Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells

Leukimia-associated inhibitory activity suppresses colony and cluster formation in vitro of cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. It was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by α-methylmannose from concanavalin A-Sepharose columns and had an apparent M_r range of 450–550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.     

Isolation of colony stimulating factor from human milk

Human milk contains colony stimulating factor (CSF), a polypeptide growth factor, which stimulates in in vitro bone marrow culture proliferation and differentiation of colony forming granulocytic macrophage progenitor cells (CFU-GM) to form colonies. This activity was not found in either bovine milk or colostrum when assayed in human or mouse bone marrow cells. The human milk CSF activity is destroyed by treatment with proteases. However, neither 6M urea, 4M guanidine hydrochloride, 5 mM dithiothreitol, nor exposure to pH 2 will inactivate the milk derived CSF. Gel filtration and isoelectric focusing indicate that human milk CSF differs biochemically from the other CSFs isolated from various sources and has a molecular weight between 250,000 and 240,000 and an isoelectric point between 4.4 and 4.9.     

Preparative Electrofocusing for Industrial Applications

Abstract

This paper presents a case study of using a multicompartment isoelectric focusing apparatus to determine the isoelectric points and focus preparative quantities of brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3). A separation of PEGylated from unPEGylated forms of granulocyte colony stimulating factor (G-CSF) is described as well. Both BDNF and NT3 have substantially higher pI values in this system than is predicted from sequenced based modeling. Although PEGylated forms of G-CSF can be separated from the unPEGylated forms, separation of protein with differing degrees of PEGylation was not achieved. The paper additionally demonstrates that this technique can be used simultaneously as an analytical and preparative tool, eliminating the need for analytical IEF gels.     

Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro

At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Müllerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Müllerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.     

Serum lipoproteins inhibitors of granulocyte-macrophage (GM) colony formation

Normal and chloroform-extracted human sera, fractionated by Sephadex column chromatography, were tested for inhibitory activity on granulocyte-macrophage (GM) colony formation. This activity was found to be connected with lipoproteins with a molecular weight of about 200,000. Serum native fractions of lipoproteins were isolated and mainly high density lipoproteins (HDL) and very low density lipoproteins (VLDL) were shown to have an unspecific inhibitory activity directed on colony stimulating factor (CSF) action.     

Identification and characterization of a T cell growth inhibitory factor produced by K562 erythromyeloid cells

Cells of the human erythroleukemia cell line K562 constitutively secrete a factor that inhibits human T lymphocyte proliferation induced via CD3/Ti. The factor, termed K-TIF (K562-derived T cell inhibitory factor) is produced in either the presence or absence of fetal calf serum in cultures of K562 cells and can be precipitated by 70% NH4SO4. Gel filtration chromatography on Superose 12 resin by FPLC showed that the inhibitory factor has a molecular weight of approximately 30-35 kDa. A protein of this size, metabolically labeled with [35S]methionine, specifically bound human peripheral blood mononuclear cells. Chromatofocusing with Mono P by FPLC (pH gradient 7.2-5) indicates that the inhibitory factor has an isoelectric point of 6.0-6.4.     

Immunocytology of cultured IgM-forming cells of mouse. II. Purification of phagocytic cell factor and its role in antibody formation

A factor required for the proliferation of IgM-forming tumor cells of mouse was purified approximately 1500-fold from culture supernatant of phagocytic cells through conventional protein fractionation procedures. The isoelectric point of the factor was pH 6.1 ± 0.1 as measured by isoelectric focusing. Its molecular weight was estimated to be approximately 5 × 10⁴ daltons. These characteristics of the factor were not identical with those of the stimulating factor for granulocyte and macrophage colony formation. Antiserum against the purified factor inhibited the growth-promoting activity of the factor. The effect of the antiserum on the normal antibody response of mouse to sheep red blood cells was examined in the tissue culture system. The antiserum inhibited only the late stage in the formation of direct plaque-forming cells.     

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.     

Antigen-dependent heterogeneity of human migration inhibitory factor

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.     

Two migration inhibitory factors with different chromatographic behavior and isoelectric points.

Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).     

A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Inhibitory activity of interleukin 2-activated lymphocytes on human granulocyte precursors (CFU-g)

Interleukin 2-activated lymphocytes (lymphokine-activated killer [LAK] cells) cultured from 2 to 14 days were added to the cultures of granulocyte precursors (CFU-g). The LAK cells inhibited colony formation of granulocyte precursors; LAK cells cultured for five days showed the strongest inhibitory activity on colony formation. The presence of cell-to-cell interaction between LAK cells and bone marrow mononuclear cells (BMNC) in CFU-g assays emphasized the LAK cell-derived colony inhibitory activity (LAK-CIA), but cell-to-cell interaction was not always a requirement for LAK-CIA, since LAK cells were also found to inhibit colony formation without such interaction. This report shows that LAK cells can inhibit in vitro colony formation of granulocyte precursors. We therefore concluded that the observed CIA is caused by soluble factor(s) derived from LAK cells, and that E-rosette-forming cells are manifesting LAK-CIA.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

Partial purification and chemical characterization of macrophage cytotoxicity factor (MCF, MAF) and its separation from migration inhibitory factor (MIF)

Macrophage cytotoxicity factor (MCF) was purified in 3 consecutive steps including adsorption chromatography on Matrex Gel Red A, hydrophobic chromatography on phenylalanine-Sepharose, and isoelectric focusing. MCF was characterized as a protein with a m.w. of approximately 30,000 by gel filtration on Sephadex G-100 with 2 isoelectric points at 7.4 and 8.4 in the presence of urea. The unpurified supernatant was fairly stable provided that manipulations favoring adsorption to membrane materials used for dialysis or ultrafiltration were omitted. The partially purified preparation was highly unstable. Trypsin treatment did not affect MCF activity, whereas chymotrypsin destroyed it. Treatment with glycosidases and neuraminidase or cultivation of cells in the presence of 2-deoxy-D-glucose or tunicamycin did not impair the MCF activity. MCF was separated from migration inhibitory factor (MIF) by 2 methods: first, isoelectric focusing in the presence of urea, and second by gel filtration on Ultrogel. MCF could be separated from interferon by chromatography on poly(I)-Sepharose.     

Human leukocyte inhibitory factor (LIF): two distinct molecular species.

Human leukocyte inhibitory factor or LIF was generated in vitro by stimulating blood lymphocytes with concanavalin A (Con A). The control and Con A active supernatants were partially purified by gel filtration on Sephadex G-100. The fraction containing LIF (68,000 daltons) activity was then subjected to isoelectric focusing (pH 3 to 10 ampholines) in a sucrose gradient. Two LIF activities were reproducibly recovered by this procedure. One molecular form was found to have an isoelectric point of approximately pH 5.0 and the other approximately pH 8.5. Both molecular species were rechromatographed on Sephadex G-75 and found to have the same apparent m.w. (68 to 75,000). Furthermore, the biologic activity of both factors was destroyed after treatment with diisopropylphosphofluoridate, suggesting that they may be esterases.     

Expression of the fusion protein recombinant human granulocyte-macrophage colony stimulating factor and leukemia inhibitory factor in a baculovirus vector system

A fusion gene coding human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) cDNAs was inserted into the transfer vector pSXIVVI⁺ X3 with the control of Syn and XIV promoters. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). The fusion protein recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) could be synthesized in cells infected with recombinant virus at a level of about 23% of their total cellular protein. Activity analysis of the fusion protein in infected cells revealed that it exhibited the dual activities of GM-CSF and LIF. Western blot analysis of the expressed fusion protein in infected larvae showed that the virus-mediated fusion protein, with a molecular weight of ∼35 kDa, is confirmed with immunoreactivity. Received 02 December 1998/ Accepted in revised form 07 May 1999     

Transforming growth factor-beta-like activity is secreted by rabbit renal cortical tubular cells in primary culture.

The activity of an autocrine growth factor in a medium conditioned by cultured rabbit renal cortical tubular cells was investigated. Little stimulatory growth activity for tubular cells was observed in the conditioned medium, and inhibitory activity was seen only in acidified conditioned medium. This factor stimulated the colony formation of NRK 49F cells in soft agar only with epidermal growth factor and inhibited the DNA synthesis of primary cultured rat hepatocytes, and its molecular weight was about 25 kDa. The factor was neutralized by the specific antibody to transforming growth factor (TGF)-beta 1. These results indicate that renal tubular epithelial cells can produce latent TGF-beta in primary culture.     

Effect of stem cell inhibition factor and macrophage inhibition factor on mouse spleen cell exocolonization and migration]

The migration activity of the spleen cells from intact mice is inhibited by the stem cell inhibitory factor (SCIF) released by lymphocytes treated with antilymphocytic globulin. The degree of the migration inhibition is proportional to the activity of SCIF in the colony-formation inhibition. The macrophage-migration inhibitory factor (MIF), obtained in the H-2 system exhibited a stimulating effect on the colony formation in mice when used in vitro for the treatment of bone marrow transplants. This activity of MIF corresponds to its migration-inhibitory effect on the spleen cells. Incubation of the bone marrow cells with MIF for 30 minutes is more effective than the 2-hour treatment. The observed effects are interpreted as an indication of non-identity of SCIF and MIF.