Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Response of lymphoid cells to thymic hormonal factors isolated from mouse thymus

Thymic hormonal factors were isolated from mouse thymus by two methods. (1) Thymic cytosols in phosphate buffer saline were filtered through Sephadex G100 with 0.1 M NH4HCO3 (pH 8.0) as buffer and the protein peaks were collected. (2) Protein having thymosin activity (F5) was isolated from thymic cytosols after heat inactivation, salt fractionation and desalting on Sephadex G25. Molecular weights of all the proteins were determined on SDS-PAGE. Biological activity of thymic proteins was studied by in vitro and in vivo assays, using synthetic thymosin alpha 1 as the standard. Thymocytes treated with different thymic proteins showed maximum stimulation at 16 h of incubation period. Preincubation of the thymocytes with the thymic proteins and subsequent incubation with Con-A decreased the stimulation index. Incubation of spleen lymphocytes with thymic proteins increased the percentage of Tdt+ cells. The antitumor effects of thymic proteins carried out on animals having leukemia, showed statistically significant results. Clinically however, the antitumor effects of the thymic proteins alone and in combination chemotherapy were negligible at 1 mg/kg body weight dose level.     

Effect of repeated stress and administration of phenobarbital on the lymphoid tissue and on protein metabolism in the lymphocytes of infant and adult rats

Further study of the response to chronic stress stimulation in the early postnatal phase showed that the i.p. injection of physiological saline (stress stimulation) induced lymphopenia, a 50% decrease in the incorporation of 3H-leucine into isolated lymphocytes and a decrease in the weight of the thymus in 7-day-old male rats. No such changes were observed in adult animals. If repeated doses of phenobarbital were administered to stressed young rats, however, lymphopenia did not occur and the rate of the incorporation of 3H-leucine into isolated lymphocytes was not different from the control value; the protein content of the lymphocytes was significantly raised, however. In adult animals, phenobarbital increased the rate of incorporation of 3H-leucine into the lymphocytes. The repeated administration of phenobarbital reduced the weight of the thymus in both young and adult animals, but a decrease in spleen weight was recorded only in the young animals. A single i.p. injection of ACTH or dexamethasone caused lymphopenia and slowed down the incorporation of 3H-leucine into the lymphocytes of both young and adult animals. The results show that the striking decrease observed in the rate of the liver metabolism of corticosterone in suckling young rats not injured by repeated stress stimulation is accompanied by significant changes in the lymphoid tissue.     

Effect of protease inhibitors on mitogen stimulation of hamster lymphoid cells

Hamster lymph node and spleen cells can be stimulated to incorporate tritiated thymidine ([³H]TdR) in vitro under serum-free conditions by the proteases trypsin and chymotrypsin. Under similar conditions, thymocytes could be stimulated by concanavalin A (ConA) but not lipopolysaccharide (LPS) or the proteases. The subpopulation of cells responding to the proteases correlated with the cells responding to LPS on fractionation of spleen and lymph node cells on discontinuous bovine serum albumin (BSA) gradients or on nylon-wool columns. The stimulation induced by trypsin was completely blocked by soybean trypsin inhibitor (SBTI) while that induced by chymotrypsin was only partially blocked. The inhibition by SBTI of protease activation was not effective when added 24 h after initiation of stimulation. On the other hand, addition of clarified isologous serum to protease activated cultures after 24 h still lead to greater than 50% inhibition of [³H]TdR incorporation.     

Mitogenic Effect of Cytoplasmic Membranes and a Cytoplasmic Fraction of Staphylococcus aureus L-Forms on Human Peripheral Blood Lymphocytes

The cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L-forms increased the incorporation of [³H] thymidine by human lymphocytes in the presence of fetal bovine serum. Both fractions stimulated cord blood lymphocytes as well as adult peripheral lymphocytes, suggesting the possibility that the observed effect was not due to an antigen-specific reaction, but to an immunologically nonspecific action. The membrane mitogen(s) was resistant to trypsin, although it was partially solubilized by trypsin treatment. The mitogen (s) could not be extracted with a chloroform-methanol mixture (2:1, v/v), although the chloroform-methanol soluble fraction was strongly mitogenic to murine splenocytes. Human serum which was added to the assay system in place of fetal bovine serum definitely suppressed the mitogenic effect of both cytoplasmic membranes and the cytoplasmic fraction, especially the latter.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

The kinetics of cellular commitment during stimulation of lymphocytes by lectins

The kinetics of cellular commitment in the stimulation of lymphocytes by concanavalin A (Con A) has been analyzed by measurement of DNA synthesis, autoradiography, and histologic staining techniques. If the competitive inhibitor α-methyl-D-mannoside (αMM) is introduced into cultures of mouse spleen cells at various times after the addition of Con A, there is a gradual decrease in its capacity to inhibit the lectin-stimulated incorporation of [³H]thymidine. Addition of the saccharide 20 h after exposure of the cells to Con A had no effect on the level of the cellular response to the lectin. With increasing periods of contact with Con A, the percentage of blast cells and the percentage of [³H]thymidine-labeled blast cells increased in parallel with the total radioactive thymidine incorporated while the average number of autoradiographic grains per labeled blast cell remained relatively constant. These observations suggest that the rising level of [³H]thymidine incorporation results from an increase in the number of cells that respond to lectin stimulation and become refractory to inhibition with αMM. Once such cells become committed, they synthesize DNA at a rate independent of the length of exposure to the lectin. The combined results indicate that mouse splenic lymphocytes are heterogeneous in their capacities to respond to Con A and that different cells require different induction periods to be stimulated.     

Mechanism of binding of soluble immune complexes to lymphocytes

Soluble immune complexes prepared with either (a) ¹²⁵I BSA and mouse antiserum to BSA in the presence of fresh mouse serum (AgAbC) or with (b) ¹²⁵I BSA and a 7S fraction of the mouse antiserum to BSA (AgAb) bind to a certain proportion of mouse lymph node and spleen lymphocytes (but not to thymocytes). The efficiency of binding is greater when complexes are prepared at defined antigen/antibody ratios and when the incubation with lymphocytes is performed at 37 °C. However, a significant degree of binding occurs at lower temperatures and even at 0 °C. Cells which bind soluble complexes overlap extensively with complement receptor lymphocytes (CRL) (B lymphocytes) since the specific elimination of CRL also depletes the population of cells which can bind soluble complexes. Two types of interactions are involved in the binding: one mediated by antibody which has been aggregated by antigen and another by complement (probably C3). They can be operationally distinguished because (1) after treatment of the lymphocytes with trypsin, the binding of AgAbC (but not of AgAb) is strongly inhibited; (2) the binding of AgAb (but not of AgAbC) is inhibited by heat-aggregated Ig; (3) the binding of AgAbC (but not of AgAb) to lymphocytes inhibits their subsequent interaction and rosette formation with erythrocytes sensitized by antibody and complement components; and (4) cobra venom factor markedly alters the binding of AgAbC to lymphocytes.     

Recruitment of null cells during graft versus host reactions in the chicken

Untreated SC (B2/B2) chicken spleen or thymus cells (2 × 10⁷) caused significantly increased [³H]thymidine incorporation in spleens of heavily irradiated FP (B15/B21) recipient chicks on Day 4 after iv injection. Mitomycin-treated SC spleen cells or spleen cells treated with rabbit anti-T-cell serum and complement failed to raise the [³H]thymidine incorporation over that in uninjected, bursa cell-injected or FP spleen cell-injected controls. However, the combination of mitomycin-treated spleen or thymus cells and anti-T-treated spleen cells caused an increased [³H]thymidine uptake, suggesting the recruitment of non-T cells into proliferation by alloreactive mitomycin-treated T cells. Bursa cells did not proliferate during GVH reactions even though they could be shown to undergo proliferation in vivo upon mitogen (lipopolysaccharide and dextran sulfate) stimulation. In contrast, anti-T-treated spleen cells from agammaglobulinemic chickens were recruited into proliferation, suggesting that the recruited cell was not only not a T cell, but also no pre-B or B cell and most likely represented a cell of the monocyte-macrophage series.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

DNA synthesis by cultured lymphocytes: a modified method for measuring 3H-thymidine incorporation

A rapid, convenient and inexpensive method for harvesting lymphocyte cultures and measuring the incorporation of ³H-thymidine into trichloroacetic acid precipitable material has been developed. The basic principle is to adsorb the entire contents of a microculture well onto the cotton applicator portion of a Q-tip, precipitate the DNA, wash away unincorporated ³H-thymidine, and count the remaining ³H in a mixture of scintillation fluid plus detergent. Data presented for mixed lymphocyte cultures between allogeneic rat lymph node cells, mixed lymphocyte cultures of human peripheral blood lymphocytes, Con A stimulated mouse spleen cells, and PHA stimulated mouse spleen cells show the method to be highly reproducible with standard deviations of less than 15% of the mean for quadruplicate mixed lymphocyte cultures and in most cases less than 5% of the mean for duplicate mitogen stimulated cultures. This culture system also gives positive values for PHA stimulated DNA synthetic responses of mouse spleen cells cultured in RPMI-1640 plus penicillin and streptomycin but without exogenous serum.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

The in vitro effect of a thymic epithelial culture supernatant on mixed lymphocyte reactivity and intracellular cAMP levels of thymocytes and on antibody production to SRBC by Nu/Nu spleen cells.

Addition of supernatants from rat thymic epithelial cultures (TES) to rat thymocytes stimulated with T-cell-mitogens or allogeneic cells leads to an increase in ¹⁴C-TdR incorporation. Furthermore, in the presence of TES, spleen cells from athymic nu/nu mice exhibit an enhanced in vitro antibody production to SRBC, whereas TES has no such effect on spleen cells from T-cell-deprived mice. If TES is added together with thymocytes to T-cell-deprived spleen cell cultures, the number of plaque-forming cells to SRBC is enhanced, suggesting that TES induces a helper cell function in thymocytes which, if added alone, have no effect. TES also increases intracellular levels of cAMP in thymocytes in vitro and appears to act on a membrane site distinct from the β-adrenergic receptor. TES fails to affect mitogen responses, MLR and cAMP levels of lymphocytes from other lymphoid organs. The biological activity of TES as compared to that of thymic extracts is discussed.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones)

Partially purified lymphocytic factors were obtained from bovine spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo:[³H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited: similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked.[³H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced.The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in culture as far as thymidine incorporation into DNA is concerned.The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F′.Both fractions F and F′ were further fractioned by chromatography on a Sephadex G75 column: 7 peaks were obtained (F₁–F₇). Biological activity was found in fraction F₁, corresponding to high molecular weight material, and in fraction F₁, corresponding to low molecular weight substances.By rechromatography on Sephadex G 75, it is easy to dissociate from F₁ a small molecular weight fraction which might be similar to F₆ as far as elution volume and biological properties are concerned.     

Effect of somatostatin on the proliferation of mouse spleen lymphocytes in vitro

The effect of somatostatin on the spontaneous proliferation of mouse spleen lymphocytes was investigated in vitro. The rate of 3H-thymidine incorporation was used as an index of lymphocyte proliferation. Somatostatin in a concentration of 10(-7) M enhanced the lymphocyte proliferation and abolished the antiproliferative effect of rat hypothalamic extract. Lower concentrations of somatostatin slightly decreased the lymphocyte DNA synthesis.     

Effect of Anti-Ia Serum In Vitro on Lipopolysaccharide-Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [³H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed.     

3H-uridine incorporation as a T lymphocyte indicator in the rat

Although about 70% of rat thoracic duct small lymphocytes labeled readily in vitro with ³H-uridine, only 3–38% of peritoneal exudate lymphocytes labeled. Since exudate cells are mostly B lymphocytes, ³H-uridine in concentrations used were presumed to label the T lymphocyte. Percentages of small lymphocytes that labeled in cell suspensions from various tissues were consistent with other estimates of T cells in those sources: 74.7% in thoracic duct, 70.2% in blood and 65.6% in spleen. When lymphopenia was induced by polyethylene ³²P strips applied to the spleen, a procedure that depletes mostly small recirculating lymphocytes, both labeled (T) and nonlabeled (B) cells were depleted in similar time sequence. Both cell types recovered at a similar rate after the spleen strips were removed. Induction of peritoneal inflammation by PPD in tubercle-bacilli immune rats caused an enhanced lymphocytic exudation but no increase in percentage of labeled (T) lymphocytes.The defect in ³H-uridine incorporation that characterizes the rat B lymphocyte seemed to be relatively specific for that RNA precurser; ³H-cytidine labeled the majority of lymphocytes in peritoneal exudate.     

Difference in genesis between antigen-induced IgM antibody-forming cells and B-cell mitogen-induced IgM antibody-forming cells in mouse spleen cell cultures.

To determine the mechanisms in the triggering of thymus-independent lymphocytes (B cells) for development into antibody-forming cells (AFC), genesis of IgM AFC elicited polyclonally by nonspecific stimulation with B-cell mitogen, such as nystatin and bacterial lipopolysaccharide, was compared with that of IgM AFC specifically elicited by antigenic stimulation, using mouse spleen cell cultures as an experimental system and sheep erythrocytes (SRBC) as a test antigen. Considering that differentiation and proliferation are necessary cellular events for precursor B cells to develop into AFC, the effect of different antimetabolic agents on the generation of each type of AFC in spleen cell cultures was examined. The generation of anti-SRBC IgM hemolysin plaque-forming cells (PFC) in B-cell mitogen-stimulated spleen cell cultures was found to be less susceptible to X-irradiation or mitomycin C than that in the SRBC-stimulated cultures. These apparently paradoxical results were affiirmed using colcemid as an inhibitor of cell mitosis and hydroxyurea (HU) as an inhibitor of cellular DNA synthesis. Thus, when spleen cell cultures responding to either SRBC or B-cell mitogen were exposed to colcemid or HU during a period from 2 days to 3 days after the stimulation, the exponential generation of anti-SRBC IgM PFC in the cultures responding to SRBC was completely halted, whereas that in the cultures responding to B-cell mitogen was not. Furthermore, N⁶, O^2′ -dibutyryl adenosine 3′, 5′ -cyclic monophosphoric acid was found to halt the exponential generation of antigen-induced anti-SRBC IgM PFC but not that of the B-cell mitogen-induced anti-SRBC IgM PFC. From these results it was suggested that B-cell mitogen might stimulate precursor Bμ cells at a late stage in the differentiative pathway to develop into AFC without cell division, and that antigenic stimulation might stimulate relatively primitive precursor Bμ cells to proliferate and then differentiate into AFC. Based on this idea, mechanisms in the triggering of B-cell activation are discussed.     

Mitogen-induced T-lymphocyte proliferation: a reappraisal of the early requirement of extracellular ionized calcium.

Mitogen-induced DNA synthesis in lymphocyte cultures requires an extracellular calcium concentration of 3 × 10^?6M or higher. When cultures of human or mouse lymphocytes were incubated with T-cell mitogens for the first 12 hours in a medium with about 3 × 10^?6M calcium ion concentration, and then the normal calcium concentration was restored, the induction of DNA synthesis in the cultures was salvaged, but it started 10–16 hours later than in control cultures. Lipopolysaccharide-induced thymidine incorporation in mouse spleen cell cultures responded to this experimental design in a more complex way. - These results support the idea that calcium ions are specifically needed for one or more of the very early steps in mitogenic activation of T-lymphocytes.