Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

B cell precursor growth-promoting activity. Purification and characterization of a growth factor active on lymphocyte precursors.

We have used a biological assay system we developed to biochemically purify a previously uncharacterized murine lymphopoietic growth factor designated lymphopoietin 1 (LP-1). This factor is capable of stimulating the proliferation and extended maintenance of precursor cells of the B lineage. A stromal cell line producing LP-1 was established after transfection of primary stromal cultures with a plasmid encoding the transforming genes of SV40. This factor was purified to a single 25-kD species from the culture supernatant of an adherent stromal cell line. This material acts on immature lymphocytes, it binds to specific receptors on cells, and is distinct from previously described hematopoietic factors. LP-1 has been purified some 10(7)-fold with an overall recovery of 35%. The purified protein exhibits a specific activity of approximately 4 X 10(6) U/micrograms of protein and is active at a half-maximal concentration of 10(-13) M.

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Role for cyclic AMP in the postreceptor control of cytokine-stimulated stromal cell growth factor production.

We examined the role of augmented formation of intracellular cyclic AMP (cAMP) in the mediation of stromal cell growth factor production that occurs constitutively or upon cytokine stimulation. Clonal murine marrow adherent cell lines were stimulated under serum-free conditions by interleukin-1 (IL-1) or lipopolysaccharide (LPS) and one (+/+ -1.LDA11) was found to produce low quantities of granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF identity was confirmed by the ability of supernatants from stromal cells to promote proliferation of the factor-dependent cell line FDC-P1, neutralization of this activity by antiserum to GM-CSF, and by Northern blot analysis. However, optimal concentrations of IL-1 and tumor necrosis factor-alpha (TNF-alpha), in combination, led to synergistic (greater than 5-fold higher quantity) GM-CSF production compared with either stimulus alone in the +/+ -1. LDA11 cell line, capable of GM-CSF production after only single stimulation with IL-1 or LPS. In addition, synergistic stimulation by IL-1 and TNF-alpha led to equivalent high amounts of GM-CSF in another cell line incapable of GM-CSF production after induction with only IL-1 or LPS. Any of several means to raise intracellular cAMP levels, including addition of 8-bromo-cyclic AMP (8Br cAMP) (0.25-1mM), pertussis toxin (20-100 ng/ml), or addition of prostaglandin E1 (PGE1) (1 microM), failed to stimulate GM-CSF production alone and strongly inhibited GM-CSF production in stromal cells stimulated by IL-1, LPS, or the synergistic combination of IL-1 and TNF-alpha. In addition, PGE1 and pertussis intoxication were agonists of adenylate cyclase in membranes of marrow adherent cells, whereas IL-1 and LPS were not. The role for regulators of intracellular cAMP was specific because any of the cAMP agonists alone, or in the presence of cytokine stimulators of stromal cells, strongly enhanced IL-6 production, an event known to be cAMP-responsive. Thus, acute formation of intracellular cAMP is a negative regulator of stromal cell GM-CSF production mediated by cytokines, but positively regulates IL-6 production and may be an important determinant of cytokine-directed marrow microenvironmental function. These findings on the requirement for augmentation versus inhibition of cytokine-mediated production of hemopoietic growth factors might be applied to an analysis of marrow stromal cell heterogeneity.

Interleukin 4 alone or in combination with interleukin 1 stimulates 3T3 fibroblasts to produce colony-stimulating factors.

Colony-stimulating activity (CSA) can be produced by fibroblasts when stimulated by interleukin 1 (IL-1). We show that like IL-1, interleukin 4 (IL-4) can stimulate 3T3 fibroblasts to produce CSA. Biological and molecular analyses show that a significant portion of the CSA is colony-stimulating factor 1 (CSF-1) and granulocyte colony-stimulating factor (G-CSF). CSF-1 production in cells stimulated with a combination of both IL-1 and IL-4 was greater than that observed when cells were stimulated with either cytokine alone. However, the data show a synergistic induction of the expression of high levels of G-CSF mRNA and protein in cells incubated in the presence of both IL-1 and IL-4. The concentration of G-CSF in supernatants from cells stimulated with both IL-1 and IL-4 was at least tenfold higher than that measured in supernatants harvested from cells stimulated with either IL-1 or IL-4 alone. Previous investigations have shown that IL-4 had direct effects on hematopoietic progenitor cell growth. The studies described herein indicate that IL-4 is also involved in the regulation of hematopoiesis in an indirect manner, that is, by playing a role in the regulation of hematopoietic growth factor production.

A novel mast cell growth factor (MCGF-3) produced by marrow-adherent cells that synergizes with interleukin 3 and interleukin 4.

A clonal marrow-adherent stromal cell line, +/+-1 LDA11, was derived and found to produce hemopoietic stimulatory activity for an interleukin 3 (IL-3)-dependent mast cell line, NFS/N1. This factor-dependent mast cell line displayed restricted growth factor responsiveness to only IL-3, interleukin 4 (IL-4), and the stromal cell-produced factor. The factor produced by stromal cells was distinguished from IL-3 and IL-4 and was characterized biochemically. This factor appears to be a novel mast cell growth factor (MCGF-3) capable of synergizing with IL-3 and IL-4. It may have broader reactivity in hemopoiesis than simply IL-3-dependent mast cells, and it may prove relevant to stromal cell-mediated hemopoiesis.

Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.

Biochemical separation of interleukin 2.

Interleukin 2 (IL-2) is a class of T cell growth factors produced by T cells of a number of species. The unique growth-promoting properties of these molecules allow the development of antigen-specific effector T cell lines which can be used to analyze the molecular basis of lymphocyte interactions. A murine T cell tumor line has been identified as a source of IL-2. The purification and biochemical properties of murine IL-2 are described, and compared with rat and human Il-2.

The treatment of induced immune deficiency with interleukin-2.

In vivo generation of alloreactive cytotoxic T lymphocytes (CTL) was found to be inhibited by treatment of mice with either cyclophosphamide or the glucocorticoid, hydrocortisone acetate. The effects of these immunosuppressive agents could be overcome, however, by the in vivo administration of IL-2 from both murine and human sources. Both human IL-2 derived by recombinant DNA techniques as well as the natural protein from mouse and man all reverse the unresponsive state. A single injection of IL-2 was sufficient to reverse the effect of cyclophosphamide treatment, while additional injections with as little as 8 micrograms of protein ablated the steroid-induced suppression. Furthermore, the responder cells generated in vivo following IL-2 therapy were shown to be antigen specific in terms of their lytic capacity. Thus, IL-2 therapy appears to restore the in vivo responsiveness of immunosuppressed recipients to allogeneic tumor cell challenge. These data demonstrate the importance of IL-2 as an immunoregulatory molecule in vivo and suggest its future use as a potent therapeutic.

Interleukin-1 is identical to hemopoietin-1: studies on its therapeutic effects on myelopoiesis and lymphopoiesis.

Conditioned medium from the human tumor cell line HBT 5637 possesses a unique hematopoietic activity, originally termed hemopoietin-1. Hemopoietin-1 alone does not stimulate bone marrow colony formation or proliferative responses in vitro, but rather potentiates responses to other hematopoietic growth factors, such as CSF-1 and GM-CSF. In studies designed to characterize the molecular nature of this factor, it was found by molecular, biochemical biological and serological criteria that all the hemopoietin-1 like activity could be attributed to IL-1 alpha. The therapeutic potential of IL-1 was then tested in a system where myelopoiesis is depressed by whole body irradiation. After 750 R irradiation, mice were administered IL-1 twice daily for the duration of the experiment. Mice which received IL-1 treatment had an accelerated recovery of marrow colony forming capacity which was also reflected by significantly higher blood neutrophil levels as compared to control irradiated mice. IL-1 treated irradiated mice also had a significant increase in resistance to bacterial challenge 14 days post irradiation. Thus, IL-1 treatment was effective in augmenting myelopoiesis following sublethal whole body irradiation. The effects of the IL-1 treatment on the recovery of lymphocyte numbers was also assessed. Here the IL-1 treated irradiated mice had fewer lymphocytes and depressed mitogen responses by spleen cells. Indeed the thymus of the IL-1 treated irradiated mice remained chronically hypoplastic for the duration of the experiment. Although IL-1 treatment increased myeloid progenitors in the bone marrow, it caused a decrease in the frequency of pre-B cells. Thus, IL-1 administration is an effective treatment for accelerating myeloid recovery following the cytoreductive effects of irradiation, but the myelopoietic augmentation may be at the expense of lymphoid recovery.

Molecular characterization of interleukin 2.

The isolation of specific T cell growth factors termed interleukin 2 (IL 2) is changing the approach to understanding T cell function. This class of growth factors has allowed te establishment of a number of human and murine T cell lines. We summarize the biochemical properties of human and murine IL 2. Studies have been initiated to isolate mRNA encoding for IL 2. Such RNA can be translated in rabbit reticulocyte lysates yielding IL. 2. This RNA may be useful for the development of probes to isolate lymphokine genes.

Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen.

A cell surface protein known as T4 (CD4, Leu3), Mr = 55,000, is expressed on the subset of human T lymphocytes which provides helper function for B cell and cytotoxic T cell activities. We show that T4 is constitutively phosphorylated and that phorbol myristate acetate (PMA) induces a rapid serine phosphorylation which is followed by a fast dephosphorylation. Within 5 min after PMA treatment, there is a 24% reduction of T4 on the cell surface, by 4 h the loss is nearly complete, and by 20 h T4 is re-expressed. Addition of antigen to a T4+ antigen-reactive T cell clone induces both phosphorylation and dephosphorylation with kinetics similar to that described for PMA. Antigen also causes reduction of cell surface T4, although to a lesser degree than stimulation with PMA. The rapid phosphorylation/dephosphorylation of T4 as well as its movement from the cell surface suggest that T4 functions as a receptor for an unknown ligand.

Clonal growth of murine pre-B colony-forming cells and their targeted infection by a retroviral vector: dependence on interleukin-7.

The cDNA for interleukin-7 (IL-7) was recently isolated from a stromal cell line derived from a long-term B-lymphoid culture. We report that purified recombinant murine IL-7 can promote the clonal growth in semi-solid culture of a subpopulation of cells expressing the B220 surface antigen from normal murine bone marrow. These colony-forming cells (CFC-Pre-B) give rise to colonies of 20 to 1,000 cells after 7 days in culture. Morphologic examination of cells within the colonies showed a characteristic lymphoid morphology, and histochemical examination demonstrated an absence of markers associated with granulocyte, macrophage, eosinophil, or megakaryocyte differentiation, as well as an absence of hemoglobinization (indicative or erythroid differentiation). IL-7 was found to specifically enhance the infection of CFC-Pre-B but not CFU-GM when the cytokine was present during a 48-hour co-cultivation period between irradiated, retrovirus-producing psi 2 clones and normal mouse bone marrow cells. In contrast, IL-3 enhanced the infection of CFU-GM but not CFC-Pre-B. Thymidine suiciding studies suggest that this targeted infection is due to specific induction of cycling of CFC-Pre-B by IL-7 and CFU-GM by IL-3. These data demonstrate that IL-7 can target retroviral infection into a specific subpopulation of early B-lymphoid cells (CFC-Pre-B), and that IL-7 cannot directly promote the in vitro clonal growth of myeloid committed progenitor cells (ie, CFU-GM).

Phosphorylation of the CD8 antigen on cytotoxic human T cells in response to phorbol myristate acetate or antigen-presenting B cells.

We have used the monoclonal antibody OKT8 to demonstrate that the cluster designation (CD)8 antigen on cytotoxic human T cells undergoes a previously unreported protein modification. Immunoprecipitation of CD8 with OKT8 from a CD8+ and CD4+ T cell line prelabeled with [32P]phosphate demonstrates that CD8 can be constitutively labeled with phosphate. CD8 undergoes rapid and intense phosphorylation in serine upon addition of phorbol myristate acetate to the cells. CD8 phosphorylation is induced upon addition of heterologous, Epstein-Barr virus-transformed B cells, which cause proliferation and are target cells for a cytotoxic CD8+CD4+ T cell line and a CD8+CD4- T cell clone. Phosphorylation induced by targets is dose-dependent, rapid, and followed by a fast dephosphorylation. Epstein-Barr virus-transformed B cells that do not induce proliferation of and are not targets for the CD8+CD4+ line and the CD8+CD4- clone do not induce CD8 phosphorylation. Cloned CD8+CD4- cells that proliferate in response to target cells, but lyse them only in the presence of lectin do not undergo target cell-induced CD8 phosphorylation. These data suggest that induction of CD8 phosphorylation is antigen-specific and is coincident with the cytotoxic response. Finally, preincubation of effector and target cells with an antibody to a monomorphic determinant of major histocompatibility complex class I antigens reduces target-induced CD8 phosphorylation to a greater extent than antibody to a major histocompatibility complex class II subregion (DR) monomorphic determinant, reinforcing the notion that major histocompatibility complex class I antigens interact with CD8+ cells.

Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression.

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration-dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage-independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL-6 may have indirect as well as direct influences on hematopoietic cell proliferation.

Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor.

The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1 beta, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum, together with recombinant GM-CSF that had been radiolabeled with 125I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis.

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain.

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.

Administration of IL-7 to mice with cyclophosphamide-induced lymphopenia accelerates lymphocyte repopulation.

Lymphopenia was induced in mice by a single injection of cyclophosphamide. IL-7 or a control protein were administered to the mice twice daily and the cellularity and composition of the spleen, lymph node, bone marrow, and thymus were determined at various time points thereafter. In comparison to the control cyclophosphamide-treated mice, animals receiving cyclophosphamide and IL-7 had an accelerated regeneration of splenic and lymph node cellularity. There was no significant difference in the rate of recovery of the bone marrow and thymus of the control and IL-7-treated mice. Assessment of the pre-B cell compartment revealed a dramatic increase in total pre-B cell numbers in the spleen and bone marrow of the IL-7-treated mice as measured by both flow microfluorimetry and a pre-B cell colony-forming assay. This was followed in a few days by a significant increase in surface IgM+B cell numbers to levels above normal values in both the spleen and lymph node. IL-7 administration to cyclophosphamide-treated mice also resulted in an accelerated recovery of peripheral CD4+ and CD8+ cell numbers in the spleen and lymph node. The numbers of CD8+ cells were increased by twofold over normal levels in cyclophosphamide-treated mice receiving IL-7. Myeloid recovery was determined in cyclophosphamide treated mice by assessing the numbers of CFU-granulocyte-macrophage and Mac 1+ cells. There was no significant difference in myeloid recovery between cyclophosphamide-treated mice receiving IL-7 or control protein. These results suggest that administration of IL-7 after chemical-induced lymphopenia may have therapeutic benefits in shortening the period required to achieve normal lymphoid cellularity.

Interleukin 1 regulates hematopoietic activity, a role previously ascribed to hemopoietin 1.

A murine in vitro assay was developed to measure potentiation of a proliferative response to suboptimal concentrations of the hematopoietic regulatory molecule granulocyte/macrophage colony-stimulating factor by an immature bone marrow population. The assay, designated the 5-fluorouracil bone marrow proliferation assay, was used to characterize potentiating activity in serum-free culture supernatants of the human tumor cell line HBT 5637. Molecular and biochemical analyses indicated that the HBT 5637-derived potentiating activity could be attributed to interleukin 1 alpha. Serologic analysis using a monoclonal antibody against purified recombinant interleukin 1 alpha proved conclusively that the potentiating activity in HBT 5637 serum-free supernatants is due to interleukin 1 alpha. From these data, the activity of interleukin 1 alpha seems to be the same synergistic activity formerly ascribed to hemopoietin 1.