Mitogenic factor from inbred guinea pigs. I. Isolation of the factor

Methods are described for the reproducible elicitation of mitogenic factor (MF) from antigen-sensitive lymphocytes of inbred strains of guinea pigs. The use of inbred animals minimizes complications due to histocompatibility factors. Each of several antigens tested was effective. Mitogenic factor is released in vitro as early as 6 hr after stimulation of lymphocytes by antigen. It was obtainable from serum-free cultures in which medium RPMI-1640 was used; this should facilitate isolation of MF. The addition of 5 mMl-cysteine to cultures substantially improved the yield of MF. MF was obtained from cultures of lymph node cells of highly purified small lymphocytes, which indicates that the small lymphocyte is the source of MF in the guinea pig. It was shown that MF can induce mitosis as well as blast transformation in non-immune lymph node cells. MF from a given strain of guinea pig is capable of stimulating lymphocytes of another strain.     

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.     

A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure.

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 X 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 X 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10⁻⁹ M) with baFGF (40 μg/ml) and EGF(0.01 μg/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E₂ release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network. © 1996 Wiley-Liss, Inc.     

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.     

Separation of lymphocyte mitogen from lymphotoxin and experiments on the production of lymphotoxin by lymphoid cells stimulated with the partially purified mitogen: a possible amplification mechanism of cellular immunity and allergy.

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration. CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence fo lymphotoxin (LT), another guinea pig lymphokine.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Studies on the role of lymphocyte-activating factor (Interleukin 1) in antigen-induced lymph node lymphocyte proliferation

The in vitro proliferation of primed lymph node lymphocytes (LNL) in response to the soluble antigen ovalbumin (OVA) was dependent upon the presence of adherent cells. Restoration of OVA-induced LNL proliferation could be achieved by addition of highly purified lymphocyte-activating factor (LAF; Interleukin 1, IL 1): LAF (IL 1) did not stimulate LNL proliferation in the absence of the priming antigen or T lymphocytes. Furthermore, treatment of the LNL with antimacrophage serum completely blocked the ability of the LNL to respond to OVA and LAF (IL 1), suggesting that the residual macrophages in the LNL population were necessary to provide an additional function or signal, possibly antigen presentation, in conjunction with LAF (IL 1). These data therefore support the two signal hypothesis of macrophage-mediated lymphocyte activation and demonstrate the ability of LAF (IL 1) to provide one of these signals.     

Partial characterization of a mitogenic factor with somatomedin-like activity produced by culture WI-38 human fibroblasts

Conditioned serum-free medium (CSFM) obtained from WI-38 human fibroblasts was found to contain a mitogenic factor(s) with somatomedin (SM)-like activity. Treatment of the cells with cycloheximide eliminated the SM-like activity in CSFM, suggesting that these cells produce and release the activity. Gel filtration revealed that the fibroblast SM-like activity (FSLA) had a molecular size near 45,000. Isoelectric focusing of this FSLA yielded 2 bands of SM activity with pIs of 4.7 and 6.1, and corresponding molecular sizes of ～29,000 and 16,500, respectively. The FSLA obtained by gel filtration revealed parallel dose response curves with a basic SM in a SM radioreceptor and radioimmunoassay and stimulated: (1) ³⁵So₄ uptake by hypophysectomized rat cartilage; (2) (U-¹⁴C) glucose oxidation is isolated rat adipocytes; and (3) (³H) thymidine uptake and cell division in these same WI-38 fibroblasts. Out studies indicate that this FSLA and basic SM are similar but not identical.     

Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: Resemblance with platelet-derived growth factor

A human osteosarcoma cell line, U-2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophoresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. ¹²⁵I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with ¹²⁵I-ODGF for binding to human-cultured glial cells.     

Inhibition of interleukin 1 activity by a factor in submandibular glands of rats

With the sequential use of ammonium sulfate precipitation, gel filtration and chromatofocusing, we have partially purified from extracts of the submandibular glands of rats a factor (referred to as submandibular gland's immunosuppressive factor or SMG-ISF) capable of inhibiting the in vitro proliferation of mitogen- and antigen-stimulated murine lymphocytes. The semi-purified suppressor fractions had an isoelectric point of 4.4 to 4.5 and consisted of at least three molecular species. These active fractions suppressed the mitogenic effects of Concanavalin A phytohemagglutinin, and lipopolysaccharide. In vitro immune reactions such as the mixed lymphocyte culture MLC reaction and the production of cytotoxic T lymphocytes (CTL) across major histocompatibility barriers in mice were also suppressed. These in vitro immunosuppressive effects required the addition of the suppressor fractions early after the initiation of the cultures and were reversed if the factor was removed from the cultures at least 48 to 72 hr before the completion of the assays. The active fractions did not affect the proliferation of CTLL 2 cells induced by interleukin 2 (IL 2), but inhibited the mitogenic and co-stimulatory effects of IL 1 on mouse thymocytes, and in this effect showed a dose-response relation suggestive of a competitive mechanism. These characteristics of SMG-ISF indicate a specific inhibition of the activity of IL 1.     

Tissue factor is not involved in the mitogenic activity of factor VIIa

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.     

A macrophage-derived factor different from interleukin 1 and able to induce interferon-gamma and lymphoproliferation in resting T lymphocytes

A novel monokine different from interleukin 1 (IL-1) is secreted from human or murine macrophages stimulated with galactose oxidase, a well-characterized enzyme able to induce marked polyclonal activation of lymphocytes. This monokine, preliminarly termed macrophage-derived blastogenic factor (MBF), stimulates resting T lymphocytes to produce interferon (IFN)-gamma and to proliferate. The apparent MW of MBF ranges between 29,000 and 35,000, although some active fractions show smaller MW ranging from 2000 to 7000, as demonstrated by gel filtration. The biological relationship between MBF and IL-1 was examined and it was found that MBF (1) is able to induce IFN-gamma production and proliferation by T lymphocytes in the absence of other inducers, (2) is not able to activate PHA-stimulated C3H mouse thymocytes, (3) is not able to induce production of IFN-beta by fibroblast cultures, (4) is resistant to proteolytic enzymes, and (5) is not neutralized by antibodies to IL-1. MBF was released in the macrophage supernatants in two waves after the oxidation process, namely, after 15 min and 2.5 hr, and each wave was capable of inducing lymphocyte activation at dilutions up to 1:32. Because the oxidation of galactose residues on cell surface structures is considered a general feature of lymphocyte activation whatever the inducer, it seems that MBF may be a mediator involved in mitogenic activation of T cells leading to IFN-gamma production and proliferation.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Platelet-derived growth factor in chemotactic for fibroblasts

Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet- derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.     

Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography

Summary A mitogenic factor which promotes quail myoblast proliferation has been purified some 10⁵-fold from chick embryo extract by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15000 to 17000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly suggest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule(s) and that it is possibly involved in the regulation of myogenesis. This work was partly supported by a grant from the National Center of Neurology and Psychiatry (NCNP grant 86-01) of the Ministry of Health and Welfare, Japan.     

Isolation and characterization of factor X activator from the venom of Vipera aspis aspis

1. A factor X activator was isolated from the venom of Vipera aspis aspis (Aspic viper) by gel filtration and ion-exchange chromatography. 2. The purified activator has a mol. wt of 75,000 and an isoelectric point of 4.6. Upon reduction, this activator migrated as two bands with mol. wts of 16,000 and 14,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. 3. The activator from V. a. aspis venom shortened activated partial thromboplastin time (APTT) of normal plasma and factor IX-deficient plasma from humans. 4. Factor X incubated with isolated activator and calcium ions drastically shortened APTT of factor X-deficient plasma and expressed hydrolytic activity against synthetic substrates for factor Xa, however no hydrolytic activity was detected with the activator alone, indicating that the activator converted factor X to the active form.     

Interleukin 1 increases collagen type IV production by murine mammary epithelial cells

The effects of human interleukin 1 (IL 1) on collagen type IV production by normal mouse mammary epithelial cells were examined. Human IL 1 was derived from the culture media of peripheral blood monocytes or placental cells that were stimulated with silica. Although crude culture media of silica-stimulated monocytes or placental cells had no enhancing activity for type IV collagen production, IL 1-containing fractions obtained by Sephacryl S-200 gel chromatography and isoelectrofocusing from such media possessed considerable activity. To confirm the effects of IL 1 on collagen production, human monocyte-derived IL 1 was highly purified by sequential isoelectrofocusing, anion-exchange (AX 300), high-performance liquid chromatography (HPLC), and HPLC gel filtration (TSK 3000). The same HPLC gel filtration fractions contained both an activity that stimulated collagen synthesis by mammary cells and thymocyte growth-promoting activity. These activities of IL 1 differed from a number of other factors, such as epidermal growth factor and another factor produced by placental cells that stimulated type IV collagen production but not thymocyte proliferation. In fact, IL 1 induced 100-fold less collagen type IV production by mammary epithelial cells than was needed to induce thymocyte proliferation. Our data suggest that IL 1-like molecules, which reportedly are produced by many tissue cell types, may therefore play a role in promoting a basement membrane formation at stromal-epithelial boundaries.