synthesis. Effects of insulin on the parameters described were less pronounced than those obtained with thyroid hormones. Cellular triacylglycerol and protein contents were not elevated significantly by thyroid hormone addition. Further, incorporation of labelled thymidine, uridine, and leucine into acid-precipitable products was not elevated by triiodothyronine above mitogen-stimulated levels. It is concluded, that rapidly dividing lymphocytes provide a suitable system for studies concerning human lipid metabolism.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

Interferon-gamma can augment expression ability of HLA-DR antigens on pokeweed mitogen-stimulated human T lymphocytes

The effect of natural interferon (IFN)-gamma on HLA-DR molecule expression of pokeweed mitogen (PWM)-stimulated T cells from cord blood and adult peripheral blood was assessed by direct immunofluorescence with fluorescein-labeled monoclonal anti-HLA-DR antibody on a flow cytometer. Although cord blood T cells showed only weak expression of HLA-DR antigens on PWM stimulation, IFN-gamma could enhance HLA-DR expression of PWM-stimulated cord blood T cells to levels comparable to those of adult ones. A similar, but slight, increase in HLA-DR expression was inducible in PWM-stimulated adult T cells by the addition of IFN-gamma, but at higher doses. This increased expression of HLA-Dr antigens on PWM-stimulated T cells was almost completely abolished by both acid treatment of IFN-gamma and neutralization of IFN-gamma with specific antiserum. In contrast to IFN-gamma, neither recombinant IFN-alpha nor IFN-beta showed any effect on HLA-DR expression of PWM-stimulated T cells. These results suggested a possible function of IFN-gamma that might modulate HLA-DR expression ability of T cells in their activation process.     

Effect of interferon alpha on pokeweed mitogen-induced differentiation of human peripheral blood b lymphocytes

In a previous publication, we reported that B-lymphocyte-deprived mice possess a heightened in vivo resistance to a methycholanthrene-induced syngeneic fibrosarcoma. The present in vitro study has disclosed that spleens of such mice possess a significantly higher cytotoxic activity against the same tumor compared to normal rabbit globulin-injected control animals, and that this increase cannot be accounted for by the mere elimination of B lymphocytes. The cell mediating this response was found to be a natural-killer-like cell on the basis of its organ distribution, its full activation without immunization, its target specificity shown directly and by cold target inhibition experiments, and by its surface markers. It is suggested that this increased activity, which does not appear to decline with age, may be responsible for the heightened in vivo antitumor resistance of T mice.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

Insulin complex binding to human peripheral and mitogen-stimulated lymphocytes

Surface labeling and internalization of insulin was demonstrated ultrastructurally with human peripheral lymphocytes and with "activated"/transformed lymphocytes from mitogen-treated cultures using the colloidal gold-labeled insulin-bovine serum albumin (GIA) procedure. The majority of peripheral lymphocytes bound only limited amounts of the insulin complex, while approximately 15% of the lymphocyte population bound modest to comparatively large quantities of the labeled hormone. Quantitative labeling data indicated a skewed GIA labeling continuum for peripheral lymphocytes rather than separate, distinct populations. Sequential labeling studies with the GIA complex followed by either the ferritin-conjugated goat anti-human immunoglobulin or the E-rosette techniques indicated that insulin labeling was neither T nor B cell specific, since extremes of GIA labeling were found in both populations. Many, but not all, circulating lymphocytes with elevated insulin binding had morphological features suggestive of "active" cells, viz., larger cell, nuclear, nucleolar, and Golgi sizes, dispersed chromatin, and greater numbers of polysomes than lymphocytes having minimal GIA labeling. Both phytohemagglutinin (PHA), a T-cell mitogen, and pokeweed mitogen (PWM), a B/T cell mitogen, induced an increase in mean GIA labeling of cultured lymphoid cells as compared to non-mitogen-treated controls. The majority of mitogen-transformed "blast-like" cells had more extensive insulin labeling than nontransformed small (medium)-size lymphocytes, although an overlap in labeling densities was noted in these two groups. PHA induced a slight increase in mean surface GIA labeling of the nontransformed lymphocyte population at 48 and 72 hr of culture as compared to similar cells in non-mitogen-treated controls and PWM cultures. We interpret these findings as indicating the emergence of increased numbers of insulin binding sites on lymphocytes, both those in the circulation and in mitogen-treated cultures, during the early response (activation) to functional and/or metabolic modulations of the cell; this surface change does not appear to be directly related to blastogenic transformation.     

Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes.

The membrane potential of human PBMC was modulated in culture by isotonic high extracellular K+ (K+e), or the K+ channel blocker, charybdotoxin (ChTX), to determine the effect of depolarization on stimulated proliferation, IL-2 elaboration, and gene expression. In serum-free cultures, ChTX and high [K+]e induced a specific dose-dependent decrease in IL-2 production. ChTX inhibited proliferation of PBMC and purified T cells, decreased IL-2 elaboration 15 h after stimulation by 78.4 +/- 5.3% (n = 5), and decreased IL-2 mRNA steady-state levels by 80% between 8 and 10 h after stimulation. The IC50 for ChTX-inhibition of IL-2 elaboration and IL-2 mRNA were both 1 nM. Similarly, high [K+]e inhibited proliferation with an IC50 of 38.9 +/- 1.1 mM (n = 13), decreased IL-2 elaboration with an IC50 of 21.3 +/- 1.2 mM (n = 6), and decreased IL-2 mRNA steady-state levels with an IC50 of 18 mM. The sensitivities of both IL-2 production and proliferation to depolarization were substantially reduced by calcium, serum, and exogenous rIL-2. From these findings we conclude that membrane potential may contribute to the control of immune responsiveness in vivo.     

Continued proliferation of mitogen-stimulated human peripheral blood lymphocytes: requirement for the restimulation of progeny

The nature and specificity of the stimuli required for the continued proliferation of lymphocytes has been studied by DNA density transfer experiments in which restimulated cells were incubated in medium containing bromodeoxyuridine and also by double label autoradiography. The progeny of cells stimulated first by concanavalin A require an additional stimulus with concanavalin A to replicate. The majority of cells stimulated with phytohemagglutinin, streptolysin O, or staphylococcal filtrate must be restimulated with mitogen in order to replicate. We attribute the quantitative difference between concanavalin A and other mitogens in our experiments to the availability of the competitor, methyl-α-d-mannoside, which permits complete removal of concanavalin A. The progeny of cells stimulated by streptolysin O or staphylococcal filtrate can be restimulated by other mitogens, although in both cases slightly greater stimulation was obtained with the homologous mitogen.     

Histamine modulates in vitro IgG production by pokeweed mitogen-stimulated human mononuclear cells

The specificity of the receptor for IgA (RFcα) on human peripheral blood monocytes and polymorphonuclear (PMN) cells was evaluated by the ability of various human IgA preparations to inhibit rosette formation between these cells and IgA-sensitized ox erythrocytes. RFc?α on PMNS and monocytes were blocked by both monomer and dimer IgA preparations indicating that multivalent expression of Fc regions does not play a major role in receptor binding and that neither secretory component nor J chain significantly influences the binding of RFcα to IgA. Immunoglobulins of both the IgA1 and IgA2 subclasses inhibited IgA rosette formation and were in fact quite similar in their efficiency of blocking of RFcα. An IgA paraprotein without a Cα₃ domain was an even better inhibitor of IgA rosette formation than the IgA1 or IgA2 immunoglobulins. This implicated the Cα₂ domain as the site on IgA which interacts with RFcα. Furthermore, that this Cα₃-deficient IgA, which exists as a half molecule, was very efficient at blocking RFcα also demonstrated that multivalent Fc expression is not important to binding of RFcα and moreover that the site on IgA which interacts with RFcα is not dependent on H-chain pairing. RFcα on both PMN cells and monocytes were susceptible to proteolysis by pronase at concentrations which did not affect the receptor for IgG on these cells. Within 18 hr after removal of RFcα these cells had resynthesized and displayed this receptor. Although it is unclear whether IgA alone can mediate the effector functions of PMNs and monocytes in mucosal areas, the present studies define more clearly the specificity and regenerative capacity of RFcα and provide a basis for understanding the role of receptors for IgA and the cells with which they are associated in immune defense especially on the mucosal surfaces.     

Regulation of human placental progesterone synthesis in vitro by naturally occurring steroids

A regulatory model of human placental progesterone synthesis is based on studies with isolated placental enzymes. Steroids causing a dose-dependent inhibition are listed in the standing order of their inhibitory potency (I50 (microM)/Ki value (microM)/type of inhibition: c = competitive and nc = non competitive). Cholesterol side chain cleavage enzyme (mitochondria): Mainly regulated by hydroxylated cholesterol derivates. No inhibition was observed by cholesterylesters and by other naturally occurring steroids tested. 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (mitochondria): 6 beta-hydroxyprogesterone (nc), dehydroepiandrosterone (0.32/0.82/c), 20 alpha-dihydroprogesterone (0.38/-/nc), progesterone (0.46/-), estrone (0.56/0.1/c), estradiol (0.1/0.8/c), 17 alpha-hydroxyprogesterone (2.1/-/nc), 17 alpha-hydroxypregnenolone (0.4/-/c), dehydroepiandrosterone sulfate (2.5/-/c), cortisone (5.0/-), cortisol (100/-). 20 alpha-hydroxysteroid dehydrogenase (cytoplasmic): estrone (0.26/0.7/c), estradiol (0.28/0.9/c), pregnenolone (4.4/9.2/c), 5 alpha-pregnan-3 beta-ol-20-one (4.6/-/nc), estriol (5.1/11.5/c); dehydroepiandrosterone (7.2/14.0/c), 5 alpha-dihydrotestosterone (26.0/-/nc), progesterone (33.0/48.0/c), dehydroepiandrosterone sulfate (50.0/23.0/nc), and testosterone (59.0/63.0/c). An autoregulatory mechanism of placental progesterone synthesis is postulated which is in good agreement with data published by others proving that placental progesterone synthesis is independent of the endocrine organs of the mother and the fetus.     

Primary in vitro antibody response from human peripheral blood lymphocytes.

A method for the induction of a primary in vitro antibody response from human peripheral blood lymphocytes is presented. Upon cultivation with trinitrophenyl conjugated polyacrylamide beads (TNP-PAA), an anti-TNP response can be obtained as indicated by the appearance of direct plaque-forming cells from day 5 of culture, with a reproducible peak on day 8. These plaques correspond to cells actively producing antibody of the IgM type, as shown by their inhibition by cycloheximide and by anti-human IgM serum, but not by anti-human Fc gamma serum. Their specificity for the TNP hapten can be demonstrated by the effector cell blockade phenomenon, with highly substituted TNP-human IgG. Although the anti-TNP response induced by TNP-PAA in mouse spleen cell cultures appears T independent the same response in human PBL may involve in addition the participation of T cells, since E-RFC depletion before culture led to a markedly decreased number of plaque-forming cells. A significant response could be obtained from the PBL of all of the 30 normal individuals tested. Importantly, the response was reproducible in its magnitude in the six individuals tested in at least three different experiments. Thus, the in vitro stimulation of human PBL by TNP-PAA can be proposed as a reliable test for the study of human B cell function in a specific primary antibody response.     

Soluble suppressor factor produced by pokeweek mitogen-stimulated human peripheral blood leukocytes

Human peripheral blood leukocytes were exposed to either PWM or Con A mitogens. Cells activated by both these mitogens were able to depress proliferation in an MLC, and to inhibit the generation of spontaneous killer cell (SK) and induced T-cell cytotoxic activity. PWM-activated cells incubated in media for 48 hr were able to elaborate a soluble factor in vitro. This factor suppressed cytotoxicity, and was active only when present at the initiation of MLC cultures. In contrast, cells exposed to Con A were able to suppress immune responsiveness, but this population did not release a soluble factor which could inhibit cytotoxicity. PWM induction appears to be dependent on phagocytic cells, while Con A activation is less dependent on this adherent population. An enriched adherent cell population, stimulated with PWM, was able to suppress cytotoxicity. Thus, the PWM-stimulated system of suppression is mediated through a soluble factor and is dependent on adherent cells.     

Bromodeoxyuridine and light treatment enhances responsiveness of pokeweed mitogen-stimulated human lymphocytes to autologous and allogeneic determinants

We have investigated a novel system whereby lymphocytes from normal human subjects can be induced to develop exaggerated reactivity to histocompatibility antigens in vitro. Peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM) showed increased and accelerated subsequent proliferation to both autologous and allogeneic stimulators. Addition of bromodeoxyuridine (BUdR) during the period of maximal PWM-induced DNA synthesis followed by light exposure caused unexpected, but marked enhancement of this secondary proliferation. While untreated cultures contained a preponderance of T8⁺ cells after PWM activation, BUdR plus light-treated cultures were largely T4⁺ cells. Because removal of suppressor cells in nonsuicided cultures with anti-T8 and complement just before restimulation failed to unmask enhanced autoreactivity, events critical in the induction of the enhanced response must have occurred during priming. Cultures of PBMC with medium alone or concanavalin A, as well as purified T cells cultured with PWM, gave no enhanced autoproliferation after BUdR and light; thus T and non-T cells must be acted on by a T- and B-cell mitogenic stimulus to prime T cells for enhanced responsiveness. The interactions between T cells and activated B cells in this in vitro system may be relevant to regulatory mechanisms important in the induction of pathological autoimmune responses.