Enhancement of B-cell stimulation by muramyl dipeptide through a mechanism not involving interleukin 1 or increased Ca2+ mobilization or protein kinase C activation

Muramyl dipeptide (MDP) enhanced mitogenic stimulation of mouse lymphocytes by polyclonal B cell activators (peptidoglycan, lipopolysaccharide, Staphylococcus aureus Cowan I cells, and pokeweed mitogen), but not by T-cell mitogens (phytohemagglutinin and concanavalin A). Only adjuvant-active MDP analogs were effective, whereas adjuvant-inactive MDP analogs, muramic acid, peptidoglycan pentapeptide, and low Mr digests of peptidoglycan were not. The half-maximal enhancement was seen at 5-10 microM MDP and occurred at both optimal and suboptimal concentrations of B cell mitogens. The enhancing effect of MDP was exerted on the B cells, since it was T cell- and macrophage-independent and was not mediated by IL-1. MDP was effective during the first 12 hrs of culture, and most strongly enhanced the mitogen-induced DNA synthesis, although significant enhancement of RNA synthesis and B cell differentiation into antibody-secreting cells was also observed. The enhancement of mitogenic response was not due to changed requirements for extracellular or intracellular Ca2+ or to increased activation of protein kinase C. These results demonstrate a novel immunoenhancing effect of MDP that should be useful in the studies on the mechanism of B cell activation.     

Expression of the p53 protein during the cell cycle of human peripheral blood lymphocytes

We have investigated the role of the cellular p53 protein in the induction of growth in size and cell DNA replication in human peripheral blood lymphocytes (PBL) and in monocyte/macrophage-depleted lymphocyte (MDL) cultures stimulated with phytohemagglutinin (PHA). Our results show that in human lymphocytes exposed to PHA, the induction of p53 protein synthesis and accumulation correlates with the extent of cellular DNA replication, rather than with growth in size. Moreover, the induction of p53 is dependent on the presence of the T-cell mitogen, Interleukin-2. A monoclonal antibody to Interleukin-2 receptors (anti-Tac) inhibits PHA-stimulated cellular DNA synthesis, and this inhibition is correlated with a reduction in the percentage of p53-positive cells. We conclude from this work that the p53 protein is a cell cycle-dependent gene whose expression can be regulated by different mitogens in different cell types.     

Fc receptors for IgE (Fc epsilon R) on human lymphoid cells: inducible expression of Fc epsilon RII (CD23) on lymphocytes and detection by monoclonal anti-Fc epsilon RII antibody

The studies presented herein describe (1) a sensitive, quantitative, and objective assay for detecting cell membrane-bound form of Fc receptors for IgE displayed on human lymphoid cells based on measuring unlabeled Fc epsilon R-bound IgE by a solid-phase RIA of cell lysate fluids; (2) the development and characterization of an IgM monoclonal antibody, termed 7E4, which is specific for human lymphocyte Fc epsilon RII (CD23) molecules; and (3) a system for reproducibly inducing de novo synthesis and expression of Fc epsilon RII proteins on human lymphocytes following exposure to the mitogenic lectin, pokeweed mitogen. The Fc epsilon RII molecules induced by exposure to PWM were proven to be present on lymphocytes, and not on other cell types in several ways, including (1) documenting sensitivity of such proteins to both acid pH and trypsin treatment, the latter manipulation being ineffective in removing Fc epsilon RII molecules on basophils and mast cells; (2) demonstrating specific reactivity of the expressed Fc epsilon RII molecules with the 7E4 monoclonal antibody, which is specific for human lymphocyte Fc epsilon RII molecules and does not react with Fc epsilon R molecules on other cell types; and (3) observing the required concomitant presence of both T and B lymphocytes during the induction process and proving that the induced Fc epsilon R+ cells are indeed B cells of the Leu-12+ phenotype by fluorescence analysis. The ability to induce expression of Fc epsilon RII molecules on human lymphocytes exposed to a mitogen such as PWM requires special technical attention to the method of preparation and isolation of human lymphoid cells from peripheral blood. This in vitro system for up-regulating Fc epsilon RII expression on human lymphocytes should provide us with an important new tool to analyze the participation of such cells in the regulatory mechanisms controlling the human IgE antibody system.     

The lack of mitogenic response of neuraminidase-treated and untreated human blood lymphocytes to divalent, hexavalent, or insoluble Helix pomatia a hemagglutinin

The mitogenic effects on human blood lymphocytes of Helix pomatia A hemagglutinin (HP) were measured by assaying incorporation of [¹⁴C]thymidine into cellular DNA. This highly purified lectin binds to human lymphocytes, treated with neuraminidase, but not to untreated lymphocytes. HP, which is hexavalent in its native form, totally failed to induce DNA synthesis in neuraminidase-treated as well as in untreated cells. Divalent HP, prepared by partial reduction and alkylation, and HP insolubilized by coupling to nylon sheets, also lacked mitogenicity. Control experiments indicated that neuraminidase-treated lymphocytes responded well to mitogenic lectins such as PHA. The lack of mitogenicity of HP was in contrast to the effects of soy bean agglutinin (SBA), which resembled HP in regard to carbohydrate specificity and ability to bind to neuraminidase-treated lymphocytes only. SBA was strongly mitogenic for neuraminidase-treated lymphocytes. The mitogenic effects of SBA were not inhibited by including varying doses of HP in the incubation mixtures. The results indicate that binding of lectin to carbohydrate receptors on the lymphocyte surface is not by itself sufficient to trigger DNA-synthesis.     

Determination of mitogenic properties and lymphocyte target sites of Dolichos lablab lectin (DLA): comparative study with concanavalin A and galactose oxidase cell surface receptors

A mannoside-directed lectin has been isolated and purified from the seeds of Dolichos lablab L. by affinity chromatography. We have established that this glycoprotein, which displays high erythroagglutinating activity without blood group specificity, highly activates murine T lymphocytes, and we have described for the first time its mitogenic properties. Although its main properties are close to those of concanavalin A (Con A), the well-known mannoside-directed mitogen devoid of sugar moiety, several differences were found in some of the early events triggered by the two lectins during lymphocyte mitogenic stimulation: higher level of interleukin-2 (IL-2) synthesis, optimal dose for IL-2 synthesis at suboptimal mitogenic concentration, lack of ecto-5' nucleotidase inhibition, and lack of mitogenic inhibition at high lectin concentration. Because the two lectins did not act on the cell surface in exactly the same way, we have compared their receptors involved in mitogenesis on the plasma membrane of murine lymphocytes. We had previously established that the polyclonal activation of these cells probably occurred through high-molecular-weight receptors (200-230 kDa). Since the mitogenic stimulation of lymphocyte by galactose oxidase (GO), like that of Con A, was inhibited by DLA, we analyzed the cell surface receptors that were common to these three polyclonal mitogens. After labeling the neuraminidase/GO-treated cell surface glycoproteins with NaB3H4, we immunoprecipitated the Con A and DLA receptors which are the target of GO mitogenic action. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the precipitates demonstrated that there exist on the lymphocyte cell surface receptors common to the polyclonal mitogens DLA, Con A, and GO. Because Con A and DLA sterically inhibit GO mitogenic stimulation, the common glycoproteins which represent the necessary sites of oxidative mitogenic action are probably those which are involved in DLA and Con A-triggered mitogenesis, despite the different properties of the two lectins. These differences could be explained by the lower molecular weight receptors of the two lectins which are not identical.     

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

Increased assembly of clathrin occurs in response to mitogenic activation of murine lymphocytes

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.     

Specific mitogenic activity of 8-Br-guanosine 3',5'-monophosphate (Br-cyclic GMP) on B lymphocytes.

8-Br-cyclic GMP has been found to be a specific B cell mitogen; it triggers athymic nude mice spleen cells and "B mice" spleen cells, nylon adherent, anti-theta and complement-treated cells to proliferate. It does not stimulate thymocytes or purified T cells. The kinetics of the response to Br-cyclic GMP and LPS are almost identical. The mitogenic effect of LPS and Br-cyclic GMP is additive when the two mitogens are given together to cells. Spleen cells (C3H/HeJ strain) that did not respond to LPS were triggered by Br-cyclic GMP to make DNA. In order to achieve maximal stimulation by Br-cyclic GMP, the drug had to be in contact with the cells for more than 24 hr. Br-cyclic GMP was found to be mitogenic for spleen cells from five different mouse strains, but not for human leukocytes. DB-cyclic AMP was found to inhibit the DNA synthesis of T lymphocytes after they interacted with Con A; DB-cyclic AMP had no effect on the ability of the B lymphocytes to be transformed by LPS. The differential effects of cyclic nucleotides on B vs. T lymphocytes are discussed.     

In vitro antigen-specific antibody response to the species-specific surface protein antigens of typhus group rickettsiae by human peripheral blood mononuclear cells: generation of an antigen-dependent suppressor T cell

We defined conditions suitable for in vitro synthesis of rickettsia-specific antibody by human PBMC cultured with the SPA of Rickettsia typhi or R. prowazekii and without addition of mitogens or polyclonal stimulators. Antibody synthesis, as measured by an enzyme-linked immunosorbent assay, was cycloheximide-inhibitable and antigen-specific. PBMC from individuals with prior rickettsial infection made antibody, whereas PBMC from those receiving vaccine or with undetectable levels of serum anti-SPA antibody did not. Antibody production was T helper cell-dependent because isolated B cells did not generate antigen-specific antibody in the absence of autologous T cells. Furthermore, prior exposure of T cells to high concentrations of SPA led to the generation of an antigen-dependent population of cells capable of suppressing the anti-SPA response when co-cultured with autologous PBMC and optimal SPA concentrations. This system should serve as an effective tool for analyzing the cellular interactions involved in the in vitro regulation of antigen-specific antibody synthesis by human PBMC.     

Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity.

Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.     

Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin.

Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.     

Capping and mitogenesis: a model implicating microfilaments in lymphocyte activation

In lymphocytes cap formation induced by concanavalin A (con A) was found to be concentration dependent on the mitogen in the presence of colchicine, a microtubule disrupting agent. The dose-respone of cap formation under these conditions was similar to mitogen dose-response. In addition, a direct correlation was found between con A capping induced in the presence of colchicine and mitogenic responses with con A alone. Agents such as dibutyryl cyclic AMP, which suppress mitogenic responses, decrease capping. Zinc increases capping when it causes enhancement of mitogenesis and decreases capping when it suppresses mitogenic response. These observations are interpreted on the basis of a model in which binding of con A to surface receptors leads to formation of microfilaments, which might be essential for capping as well as the initiation of DNA synthesis. Thus, the experimental observations in this report lend support to a model implicating the formation of microfilaments as a crucial event in triggering a variety of cellular responses following ligand binding.     

"Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes

To detect nuclear proteins that might be involved in induction of cellular mitogenesis, we examined the effect of various mitogens on early changes in synthesis of nuclear proteins in murine B lymphocytes. Using two-dimensional gel electrophoresis, we found that activation of B cells by mitogens (anti-immunoglobulin antibody, lipopolysaccharide, phorbol 12-myristate 13-acetate (PMA)/A23187) was associated with a rapid and prominent (5-20-fold) increase in the synthesis of a 40-kDa/pI 5.0 nuclear protein, here termed numatrin. Numatrin was found to be absent from the cytosol (soluble fraction) of resting as well as activated B cells and was markedly resistant to DNase/RNase digestion and 2 N NaCl extraction, indicating that this protein is tightly bound to the nuclear matrix. Kinetic studies showed that the increase in synthesis of numatrin was detected 60-120 min following mitogen activation, reached a peak at 16 h, and declined to almost control level by 48 h, correlating with the peak of cellular DNA synthesis. The increase in synthesis of numatrin in normal B cells was found to be associated exclusively with cellular commitment for mitogenesis because activation of B cells by stimuli such as B cell stimulating factor 1, PMA alone, and calcium ionophore A23187, which do not stimulate an increase in DNA synthesis, also failed to induce an increase in the synthesis of numatrin. Inhibition of anti-Ig-induced proliferation (by PMA pretreatment) was associated with a 63% inhibition in the synthesis of numatrin. Addition of 8-mercaptoguanosine to these PMA-treated cells was associated with restoration of the increase in synthesis of numatrin, concomitant with induction of proliferation. Elevated synthesis of numatrin was also detected in the malignant B lymphoma cells: Raji, BAL-17, and WEHI-231. Taken collectively, these results suggest that numatrin, a tightly bound nuclear matrix protein, is a growth-regulated protein which might have an important role in regulation of cellular mitogenesis in normal and malignant B lymphocytes.     

A B Lymphocyte Mitogen Extracted from a Fungus Peziza vesiculosa1

A water-soluble mitogen was extracted with hot-water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable (less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B. Vesiculogen increased antigen-nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated-horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.     

Ionophorous activity and murine B lymphocyte mitogens.

The relationship between ionophorous and B cell mitogen activity has been investigated. Most known ionophores were nonmitogenic for mouse spleen cells. In addition, when tested in a bilayer lipid membrane (BLM) apparatus, most types of B cell mitogens were nonionophorous. However, excitability-inducing material (EIM), a high m.w. polymeric protein, which is a channel-forming ionophore, was a potent mitogen for mouse B lymphocytes. Similarly, keyhole limpet hemocyanin (KLH), a high m.w. polymeric protein, which is a B cell mitogen, is a channelforming ionophore. The mitogenic activities of these two compounds were not due to contamination with endotoxin since they produced weak or absent responses in the limulus lysate clotting and rabbit pyrogenicity assays, and were also mitogenic for spleen cells of endotoxin-low responder C3H/HeJ mice. Both the mitogenic and ionophorous activities of EIM and KLH were dependent on their polymeric structure since dissociation of these compounds into monomeric subunits markedly decreased both activities. However, heat denaturation destroyed their ionophorous ability but preserved their mitogenicity, thereby demonstrating that ionophorous activity was not essential for B cell activation. These data suggest that B cell mitogens do not necessarily act as primary ionophores. However, we propose that these molecules intercalate into the lipid portion of the cell membrane, and that this interaction initiates the process of B cell activation.     

Divalent mitogens as tools in the study of commitment of lymphocytes to proliferation

We have reinvestigated the question of whether lymphocytes are committed to proliferation by an early, relatively brief, exposure to mitogens with conventional multivalent lectin, concanavalin A, and antigen, dinitrophenyl bovine serum albumin and with strictly divalent lectin, succinyl concanavalin A and antigen, di-dinitrophenyl polyethylene oxide. Whereas very brief incubation with multivalent mitogens leads to substantial irreversible stimulation, much longer exposure to divalent mitogens is required. It appears that the stimulatory signal to any given cell is reversible until the onset of DNA synthesis.     

Comparison of intracellular and extracellular mitogenic activity

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.     

Interleukin 2 and a lactogen regulate proliferation and protein phosphorylation in Nb2 cells.

Cell proliferation and protein phosphorylation in response to activation of lactogenic and interleukin 2 (IL-2) receptors were studied in Nb2 cells, a rat T-lymphocyte cell line. Human growth hormone (hGH) and rat IL-2 stimulated Nb2-cell proliferation to approximately the same degree, and the actions of both mitogens were potentiated by phorbol 12-myristate 13-acetate (PMA). A monoclonal antibody specific for the rat IL-2 receptor inhibited the mitogenic actions of rat IL-2, but not those of hGH. Exposure of Nb2 cells to either mitogen for 2-3 h increased phosphorylation of an 18,600-Da protein and decreased phosphorylation of a 15,600-Da protein. PMA also inhibited phosphorylation of the latter protein, but, by itself, PMA did not stimulate phosphorylation of the 18,600-Da protein. Overall, the results suggest that hGH and IL-2 act through separate receptors to stimulate proliferation of Nb2 cells, and that some of the actions of both mitogens may be mediated, in part, through regulation of protein phosphorylation.     

Classification system based on the functional equivalency of mitogens that regulate WI-38 cell proliferation

Analysis of the proliferative response of WI-38 cells to nine mitogens, which in various specific combinations stimulate DNA synthesis in these cultures, delineated three classes of mitogens. Class I includes epidermal growth factor (EGF), fibroblasts growth factor (FGF), platelet-derived growth factor (PDGF), and thrombin (THR); Class II includes insulin-like growth factor I (IGF-I), multiplication stimulating activity (MSA) (the rat homolog of human IGF-II), and insulin; and Class III includes hydrocortisone (HC) or the synthetic analog dexamethasone (DEX). In cultures arrested at low density, members of each of the three classes act synergistically in stimulating DNA synthesis. Any Class I mitogen in combination with any Class II and either Class III mitogen stimulated DNA synthesis of levels observed in 10% serum-supplemented medium. At least some (EGF, FGF, PDGF) and possibly all (THR) of the Class I mitogens are known to act through separate receptor systems. Our experiments using blocking antibodies to the IGF-I receptor confirm that the Class II mitogens all act by binding to IGF-I receptors. Use of the inhibitory synthetic glucocorticoid analog RU 486 confirmed that the Class III mitogens act via the glucocorticoid receptor. Thus, growth factor-induced DNA synthesis in WI-38 cells is apparently mediated by the glucocorticoid receptor (Class III), the IGF-I receptor (Class II), and most interestingly any one of several Class I growth factor receptors.