Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.     

Sulfation and mitogenic activities of growth factors in human serum: importance of compounds of molecular weight lower than 1,000 in the global expression of activity of this serum on 3 different cellular types

A human serum ultrafiltrate contains compounds needed for a maximal expression of sulfation and mitogenic activities of the corresponding retentate. These low molecular weight (less than 1,000) molecules have no effect, by their own, on 35SO4(2-) uptake in chick embryo cartilage, but show a significant synergistic effect with serum growth polypeptides. Tested alone, they display a slight mitogenic activity as measured by 3H-thymidine incorporation in chick embryo fibroblasts or in lymphocytes activated by phytohemagglutinin. Here they also act in a synergistic way on mitogenic activities of growth factors from human retentate. A fraction (P2B), partially purified from human plasma ultrafiltrates, produces the same synergistic response with human retentate in the three cellular systems (cartilages, fibroblasts, activated lymphocytes). However, concentrations which lead to optimum responses are very different according to the cell type studied. These results suggest the existence of several compounds which can each act specifically on a cellular system.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.     

Dextran-sulfate: a mitogen for human T lymphocytes

Dextran-sulfate (DxS) induced proliferation of human peripheral blood T lymphocytes but not of adult or neonatal B lymphocytes. The mitogenic activity on T cells by DxS required the presence of accessory cells because DxS was unable to trigger T cells to DNA synthesis in the absence of accessory cells. In addition, DxS stimulated OKT4+8- T cells to produce interleukin 2, a process that also occurred only in the presence of accessory cells. Cyclosporin-A strongly suppressed T cell proliferation induced by DxS by rendering T cells unresponsive to interleukin 2 and by inhibiting the synthesis of this T cell growth factor by OKT4+ T cells. These results indicate that DxS is a mitogen for human T lymphocytes but not for adult or neonatal B lymphocytes. The mechanism by which DxS triggers T cells is discussed.     

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

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.     

Lymphokines: their role in lymphocyte responses. Properties of interleukin 1

Interleukin 1, or IL 1, otherwise known as lymphocyte-activating factor, is a macrophage-derived 12,000- to 15,000-dalton polypeptide. Isoelectric focusing of human IL 1 reveals three peaks at pI's of 5.2, 6.0 and 6.9 respectively. IL 1 can be depleted of lymphocyte-derived IL 2 by SP-Sephadex chromatography. IL 1 augments the mitogenic response of PNA- Lyt 1+ thymocytes, and promotes thymocyte helper functions and B cell antibody production. IL 1 induces stable E rosette formation and the production of lymphokines such as T cell growth factor (IL 2) by peripheral T lymphocytes. Others have shown that IL 1 or closely related factors also stimulate hypothalamic cells to induce fever; induce in vitro fibroblast growth, prostaglandin, and collagenase production; and stimulate hepatocytes to produce acute phase proteins such as serum amyloid A. Murine epidermal cells also produce a 15,000-dalton factor that is mitogenic for thymocytes and may be similar to IL 1. We have recently hybridized spleen cells from mice sensitized with partially purified human IL 1 with a myeloma cell line. Clones have been isolated that produce supernatants that partially inhibit the thymocyte proliferative response to IL 1 but not the T cell growth factor activity of IL 2. Should these hybridoma products prove to be monoclonal anti-IL 1 antibodies, they will facilitate the further purification and characterization of IL 1.     

Mitogenic and comitogenic properties of the indole alkaloid class of tumor promotors

We determined the mitogenic and comitogenic properties of tumor promoters in the indole alkaloid series; agents that differ structurally from 12-0-tetradecanoylphorbol-13-acetate (TPA), which is known to be a lymphocyte mitogen. Teleocidin and dihydroteleocidin B were mitogenic for human lymphocytes, and lyngbyatoxin A elicited little or no mitogenesis. Catalase enhanced the mitogenicity of teleocidin and dihydroteleocidin B, and lyngbyatoxin A was also mitogenic in the presence of catalase. The potency of the agents was TPA = teleocidin greater than dihydroteleocidin B greater than lyngbyatoxin A. Dimethylsulfoxide and butyric acid markedly inhibited proliferation induced by these agents in human lymphocytes. The indole alkaloid tumor promoters were all comitogenic for murine thymocytes and induced production of interleukin-2. While the comitogenic effect of teleocidin was similar to that of TPA in its susceptibility to inhibition by dimethylsulfoxide and butyric acid, the comitogenic effect of dihydroteleocidin B and lyngbyatoxin A were less susceptible to these agents. These findings may facilitate the identification of cellular sites responsible for the inhibitory effect of differentiating agents on proliferative responses of lymphocytes.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

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.     

The reactivity of frozen B lymphocytes to B cell mitogens and human B cell growth factor: A study of step 1 and step 2 activators

Human B lymphocytes, purified from the peripheral blood of several different donors can be pooled, frozen, and stored in liquid nitrogen to provide an easy and reproducible source of cells for mitogenic assays. These B cell preparations did not show any reactivity to T cell mitogens, but responded to Staphylococcus aureus Cowan strain 1 (SAC) and anti-IgM antibodies to the same extent as freshly purified B cells. When stimulated with either anti-IgM antibodies or SAC, these B cells became responsive to B cell growth factor (BCGF), allowing a quantitative measurement of this important lymphokine activity. In addition, we have studied the reactivity of frozen B lymphocytes to various combinations of activators. We have confirmed that phorbol myristate acetate (PMA) was a very potent mitogenic agent for preactivated human B cells and shown that bacterial lipopolysaccharide (LPS), although not mitogenic by itself, could synergize with anti-IgM antibodies to yield increased levels of stimulation. Furthermore experiments using the lysosomotropic agent leucine methyl ester showed that the action of LPS on anti-IgM-stimulated B cells did not require the presence of functional monocytes. Neither PMA nor LPS could induce BCGF responsiveness and thus these two compounds can be considered exclusive step 2 activators for human peripheral blood B cells.     

Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide.

The lipid A moiety of bacterial lipopolysaccharide (LPS) elicits several types of responses in murine B lymphocytes. First, lipid A induces the nonproliferative expression of cell surface antigens in more immature cell types. Second, lipid A induces a mitogenic response in more mature B cell types. Lipid A induces the expression of Ia antigens on bone marrow cells from C3H/DiSn but not C3H/HeJ mice. The Ia-inducible cells possess surface immunoglobulin. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) induce the appearance of Ia antigens on B lymphocytes from both C3H/HeJ and C3H/DiSn mice, suggesting that lipid A exerts its inductive effects by increasing cyclic AMP levels in cells. In contrast to what is observed by using other strains of mice, mature B lymphocytes from C3H/HeJ mice do not support a mitogenic response to lipid A. The subpopulation of B lymphocytes in C3H/HeJ mice that normally respond mitogenically to LPS not only appear to lack an LPS-response mechanism utilized in the mitogenic pathway, but they lack the LPS-response pathway of the immature B cell types. A lipid A-bound protein (LAP) induces both the expression of Ia and a mitogenic response in the different subpopulations of B lymphocytes from C3H/HeJ and C3H/DiSn mice. The genetic defect in C3H/HeJ mice that limits responses to lipid A may be associated with a receptor that is normally expressed on many different cell types.     

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.     

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.     

The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-inducing activities of AEF can be experimentally dissociated, indicating that different subpopulations of T lymphocytes may be involved in the response to AEF. In contrast to similar soluble helper factors derived from allogeneic cell interactions, AEF appears to be unique in its ability to autonomously induce a primary CTL response in vitro.     

Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells

A growth factor for vascular endothelial cells was identified in the media conditioned by bovine pituitary follicular cells and purified to homogeneity by a combination of ammonium sulfate precipitation, heparin-sepharose affinity chromatography and two reversed phase HPLC steps. The growth factor was a cationic, heat stable and relatively acid stable protein and had a molecular weight, as assessed by silver-stained SDS-PAGE gel, of approximately 45,000 under non reducing conditions and approximately 23,000 under reducing conditions. The purified growth factor had a maximal mitogenic effect on adrenal cortex-derived capillary endothelial cells at the concentration of 1-1.2 ng/ml (22-26 pM). Further characterization of the bioactivity of the growth factor reveals that it exerts mitogenic effects also on vascular endothelial cells isolated from several districts but not on adrenal cortex cells, lens epithelial cells, corneal endothelial cells, keratynocytes or BHK-21 fibroblasts, indicating that its target cells specificity is unlike that of any previously characterized growth factor. Microsequencing reveals a unique N-terminal amino acid sequence. On the basis of its apparent target cell selectivity, we propose to name this factor vascular endothelial growth factor (VEGF).     

1 alpha,25-Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production

Previous studies have indicated that upon in vitro activation with mitogenic lectins, human peripheral blood T lymphocytes express receptors for the steroid hormone 1 alpha, 25-dihydroxyvitamin D3(1,25(OH)2D3). Furthermore, the hormone can inhibit interleukin 2 production by the activated cells. In this investigation, we report that human peripheral B lymphocytes activated in vitro with the B lymphotropic Epstein-Barr virus (EBV) also express 1,25(OH)2D3 receptor-like macromolecules. These receptors are localized in the cell nucleus and exhibit properties similar to those found in classical target tissues for 1,25(OH)2D3. They sediment on sucrose gradients at 3.3 S, display a dissociation constant (Kd) of 4 X 10(-10) M, and can bind to DNA. In addition to the 1,25(OH)2D3 receptors, however, EBV-activated lymphocytes express a second class of 1,25(OH)2D3-binding proteins that appear to occur mainly in the cell cytosol and exhibit distinct biochemical properties from the receptor, including higher sedimentation coefficients (3.7 S to 4 S) and the lack of ability to bind to DNA. The addition of 1,25(OH)2D3 to cultures of EBV-infected cells inhibited the production of IgM and IgG by the B cells. The vitamin D3 analog 24,25(OH)2D3 did not inhibit Ig production, thus suggesting that the effect is probably mediated through the high affinity receptor macromolecule localized in the nucleus. Because the EBV-induced Ig production is independent of T cell participation, the data also suggest that the effects of 1,25(OH)2D3 are exerted directly on the B cell. The present results add to the evidence of the importance of 1,25(OH)2D3 as an immunoregulatory hormone.