Proliferation of human peripheral lymphocytes : Characteristics of cells once stimulated or re-stimulated by concanavalin A

About 20% of human blood lymphocytes will subsequently synthesize DNA after culturing with concanavalin A (conA) for 24 h. The stimulated cells go through one but only one round of DNA synthesis unless restimulated. We used the techniques of velocity sedimentation and auto-radiographic grain counts to demonstrate that stimulated cells do divide and return to a mitogen dependent, arrested G 1 state. Once-stimulated lymphocyte populations show the same time course for restimulation as do cultures which are being stimulated for the first time, but require at most one-fifth as much conA added to the medium in order to be maximally stimulated. We have shown by density transfer experiments that all of the progeny of cells which require up to 10 μg/ml of conA for the first DNA replication respond to 2 μg/ml with a second round of replication.     

Concanavalin A promotes bromodeoxyuridine induction of enodgenous C-Type virus in B cells

Spleen lymphocytes stimulated in vitro by concanavalin A, aT-cell mitogen, in the presence of bromodeoxyuridine, release C-type viruses. Since spleen cultures are noninducible by other T-cell mitogens in contrast to several inducing B-cell mitogens, we have analyzed the subpopulation of cells releasing virus following concanavalin A stimulation. In one approach lymphocyte suspensions were enriched for B or T cells by treatment with anti-Thy 1.2 plus complement or by nylon wool filtration, respectively. Mitogen-triggered virus release in these two enriched, as well as in reconstituted populations, was then measured. In a second line of investigation, increasing doses of X irradiation were used to preferentially block B- but not T-cell proliferation. Mitogen-stimulated virus release was also determined in these irradiated cultures. These methods independently demonstrated that the presence of both B and T cells was necessary for virus induction by concanavalin A in the presence of bromodeoxyuridine. Results of these experiments suggested that concanavalin A/bromodeoxyuridine-induced virus release occurred in a B-cell- through T-cell-dependent stimulation.     

Control of proliferation in mitogen responsive human peripheral blood lymphocytes: restimulation of cells stimulated by sodium periodate.

Human peripheral blood lymphocytes are stimulated to a greater extent by sodium periodate when cells are incubated in medium containing human serum than when incubated in medium with fetal calf serum. NaIO4 STIMULATION CAN BE REVERSED BY TREATMENT WITH SODIUM BOROHYDRIDE BUT CELLS ALREADY COMMITTED TO DIVISION ARE NOT AFFECTED BY BOROHYDRATE TREATMENT. Maximal commitment to DNA synthesis of a NaIO4 oxidized cell suspens-on occurs after about 28 hr of incubation in medium. The committal time after periodate stimulation is identical to that after stimulation with concanavalin A. Cells treated with periodate and then reduced with borohydride immediately after oxidation are refractory to further per-odate stimulation. Cells stimulated with periodate and then incubated for 6 hr before treatment with borohydride can be restimulated with periodate, indicating a turnover of membrane sites in the 6 hr period. Periodate-stimulated cells divide only once in response to the stimulation. The progeny of cells which were stimulated with periodate can be restimulated by treatment with either periodate or concanavalin A.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Evaluation of IgM,IgG, IgA,IgD, and IgE secretion by human peripheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aureus Cowan I

Sheep erythrocytes coated with staphylococcal protein A were used as target cells in a reverse hemolytic plaque assay. Monospecific antisera to human immunoglobulins G, M, A, D, and E were used to detect each of these classes in cultures of human peripheral blood mononuclear cells stimulated with the polyclonal B-cell activators pokeweed mitogen and Staphylococcus aureus (Cowan I). Both mitogens induced substantial increases in the numbers of cells actively secreting immunoglobulins; in time-course experiments, the peak response was seen on Day 5. The numbers of cell secreting IgM, IgE, and IgD were usually higher in cultures stimulated with S. aureus than with pokeweed mitogen.     

Cell matrix adhesion-related proteins VLA-1 and VLA-2: regulation of expression on T cells

The mitogens phytohemagglutinin (PHA) and concanavalin A inhibited the appearance of the very late activation antigen (VLA)-1, but did not inhibit VLA-2 expression on cultured activated T cells. In contrast to diminished VLA-1 expression, mitogen treatment caused increased cell surface expression of other activation antigens such as T10, HLA-DR, interleukin 2 (IL 2) receptor, and 4F2, and greater cell proliferation. Conversely, when T cells were not repetitively restimulated with mitogen, these less proliferative "postactivated" T cells had elevated VLA-1 expression. The diminished expression of VLA-1 caused by PHA was reversible since subsequent removal of mitogen was associated with increased VLA-1, paralleled by a decrease in interleukin 2 receptor levels. In addition to preventing or delaying the initial appearance of VLA-1, PHA stimulation also was somewhat effective in causing the disappearance of VLA-1 already present, especially on recently established cultures. However, cultures that had either never seen PHA, not seen PHA for several weeks, or been stimulated regularly with PHA, but were several months old, did not lose VLA-1 in response to PHA stimulation, suggesting that a state of insensitivity to PHA effects could be attained. Unlike PHA-stimulated T cells, T cells repetitively restimulated with alloantigen or the monoclonal antibody T3 did not show a marked absence of VLA-1 but rather showed an increased level of VLA-2 relative to VLA-1. Taken together, results of stimulation by either mitogen, alloantigen, or anti-T3 monoclonal antibody support the conclusion that T cell stimulation in general can cause a decreased VLA-1:VLA-2 ratio, whether by decreased VLA-1 or increased VLA-2. These shifts in VLA-1:VLA-2 ratios are probably not simply the result of shifts in the relative proportions of different subpopulations, because similar growth-related changes in this ratio were observed on the T cell line ANITA, which is a homogeneous population of cells. Because both VLA-1 and VLA-2 are differentially regulated on cultured, long term activated T cells depending on stage of activation and growth conditions, and are members of a family of at least five heterodimers that includes cell matrix adhesion molecules, we suggest that these studies will provide clues to novel aspects of T cell growth regulation, perhaps relating to T cell-matrix adhesion.     

Induction and suppression of immunoglobulin synthesis in cultures of human lymphocytes: effects of pokeweed mitogen and Staphylococcus aureus Cowan I

The synthesis and secretion of immunoglobulins by human peripheral blood mononuclear cells in cultures stimulated with pokeweed mitogen or Staphylococcus aureus Cowan I were evaluated by enumeration of cells containing cytoplasmic immunoglobulins and cells actively secreting immunoglobulins, and by quantitation of immunoglobulins released into culture supernatants. The two mitogens caused comparable stimulation of immunoglobulin production; however, in contrast to pokeweed mitogen, S. aureus was active in cultures depleted of T lymphocytes, and its stimulatory effects were resistant to the influence of suppressor T cells generated by co-stimulation with concanavalin A or by preincubation without mitogenic stimulus. These results indicate distinct pathways of induction and suppression of immunoglobulin synthesis for these two polyclonal B cell activators, and suggest that stimulation by S. aureus is less thymus dependent than that induced by pokeweed mitogen.     

Role of non-RT1 genes in the response of rat lymphocytes to Mycoplasma arthritidis T cell mitogen, concanavalin A and phytohemagglutinin

A comparison of splenic cells from various inbred rat strains indicated that DA, Lewis, Buffalo, August, Wistar Furth, and (LEW X BN)F1 all responded well to the Mycoplasma arthritidis T cell mitogen, phytohemagglutinin and concanavalin A, but cells from BN and MAXX rats were very weakly or nonresponsive. Cells from congenic strains expressing nonresponder background genes, and responder haplotypes at RT1 (BN.1L(LEW), RT1; BN.1A(DA), RT1av1) failed to respond significantly to the mitogens. Rats expressing responder background genes but the nonresponder haplotype at RT1 at RT1 (WF.1N-(MAXX), RT1n) exhibited high responses to all mitogens. The controlling role of non-RT1 genes was confirmed by testing tissue-typed (DA X BN)F2 progeny and (DA X BN)F1 X DA and (DA X BN)F1 X BN progeny. No association was seen between the expression of a/a, a/n, or n/n at RT1 and the degree of response to the mitogens. In contrast, as the proportion of DA non-RT1 genes increased, so did the degree of mitogenic responsiveness. Similar results were obtained by using a partially purified preparation of the mycoplasma T cell mitogen. The results indicated that in the (DA X BN)F1 hybrids, responsiveness to all mitogens was recessive: this contrasts with the (LEW X BN)F1 hybrids in which responsiveness was dominant. Finally, we showed that both responder and nonresponder splenic cells were capable of binding the M. arthritidis mitogen. The data contrast with those obtained with nonresponder mouse strains the cells of which failed to bind mitogen due to the absence of the E alpha chain of the I-E-coded molecule.     

Mitogenic responses of peripheral blood mononuclear cells of vervet monkeys (Cercopithecus aethiops): Apparent role of adherent cells

Peripheral blood mononuclear cells (PBM) from normal vervet monkeys (Cercopithecus aethiops) were examined for blastogenic responses to concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). The mitogen stimulated PBM in a dose-dependent manner. Response to ConA was apparently higher than for the other two mitogens. Cell density and mitogen concentration were critical parameters for optimal lymphocyte proliferation, an observation in line with that reported for other mammalian species. Depletion of an adherent cell population probably of monocyte/macrophage lineage from vervet PBM gave higher proliferative responses to both ConA and PHA, but the response without adherent cells to ConA was greater than the response without adherent cells to PHA. This latter finding is in contrast to what has been reported in many other species.     

Human lymphocyte subpopulations: giant SRBC rosettes.

Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.     

Elaboration of leukocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens.

This paper describes experiments to determine whether human lymphocyte sub-populations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-β₂-microglobulin (anti-β₂m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-β₂mm were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.     

Low responsiveness of human neonatal lymphocytes to a B-cell mitogen, Staphylococcus aureus Cowan I (SpA CoI)

Responses of neonatal and adult lymphocytes to various mitogens were studied. Lymphocytes from umbilical cord blood (UCB) responded well to both phytohemagglutinin and concanavalin A, and also to pokeweed mitogen and Staphylococcus aureus Protein A. The responses of UCB lymphocytes to these mitogens were not significantly lower than those of adult peripheral blood lymphocytes (PBL). In contrast, UCB lymphocytes showed only a minimal response to killed Staphylococcus aureus Cowan I (SpA CoI), a potent B-cell mitogen for human PBL, although the proportion of B cells in UCB was not less than that in PBL. The low level of response of lymphocytes from UCB to SpA CoI was not ascribed to differences in dose response or kinetics. Purified B cells from UCB were not stimulated by SpA CoI either, suggesting tht the low responsiveness was not due to the suppressive effect of T cells or macrophages, but to some intrinsic defect in B cells in UCB. These results suggest that the B cells in neonates may be more immature than the T cells.     

Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses.

Leukocytes from the various lymphoid tissues of rainbow trout (RBT) were tested for their capacity to respond to the lymphocyte mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and purified protein derivative of tuberculin (PPD). Thymocytes responded to Con A but not to LPS or PPD. In contrast, leukocytes from anterior kidney were stimulated with LPS but not with Con A or PPD. Cells from spleen and peripheral blood were stimulated by each mitogen. However, the degree of stimulation at optimally stimulatory concentrations of each mitogen was distinctive. The finding that the patterns of mitogenic responses of cells from each tissue were significantly different suggested that there is lymphoid heterogeneity in the RBT with a unique tissue distribution. The species source of serum utilized as a medium supplement appeared to be capable of markedly affecting mitogenesis. Thus, LPS and PPD stimulation occurred in medium supplemented with rainbow trout serum (RBTS). On the other hand, LPS and PPD stimulation was not observed in medium supplemented with fetal bovine serum (FBS), with the exception of peripheral blood leukocytes which were stimulated by LPS in culture medium supplemented with FBS. Con A stimulated leukocytes from each lymphoid tissue in medium supplemented with RBTS and, with the exception of cells from anterior kidney, also stimulated cells from each tissue in medium supplemented with FBS. The kinetic profiles of the responses of peripheral blood leukocytes to Con A, LPS, and PPD suggested that the extent as well as the time required for maximal stimulation was dependent on the dose of mitogen.     

INHIBITORY EFFECTS OF MITOGENS ON ADENOIDAL LYMPHOCYTES IN VITRO

Cultures of human adenoidal lymphocytes exposed briefly to either phytohemagglutinin (PHA), Staphylococcus filtrate (Staph-F), concanavalin-A(Con-A), or pokeweed mitogen (PWM) incorporate increased amounts of thymidine earlier than replicate cultures exposed continuously to the mitogens. These effects can begin in the first 24 hr of culture and are seen maximally between 36 and 72 hr. Once a blastogenic response is established, PHA or PWM can diminish that response. Inhibition with PWM requires that the initial stimulation was with this mitogen, while PHA can inhibit blastogenesis to both PHA and PWM-stimulated cells. Because these mitogens can have a paradoxical effect on adenoidal lymphocytes, being capable of both initiating and inhibiting DNA synthesis, this phenomena should be kept in mind when such systems are utilized for the evaluation of antigens and drug effects.     

Effects of retinoic acid on the human lymphocyte response to mitogens

Nontoxic concentrations of retinoic acid enhance DNA synthesis of human peripheral blood lymphocytes in response to phytohemagglutinin or rabbit-antihuman thymocyte globulin, whereas the response to concanavalin A or pokeweed mitogen remained unaffected. Retinoic acid-induced stimulation of lymphocyte reactivity to phytohemagglutinin or antithymocyte globulin was most evident in T cell-enriched subpopulations and required the near-concurrent addition of retinoic acid and mitogens. Retinoic acid-mediated enhancement of lymphocyte proliferation in response to phytohemagglutinin or antithymocyte globulin was paralleled by a concomitant suppression of immune interferon production of lymphocytes stimulated with these mitogens. These findings allow further studies on the immunoregulatory action of retinoids in vitro.     

Suppression of DNA synthesis in mitogen stimulated leukocytes with partial trypsin digests of Staphylococcus aureus protein A

Staphylococcus aureus protein A (SPA) acts as a mitogen stimulating DNA synthesis and is not antagonistic to the mitogenic activity of other mitogens. The mitogenicity of SPA is due to the protein's multivalent Fc antibody receptors. Partial trypsin digestion of SPA yields monovalent Fc antibody receptors which are not mitogenic. Using a thymidine incorporation assay, results indicated that tryptic digests of SPA added to mouse splenic leukocytes 12 hours after stimulation with either concanavalin A, lipopolysaccaride or SPA suppressed the DNA synthesis normally elicited by these mitogens. SPA was digested with 1% trypsin at pH 8 for varying periods of time. As the digestion time increased from 0 to 60 minutes the mitogenicity of the SPA-trypsin digests decreased indicating cleavage from multivalent to monovalent SPA Fc antibody receptors. The decrease in mitogenicity of the SPA digests was directly associated with increased ability to inhibit DNA synthesis in mitogen stimulated leukocytes. A proposed mechanism for the inhibition of DNA synthesis suggests that SPA-monovalent Fc antibody receptors mimic cellular Fc receptor function thereby influencing cellular gene expression.     

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Kinetics of lymphocyte stimulation in vitro by non-specific mitogens

The role of mitogens during lymphocyte activation was studied with kidney bean leucoagglutinin, concanavalin A and kidney bean phytohemagglutinin. The mitogens were removed by treatment with appropriate antisera, which was demonstrated to remove also mitogens already attached to the cells. The process of lymphocyte activation in vitro can be divided into four distinct steps, three of which are mitogen-dependent and the fourth is mitogen-independent. The first step consists of attachment of the stimulatory molecules to the cell membrane. The second step consists of reaction between mitogen and an activating system. During these two phases the cells become preactivated. The establishment of a preactivated state involves at least some synthesis of cytoplasmic RNA. The preactivated state is reversible and during the third step of lymphocyte activation the final result of preactivation is determined. Depending on the presence or absence of mitogen the cells may remain preactivated for over 60 h, they may return to the resting state or they may proceed through the final stages of the proliferation cycle and eventually divide. This fourth step is independent of the presence or absence of mitogen. A prolonged contact between cells and mitogen is required during the mitogen-dependent steps. The process of lymphocyte activation by mitogen is thus continuously being regulated by the stimulatory molecules on the lymphocyte membrane, which may be of considerable significance also for in vivo immunologicai reactions at the cellular level.     

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth.

Human fibroblast and leukocyte interferons were found to suppress lymphocyte mitogenesis induced by optimal doses of phytohemagglutinin and concanavalin A. In certain situations (low doses of mitogen and/or low doses of interferon), however, interferon significantly enhanced mitogenesis. In experiments using varying concentrations of interferon, dose-response curves with different slopes were obtained for fibroblast and leukocyte interferons. The effect of interferon was apparently exerted during early stages of the lymphocyte cell cycle. There was no inhibitory effect of interferon if the lymphocytes were washed with medium before being exposed to mitogen. Interferon increased the binding of radiolabeled mitogens to cells. The results suggest that the immunological effects of interferon are consequences of actions on lymphoid cells. Fibroblast and leukocyte interferons seem to have different modes of action, or to bind differently to target cells. Possible mechanisms for the suppressive and enhancing effects of interferons on lymphoid cells are discussed.