Purification of MEA, a mast cell growth-enhancing activity, to apparent homogeneity and its partial amino acid sequencing

A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9.     

The human lung fibroblast cell line, MRC-5, produces multiple factors involved with megakaryocytopoiesis

The human lung fibroblast cell line MRC-5 constitutively produces a megakaryocyte potentiator activity, identified in murine bone marrow liquid culture assays for acetylcholinesterase and megakaryocyte colony assays in the presence of low concentrations of IL-3. The production levels of this activity were increased after stimulation with the phorbol ester analog, mezerein, and the calcium ionophore, A23187. Complete purification of a protein having this activity from conditioned media of induced MRC-5 cells was achieved using gel filtration and ion exchange chromatography. The first 14 residues of the purified protein identified by amino-terminal sequencing were identical to the first 14 residues of IL-6. Recombinant human IL-6 was tested and found to promote megakaryocyte growth. IL-1 beta, another component detected in MRC-5 conditioned media, was unable to promote megakaryocyte colony formation but did reduce the concentration of IL-6 necessary to support megakaryocyte colony formation. Immunoprecipitation using rabbit antiserum prepared against IL-6 removed the megakaryocyte growth activity found in MRC-5 conditioned media. Thus, connective tissue cells such as fibroblasts in the bone marrow may co-stimulate thrombocytopoiesis via IL-6 and, possibly, via IL-1 production.     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Terminal deoxynucleotidyl transferase (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.     

Interaction of interleukin-5 with its receptors on murine leukemic BCL1 cells and its implication in biological activity

Interaction of interleukin (IL)-5 with its receptors on murine leukemic cell line, BCL1 cells was examined. 125I-labeled recombinant murine IL-5(rmIL-5) bound specifically to high-affinity receptors on BCL1 cells. rmIL-5, which was about 2500-fold more active than recombinant human IL-5(rhIL-5) in IgM-inducing activity on BCL1 cells, also showed about 5000-fold higher affinity to receptors. These results suggest that the bioactivity of IL-5 correlates with its receptor-binding activity. When disulfide bond formation was blocked, rmIL-5 dissociated into a monomer and lost its biological activity. This monomeric form of rmIL-5 also lost its ability to bind to cells, suggesting that dimer formation is essential for the biological activity of IL-5.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Interleukin 3 enhanced interleukin 2-dependent maturation of NK progenitor cells in bone marrow from mice with severe combined immunodeficiency

Human recombinant interleukin 2 (IL 2) and highly purified murine interleukin 3 (IL 3) were tested for their ability to generate NK activity in bone marrow cells from mice with severe combined immunodeficiency. IL 2 alone could dose dependently induce NK activity in marrow cells as determined by cytotoxicity against YAC-1 target cells. It was demonstrated that IL 3 had dual effects on the generation of NK activity in this culture system. The addition of IL 3 resulted in inhibition of NK cell activity seen at high concentrations of IL 2. In contrast, when IL 3 was added together with low concentrations of IL 2, the generation of NK cells as judged by cytotoxicity assay as well as the appearance of cells with NK phenotypes was markedly augmented. In some experiments, mice were treated with 5-fluorouracil (5-FU) to eliminate relatively differentiated NK precursors from bone marrow cells. It was noted that the residual immature marrow cells from 5-FU-treated mice showed little NK activity even after the culture with high concentrations of IL 2. Importantly, IL 3 could induce the generation of NK activity from 5-FU-treated marrow cells in the presence of IL 2. Kinetic studies indicated that NK activity was appreciably generated from 5-FU-treated marrow cells when preincubated with IL 3 at least for 12 hr and subsequently cultured with IL 2. The cells bearing IL 2 receptors appeared in 5-FU-treated marrow cells, even though cultured only with IL 3, which implied that IL 3 could support the development of very primitive NK cells from IL 2-unresponsive to IL 2-responsive states. These results suggested that IL 3 might play a crucial role for the IL 2-induced generation of NK cells in bone marrow through promoting the expression of IL 2R on NK progenitor cells.     

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes.

Recent reports have demonstrated fusion of the TEL gene on 12p13 to the JAK2 gene on 9p24 in human leukemias. Three variants have been identified that fuse the TEL pointed (PNT) domain to (i) the JAK2 JH1-kinase domain, (ii) part of and (iii) all of the JH2 pseudokinase domain. We report that all of the human TEL/JAK2 variants, and a human/mouse chimeric hTEL/mJAK2(JH1) fusion gene, transform the interleukin-3 (IL-3)-dependent murine hematopoietic cell line Ba/F3 to IL-3-independent growth. Transformation requires both the TEL PNT domain and JAK2 kinase activity. Furthermore, all TEL/JAK2 variants strongly activated STAT 5 by phosphotyrosine Western blots and by electrophoretic mobility shift assays (EMSA). Mice (n = 40) transplanted with bone marrow infected with the MSCV retrovirus containing either the hTEL/mJAK2(JH1) fusion or its human counterpart developed a fatal mixed myeloproliferative and T-cell lymphoproliferative disorder with a latency of 2-10 weeks. In contrast, mice transplanted with a TEL/JAK2 mutant lacking the TEL PNT domain (n = 10) or a kinase-inactive TEL/JAK2(JH1) mutant (n = 10) did not develop the disease. We conclude that all human TEL/JAK2 fusion variants are oncoproteins in vitro that strongly activate STAT 5, and cause lethal myelo- and lymphoproliferative syndromes in murine bone marrow transplant models of leukemia.     

Strong immunostimulation in murine immune cells by Lactobacillus rhamnosus GG DNA containing novel oligodeoxynucleotide pattern

Whole cells, cell wall components and some soluble factors from Lactobacillus rhamnosus GG (LGG) are known to invoke immune responses as they interact with animal and human immune cells. In the present study, we found that chromosomal DNA from LGG is a potent inducer of splenic B cell proliferation, CD86/CD69 expression and cytokine production in mice. In the genomic DNA of LGG we discovered TTTCGTTT oligodeoxynucleotide (ODN) ID35, which has a potent activity in a number of immunostimulatory assays. Phosphorothioate backbone is not required for the activity of ID35. The ODN ID35 showed levels of activity comparable with those induced by the murine prototype ODN 1826 in B cell proliferation, CD86/CD69 expression, interleukin (IL)-6, IL-12, IL-18, interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) mRNA expression and IFN-gamma/IL-12p70 protein production assays. Additionally, ID35 appeared to be equally active in both murine and human immune cells. These stimulatory effects are due to TTTCGTTT motif located in the 5' end of ID35. In this study we demonstrate for a first time that, DNA from LGG is a factor of immunobiotic activity. Furthermore, ODN ID35 is the first ODN, with such a strong immunostimulatory activity to be found in immunobiotic bacterial DNA.     

Generation of NK cell activity from human bone marrow

This study was designed to examine the effect of interleukin 2 (IL 2) on cytotoxic activity of human bone marrow cells and to characterize the IL 2-dependent killer cells and the cell population required for their induction. We have demonstrated that the most aggressive IL 2-dependent killer cells (directed against leukemic and solid cancer targets) exhibited LGL morphology and expressed NK cell-associated antigens NKH1 and CD16, but not T cell-associated antigens CD3, CD4, CD5, or CD8. Similarly, the bone marrow cell population necessary for induction of killer cells with highest cytotoxic activity displayed NK cell surface characteristics, as exemplified by CD16 and Leu-7 antigens. On the contrary, very low or no lytic activity was generated from the bone marrow cell population expressing T cell markers CD3 and CD5. These data indicate that the IL 2-dependent bone marrow-derived killer cells with antitumor activity were activated NK cells. If T cells are involved at all in IL 2-dependent bone marrow killing, their potency is inferior to that of activated NK cells. The clinical applications of these studies are discussed.     

Ribonucleotide reductase M2 subunit sequences mapped to four different chromosomal sites in humans and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines

The sites of sequences homologous to a murine cDNA for ribonucleotide reductase (RR) subunit M2 were determined on human and murine chromosomes by Southern blot analysis of interspecies somatic cell hybrid lines and by in situ hybridization. In the human genome, four chromosomal sites carrying RRM2-related sequences were identified at 1p31----p33, 1q21----q23, 2p24----p25, and Xp11----p21. In the mouse, M2 sequences were found on chromosomes 4, 7, 12, and 13 by somatic cell hybrid studies. By Southern analysis of human hydroxyurea-resistant cells that overproduce M2 because of gene amplification, we have identified the amplified restriction fragments as those that map to chromosome 2. To further confirm the site of the functional RRM2 locus, two other cDNA clones, p5-8 and S7 (coding for ornithine decarboxylase; ODC), which are coamplified with RRM2 sequences in human and rodent hydroxyurea-resistant cell lines, were mapped by Southern and in situ hybridization. Their chromosomal map positions coincided with the region of human chromosome 2 (p24----p25) that also contains one of the four RRM2-like sequences. Since this RRM2 sequence and p5-8 and ODC are most likely part of the same amplification unit, the RRM2 structural gene can be assigned to human chromosome 2p24----p25. This region is homologous to a region of mouse chromosome 12 that also carries one of numerous ODC-like sequences. In an RRM2-overproducing mouse cell line, we found amplification of the chromosome 12-specific restriction fragments. Thus, we conclude that mouse chromosome 12 carries the functional locus for RRM2.     

Recombinant murine and human IL 1 alpha bind to human endothelial cells with an equal affinity, but have an unequal ability to induce endothelial cell adherence of lymphocytes

Consistent with the reports of others, we have demonstrated that human peripheral blood lymphocytes adhere to cultured human umbilical vein-derived endothelial cells (EC) in vitro. In our studies adherence was increased twofold to threefold by a 6-hr preincubation of the EC with IL 1. Recombinant human IL 1 alpha induced a maximal adherence response at less than 1 U per 2 X 10(4) EC. In contrast, recombinant murine IL 1 alpha was found to be 250- to 1250-fold less active in the adherence assay, based on units of IL 1 activity defined by the murine thymocyte proliferation assay. Moreover, when EC were preincubated with excess murine IL 1, no inhibition of the adherence-inducing effect of human IL 1 was noted. To characterize further this dichotomy of biological potency of murine and human IL 1 on the adherence assay, IL 1 binding studies were initiated. Recombinant human and murine IL 1 alpha were equally effective in inhibiting the binding of 125I-labeled human and murine IL 1, based on both micrograms of protein and units of IL 1 activity. The results of this study demonstrate that although human and murine IL 1 bind with equal affinity to receptors on human EC, human IL 1 is significantly more potent at inducing the increased EC adhesiveness for lymphocytes. The implications of these results for endothelial cell IL 1 receptor function are discussed.     

Modulation of the adhesion of hemopoietic progenitor cells to the rgd site of fibronectin by interleukin 3

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions and also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by α5b?1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of α5 and one antibody against b?1 were used to study expression of the integrin. By flow cytometry the expression of α5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation. © 1995 Wiley-Liss, Inc.     

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.     

A short synthetic peptide fragment of human interleukin 1 with immunostimulatory but not inflammatory activity

Short peptide fragments of human and murine interleukin 1 (IL 1) were synthesized on the basis of their predicted exposure on the surface of the molecule in an attempt to identify the minimal structure responsible for the immunostimulatory activity of IL 1. One of these peptides, a fragment of nine residues of human IL 1 beta (VQGEESNDK, fragment 163-171), showed high T cell activation capacity, as judged by its ability to stimulate murine thymocyte proliferation and to potently induce interleukin 2 production in spleen cells. On the other hand, the 163-171 peptide was devoid of prostaglandin-inducing capacity in vitro and pyrogenic activity in vivo, two inflammatory features peculiar to the entire hu IL 1 beta molecule. Thus we propose that this peptide may represent one of the portions of hu IL 1 beta responsible for its immunostimulatory capacity.