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.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Mitogen activation of human lymphocytes from normal and acute lymphocytic leukemia (ALL) subjects

Lymphocytes from acute lymphocytic leukemia (ALL) subjects were converted by mitogens to blast-like cells whose microscopic appearance and rate of formation was indistinguishable from those in mitogen incubated control lymphocytes. In ALL lymphocytes, however, pokeweed mitogen (PWM) failed to stimulate GGT expression; the mean increase it caused in thymidine kinase (TK) activity and thymidine incorporation was normal, though there were appreciable individual variations. These variations were also apparent with concanavalin A (Con A) but, in most ALL cases, TK and thymidine incorporation rose to much higher levels than in Con-A-treated control lymphocytes. The results indicate that evaluation of the response to mitogens by quantitative biochemical criteria provides a sensitive method for revealing functional impairments in microscopically normal ALL lymphocytes.     

IMMUNOLOGICAL AND HEMATOLOGICAL PARAMETERS IN CAPTIVE HARBOR SEALS (PHOCA VITULINA)

Peripheral blood was collected from 13 captive seals (12 harbor seals and 1 gray seal) of various ages to study different immunological and hematological parameters. In vitro mitogenic reactivity of blood lymphocytes was measured by means of a microculture lymphocyte transformation test (LTT). After stimulation with different doses of the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM), all examined seals showed significant proliferative responses to each mitogen. Furthermore, mitogenic reactivity significantly decreased with animal age suggesting this parameter of seal lymphocyte function is age-related. The present experiments support that the LTT is a suitable tool to monitor the functional capacity of seal lymphocytes. By means of the erythrocyte-rosette (E:-rosette) test it was demonstrated that a subpopulation of mononuclear seal bloosd leucocytes formed rosettes with sheep red blood cells (Srbc). This observation indicates that the phenomenon probably represents a marker for T lymphocytes in the seal as in several other mammalian species. Furthermore, the percentage of Srbc rosette-forming cells decreased with the age of the animals. Total blood leucocyte counts and differential leucocyte counts were determined by light microscopy. The number of leucocytes varied considerably among individual animals. Parallel to a significant decline of the percentage of lymphocytes with age, a corresponding increase in the percentage of neutrophils was demonstrated. In all of the seals, the percentages of monocytes and eosinophils were found to be low with only minor individual variation. The LTT and the E-rosette test are suggested as potential tools to elucidate immunological disorders in the seal.     

Lymphocyte transformation in casein-induced murine amyloidosis.

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.     

Decrease in mitogen responsiveness of mononuclear cells from peripheral blood after epinephrine administration in humans

A single subcutaneous injection of 0.2 mg epinephrine into healthy human subjects caused a transient lymphocytosis in peripheral blood. Mononuclear cells (MNC), isolated at various times after epinephrine administration, were cultured in the presence of mitogens. The blastogenic responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were significantly reduced for up to 60 min post-epinephrine (p less than 0.05); the response to concanavalin A (Con A) was reduced in the 15-min samples only. All responses returned to pre-injection levels by 120 min post-injection. Removal of adherent monocytes from MNC isolates before culture did not restore normal mitogen responsiveness. When MNC were cultured in the absence of mitogens, there was no difference in survival between pre- and post-epinephrine samples. Incubation of untreated MNC for 2 hr or 18 hr in vitro with various concentrations of epinephrine (10(-5) to 10(-1) mg/ml) had no effect upon the subsequent blastogenic response to mitogens. Other workers have reported that epinephrine administration causes alterations in the composition of the circulating lymphocyte pool. Taken together, these data suggest that the reduction in mitogen responsiveness after epinephrine is the result of changes in the distribution of lymphocyte subclasses in peripheral blood.     

Comparative mitogen responses of lymphocytes from Colombian Panamanian, and Peruvian owl monkeys.

A comparative study of lymphocyte responses to various mitogens, an index of cell-mediated immune competency, was made under various culture conditions using peripheral blood lymphocytes from Colombian, Panamanian, and Peruvian Aotus monkeys. Dose response curves were determined for each primate group to phytohemagglutinin, concanavalin A, and pokeweed mitogen stimulation. Considerable variation in mitogen response was observed. The influence of culture media supplemented with fetal calf serum or autologous plasma, the number of cells per culture, and the duration of incubation on mitogen responsiveness was also evaluated. Optimal lymphocyte culture conditions also differed among the three groups. Owl monkeys from the same location in South and Central America showed similar responses to physical condition and free of detectable disease, these differences probably reflect fundamental inherent biological differences between subpopulations of owl monkeys.     

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.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.     

Role of calcium and cyclic AMP on the activation of lymphocyte glycogen phosphorylase by mitogens

1. Various mitogens such as concanavalin A, phytohaemagglutinin, the pokeweed mitogen and trypsin were found to produce a rapid and transient activation of glycogen phosphorylase activity of lymphocytes incubated in a Krebs-Ringer-bicarbonate-glucose buffer. 2. Activation of the enzyme by these mitogens was always accompanied by an increase in the intracellular cyclic AMP concentration. 3. The presence of calcium ions in the incubation buffer was essential for obtaining the mitogen effects. Addition of ionophore A-23187 also produced an activation of glycogen phosphorylase, similar to that found in mitogen activation but without increase in intracellular cyclic AMP concentration. Dibutyril cyclic AMP also produced lymphocyte phosphorylase activation, even in the absence of extracellular calcium ions. 4. It is proposed that phosphorylase activation by mitogens occurs through a mechanism that involves the participation of both calcium ions and cyclic AMP.     

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Effect of human leukocyte interferon on human lymphocytes in vitro: cytogenetic studies

Mitogen-stimulated lymphocytes from 8 healthy donors were exposed to interferon, and cytogenetic studies were preformed. The response of lymphocytes to the mitogens phytohemagglutinin (PHA), concanavalin A (con A) and pokeweed mitogen (PWM) was inhibited by interferon, whereas an increased number of structural chromosomal aberrations was not detected. Further investigations of the cytogenetic effects of interferon are needed.     

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.     

Human lymphocyte responses are enhanced by culture at 40 degrees C.

In vitro responses of human peripheral blood lymphocytes (PBL) were found to be markedly enhanced by culture at 40 degrees C rather than at the conventional temperature of 37 degrees C. We studied proliferative responses of lymphocytes by activation by phytohemagglutinin (PHA) concanavalin A (Con A), pokeweed mitogen (PWM), and allogeneic lymphocytes in mixed lymphocyte culture (MLC) and found enhancement of DNA synthesis at the higher temperature. Cytotoxic T cell responses to allogeneic cells were also enhanced when MLC was done at 40 degrees C. These enhanced immune responses appear to be due in part to increased numbers of participating cells. If in vitro lymphocyte responses correlate with in vivo responses, then fever associated with infection or tumor may be beneficial whereas that associated with autoimmune disorders may have a detrimental effect.     

Suppressor cell influence in selected strains of inbred rats: I. Strain-dependent mitogen- and antigen-related low responsiveness

Lymphocytes derived from Lewis (LE), Brown-Norway (BN), or the F₁ hybrid (LBNF₁) rats respond in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. The magnitude of the response, as determined by incorporation of ³H-thymidine, ¹⁴C-adenine, and ³H-leucine, was highest for LE and lowest for BN animals. These proliferation response differences were observed for lymph node lymphocytes and peripheral blood lymphocytes. The response to antigen, as measured by lymphocyte transformation, reflected the mitogen responsiveness of the strains tested, i.e., LE animals responded to a higher level than did BN animals. Equivalent levels of antibody were found in all animals immunized with antigen. In addition, BN rats are suppressed to a greater magnitude than are LE rats when both strains are primed, rechallenged, and assayed via lymphocyte transformation to the test antigen.     

Mitogen- and antigen-responsive milk lymphocytes.

Bovine and canine milk contained lymphocytes that responded to the nonspecific mitogens; phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and gram negative bacterial lipopolysaccharide (LPS). It also was found that animals specifically sensitized with tuberculin or infected with infectious bovine rhinotracheitis virus (IBR) had antigen sensitive lymphocytes in their milk. In general, the responses of the milk lymphocytes from an individual animal were not identical to responses for the blood lymphocytes. Marked variation was observed in the daily responses of cells from milk samples from different quarters of the same animal, and between animals. The implications of antigen and mitogen responsive milk lymphocytes are discussed, in relation to their possible role in protective immunity.     

Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E

Abstract Syringomycin E (SR-E) is low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.     

Ability of FAB-fragments of normal rabbit and human gamma globulins to suppress human lymphocyte blast transformation induced by phytomitogens]

Monovalent and bivalent Fab-fragments of normal human or rabbit gamma-globulin suppressed blasttransformation of human lymphocytes induced by phytohemagglutinin and concanavalin A. Peptic F(ab)2-fragments from highly-purified rabbit anti-DNP antibody displayed suppressing activity similar to that of the fragments of normal gamma-globulin. Fab-fragments affected blasttransformation when added to lymphocytes either simultaneously with the PHA or 24 and 48h after the mitogen. The data obtained may indicate that the inhibiting of lymphocyte blasttransformation produced by the gamma-globulin fragments was not caused by their competing with mitogens for the receptors on the target-cell; the Fab-fragment activity was probably determined by the structures located outside the antibody active centre.