Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Manifestation of radiation injury of human lymphocytes using PHA mitogenic stimulation in different culture systems

The proliferative response of human lymphocytes to PHA in vitro is affected by X-irradiation. Dose-related changes of mitogenic stimulation of irradiated lymphocytes were compared in two culture systems--cultivation of separated lymphocytes and cultivation of whole blood. In whole blood cultures, the proliferative activity of stimulated lymphocytes was markedly and reproducibly depressed by irradiation. The values of mitogenic response within a dose range from 0 to 2.5 Gy could be fitted with high correlation by an exponential curve. In a modified test where the mitogenic stimulus was given after 24 h delay, depression of the response was even more pronounced. Radiosensitivity of human lymphocytes as determined by means of mitogenic stimulation in whole blood cultures appears to be a characteristic individual feature. The mean D37 value of the radiation-induced depression of mitogenic response in a group of 20 healthy donors was 2.5 Gy in the standard test and 2.0 Gy in the test with a delayed mitogenic stimulus. In contrast, the data obtained from separated lymphocyte cultures were characterized by a high degree of the test-to-test variability and by much lower radiosensitivity. The possible mechanisms of these distinctive manifestations of the same primary radiation injury are discussed.     

The phylogenetic structure of the genus Acinetobacter

Abstract A N-acetyl-D-galactosamine (GalNAc) specific bacterial lectin-like substance from Eikenella corrodens 1073 (EcLS) was found to have potent mitogenic activity when cultured with splenocytes from BALB/c mice. The results indicated that B lymphocytes are the major cell type responding to EcLS. The mitogenic activity of EcLS was dose-dependent, and the optimal concentration was around 5 μg/ml. The mitogenic activity did not appear to be due to a bacterial endotoxin, as GalNAc inhibited the mitogenic activity of EcLS, but did not inhibit the activity of lipopolysaccharide isolated from E. corrodens . EcLS stimulated murine B lymphocytes not only to proliferate, but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by B lymphocytes stimulated with EcLS. These findings suggest that EcLS is a novel lectin that not only induces B lymphocyte proliferation, but also differentiation.     

Some properties of the mitogenic factor induced in human lymphocytes under the influence of phytohemagglutinin]

Production of the PHA-induced human lymphocyte mitogenic factor (MF) previously described by the author and its influence on the lymphoid cells were investigated. The PHA-antiserum and immunosorbents were used for the PHA inactivation. The PHA-stimulated lymphocytes produced the mitogenic factor (MF) during the G1 period and the beginning of the S period of the cell cycle. When the cells were cultivated in the protein-free medium, the level of the DNA and the protein synthesis was significantly decreased, but the level of the MF production was 1.6 times greater than that of the MF production in the protein-containing medium. Kinetics of the lymphocyte reaction to the MF is described: DNA synthesis (as judged by the H3-thymidine uptake) began on the 5th--6th day and reached the maximum on the 6th--7th day.     

Thymus and Bursa Dependence of Lymphocyte Mitogenic Factor in the Chicken

AMONG the soluble products generated during activation of rodent or human lymphocytes¹, lymphocyte-stimulating (‘mitogenic’) factors are detected by their ability to accelerate DNA metabolism of freshly cultured allogeneic or syngeneic lymphocytes^2–8. If such lymphocyte activation products function as mediators of cellular immunity^1,9–11, their activities will be generated independently of the antibody-forming system. In the chicken, antigen-stimulation of sensitized lymphocytes in vitro involves both thymus (T-) and bursa-(B-)-dependent cells^12–14. If antigen-induced lymphocyte transformations are generally mediated by lymphocyte mitogenic factors, both T-cells and B-cells should produce lymphocyte mitogenic factors when stimulated by specific antigen. We have therefore determined whether antigen-stimulated chicken lymphocytes generate a lymphocyte mitogenic factor and whether this response is affected by neonatal thymectomy or bursectomy.     

Proliferation of human B lymphocytes mediated by a soluble factor

Recent studies have established the ability of a proportion of activated human B lymphocytes to undergo G1 phase cell cycle progression and subsequent S phase entry on exposure to factor(s) present in lectin-stimulated mononuclear cell-conditioned media. One factor capable of stimulating activated human B lymphocyte proliferation may be separated from peripheral blood lymphocyte-conditioned media by successive ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The isolated factor is distinct from the other well-described cytokines, possesses a molecular weight of 12,000-13,000, has a mildly acidic isoelectric point (at pH 6.3-6.6), is protease sensitive, and is relatively heat sensitive. The human B cell mitogenic factor possesses functional and cellular specificity in that its action is restricted to B lymphocytes and its function is proliferative. The production of the B cell mitogenic factor by T lymphocytes is augmented by the presence of a macrophage and further stimulated by syngeneic B cells.     

Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.     

Inhibition of phytohemagglutinin-stimulated lymphocyte transformation by neutral SH-dependent proteinase of the spleen

The effect of neutral SH-dependent proteinase of the spleen on transformation of human peripheral blood lymphocytes has been studied. Proteinase (1--10 micrograms/ml) inhibits phytohemagglutinin-stimulated (PHA) lymphocyte transformation and has no effect on spontaneous transformation. Proteinase does not alter the mitogenic properties of PHA and possesses no cytotoxicity. Therefore, inhibition of stimulated lymphocyte transformation is either resultant of proteinase direct action on the cell or associated with the inhibitor factor formation.     

Isolation by isoelectric focusing of a mitogenic substance released by stimulated human lymphocytes

Supernatants of human lymphocytes incubated for 4 hr with extracellular products of group A streptococci (streptococcal filtrate) and then washed and reincubated had a mitogenic activity for homologous lymphocytes. Fractionation by isoelectric focusing of the lymphocyte supernatants and of the streptococcal filtrates showed mitogenic activity in fractions with different isoelectric points. Moreover, human lymphocytes stimulated either with a mitogenic fraction of the lymphocyte supernatant or with a mitogenic fraction of the streptococcal filtrate, after treatment with bromodeoxyuridine (BUdR) and reciprocal restimulation, were responsive only to the heterologous stimulant.     

Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL-60 cells. A flow cytometric assay

Nucleolin and fibrillarin are two histone-like major proteins in the nucleolus that were found to be overexpressed in proliferating cells. Using specific antibodies to either nucleolin or fibrillarin flow cytometric, measurements were carried out to demonstrate quantitative changes of these proteins during lymphocyte mitogenic activation and differentiation of HL-60 promyelocytic leukaemia cells. The expression of nucleolin increased during lymphocyte stimulation and decreased slowly but constantly in the course of differentiation of HL-60 cells. Expression of fibrillarin reached a maximum in the first cell cycle and then dropped to a basic level in stimulated lymphocytes. Compared to nucleolin, the level of fibrillarin decreased more rapidly and more extensively in differentiating HL-60 cells. The data support other observations that nucleolin is a stabile structural protein at the ribosomal genes while fibrillarin may have a more specific functional role in nucleologenesis and ribosome production.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Components of mycobacteria and muramyl dipeptide with adjuvant activity induce lymphocyte activating factor

Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6-O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D₁ cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D₁ macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.     

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Tumour necrosis factor (TNF) is a potent mitogen for some fibroblast cell lines. Here we have examined the TNF-mediated changes in protein phosphorylation in Swiss 3T3 and human FS-4 fibroblasts, and compared them with changes observed after the treatment of cells with other mitogens, such as platelet-derived growth factor (PDGF) and bombesin. TNF stimulated the rapid phosphorylation of two 41,000-Mr and two 43,000-Mr cytosol proteins on tyrosine, threonine and/or serine, as did PDGF, epidermal growth factor and fibroblast growth factor; the increased levels of this mitogen-induced protein-tyrosine phosphorylation correlated well with the extent of mitogen-induced DNA synthesis as determined by the percentage of labelled nuclei. In contrast, bombesin, which is an even better mitogen for Swiss 3T3 cells than TNF, stimulated the tyrosine phosphorylation of 41,000-Mr and 43,000-Mr proteins only to a limited extent. On the other hand, bombesin and PDGF stimulated the rapid serine phosphorylation of an 80,000-Mr acidic protein, a major substrate for protein kinase C; increased phosphorylation of the 80,000-Mr protein was not observed at all when cells were stimulated with TNF. These results suggest significant differences among the mitogenic signalling pathways of TNF, PDGF and bombesin as regards the involvement of protein kinases; the mitogenic signalling pathway of TNF involves the activation of tyrosine kinase, but not of protein kinase C, whereas bombesin seems to transduce its mitogenic signal mainly through the activation of protein kinase C, and the activation of both kinases seems to be involved in the mitogenic signalling pathway of PDGF.     

Characterization of fibroblast proliferation factors elaborated by antigen- and mitogen-stimulated guinea pig lymph node cells: Differentiation from lymphocyte-derived chemotactic factor for fibroblasts,lymphocyte mitogenic factor,and interleukin 1

Guinea pig lymph node cells stimulated in culture by T-cell mitogens or sensitizing antigens release ~60,000- and ~16,000-mol wt proteins that induce normal guinea pig fibroblasts to proliferate in vitro. These fibroblast proliferation factors can be separated from lymphocytederived chemotactic factor for fibroblasts and from lymphocyte mitogenic factor by gel filtration employing Sephadex G-100. The 16,000-mol wt fibroblast proliferation factor was found to coelute with interleukin 1 (IL 1) from gel filtration columns. When the 16,000 molecular weight factor was further analyzed by anion exchange-high-performance liquid chromatography five major peaks containing IL 1 activity were obtained, only one contained fibroblast proliferation activity, suggesting forms of IL 1 exist that are not mitogenic for fibroblast. Occasionally, a large-molecular-weight inhibitor of fibroblast proliferation was detectable in void volume fractions from gel filtration of supernatant from antigen-stimulated lymph node cell cultures. This inhibition was accompanied by gross aggregation of fibroblasts. These studies suggest that fibroblast accumulation at sites of certain cell-mediated immune reactions in vivo may in part be attributable to the release of mediators by lymphocytes and, or macrophages that induce fibroblast growth.     

Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) stimulated growth of confluent human diploid fibroblasts (FS-4 cells) in the presence of fetal calf serum. TGF-beta synergistically enhanced both the TNF- and EGF-stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS-4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF-beta-induced growth of FS-4 cells. Mitogenic responses of indomethacin-treated cells to EGF, TNF, and TGF-beta were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2 produced less inhibition of the cell growth.     

Effect of chitin heparinoids on the activation of peritoneal macrophages and on the production of monokines in mice

Sulfonated derivatives of chitin which showed anticoagulant activity (chitin heparinoids) were studied with regard to the activation of mouse peritoneal macrophages and the production of monokines. In comparison with 70% deacetylated chitin (DAC-70), which was the most adjuvant-active derivative of chitin, all chitin heparinoids were less effective for the augmentation of cytolytic activity of peritoneal macrophages. The number of macrophages was hardly increased or decreased by intraperitoneal injection of chitin heparinoids, and the activity of circulating colony-stimulating factor was not changed by their treatment. Only N-sulfonated DAC-70 stimulated the production of interleukin-1 by thioglycolate-induced peritoneal macrophages in vitro. However, its effect was weaker than that of DAC-70. Chitin heparinoids showed no or weak mitogenic activity on normal mouse spleen cells.     

Regulation of lipopolysaccharide-induced granulopoiesis and macrophage formation by spleen cells. I. Relationship between colony-stimulating factor release and lymphocyte activation in vitro.

Addition of bacterial lipopolysaccharide (LPS), a B cell mitogen, to mouse spleen cultures strongly stimulated production of colony-stimulating factor (CSF), the humoral regulator of granulopoiesis, and macrophage formation in vitro. Secretion of CSF from LPS-stimulated spleen cells coincided with cellualr DNA synthesis and cell transformation and both activities could be attributed to the lipid A moiety of the molecule. Different experimental approaches were used to study the relationship of CSF release and lymphocyte activation in response to LPS: a) modification of LPS with polymyxin B, an antibiotic bactericidal for most Gram-negative bacteria, caused a marked reduction in mitogenic activity, although the ability to induce CSF was not significantly altered; b)spleen cells from CBA/N mice, a mutant strain with an x-linked genetic defect in immunologic and mitogenic responses to polyclonal activators including LPS, showed diminished mitogeinc responses; however, high levels of CSF were produced; c) mitotic and DNA inhibitors (colchicine and cytosine arabinoside) did not affect CSF release although they completely inhibited mitogenicity. Thus, the spleen cell population participating in the process of LPS-induced CSF generation is probably a nondividing, terminally differentiated one without need for DNA synthesis. In addition, it was also shown that active RNA and protein synthesis are needed in this process.     

Pertussis toxin triggers rapid second messenger production in human T lymphocytes

Pertussis toxin (PT) is a known mitogen for T lymphocytes. The mechanism by which the toxin stimulates proliferation has remained obscure and paradoxical because, in some types of cells, the toxin also inhibits growth factor-mediated signal transduction. It has previously been shown that the adenosine-diphosphate ribosyltransferase activity of the toxin is not required to produce the mitogenic effect. A biochemical explanation for the mitogenic activity has therefore remained obscure. We investigated the biochemical basis for the mitogenic activity of PT by using the transformed human T cell line, Jurkat. PT stimulated a rapid rise in cytosolic-free [Ca2+] from both intra- and extracellular sources. This was associated with an increase in the cellular diacylglycerol and inositol triphosphate levels with a concomitant decrease in the levels of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. The half-maximal effective dose of PT was 1.7 nM. PT also stimulated the production of interleukin 2. Only the holotoxin or B-oligomer (the presumptive membrane-binding subunit) was capable of stimulating an increase in [Ca2+] in these cells. This activity of PT mimicked that of some anti-T3-T cell antigen receptor complex monoclonal antibodies that also stimulate increases in the second messengers, diacylglycerol and Ca2+. The effects of PT and anti-T3 complex antibody were identical and not additive in Jurkat cells, suggesting that both agents were activating the same signal transduction pathway. These data provide a mechanistic explanation for the mitogenic effects of PT and suggest that the toxin may be interacting with a specific receptor in the T lymphocyte plasma membrane.