Establishment of an interleukin-5-dependent subclone from an interleukin-3-dependent murine hemopoietic progenitor cell line, LyD9, and its malignant transformation by autocrine secretion of interleukin-5.

An interleukin-5 (IL-5)-dependent subclone, K-5, was established from an IL-3-dependent murine hemopoietic progenitor cell line by co-culturing with bone marrow stroma cells. K-5 cells were induced to differentiate into myeloid lineage cells by co-culturing with cloned PA6 stroma cells. By co-culturing with another cloned stroma cell (ST-2s10), K-5 cells gave rise to a factor-independent transformant cell line LT-5 which proliferated in an autocrine manner by secretion of IL-5 and produced tumors in nude mice. Molecular cloning of the IL-5 gene of LT-5 cells and the nucleotide sequencing of its 5' flanking region indicate that a transposition of an intracisternal A-particle (IAP) element to the 5' flanking region of the IL-5 gene is responsible for the constitutive expression of IL-5 mRNA of an aberrant size in LT-5 cells.     

Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.     

Thec-kit receptor transduces the stem cell factor-triggered growth signal in murine interleukin-3-dependent cell line

The stem cell factor is a glycoprotein hormone which regulates the proliferation and differentiation of primitive hematopoietic cells through its interaction with a tyrosine kinase transmembrane receptor which is encoded by thec-kit proto-oncogene. To examine whether a murinec-kit receptor can be functional in murine interleukin-3 (mlL-3)-dependent hematopoietic cell line, we introduced the murinec-kit cDNA into mlL-3-dependent pro-B cell line Ba/F3. One of the resulting clones, Ba/F3 clone BF-K96, expressed the 140 kDa protein recognized by anti-c-kit monoclonal antibody and the expressedc-kit receptor protein on the cell surface bound to a radiolabeled soluble form of murine stem cell factor (mSCF) with high affinity. BF-K96 clone expressing thec-kit receptor could proliferate in response to mSCF in the absence of mlL-3. The cell clone could also grow in co-culture with mouse 3T3 cells which are endogeneously expressing a membrane-associated type of mSCF on their cell surfaces. These findings demonstrate that thec-kit receptor expressed on mlL-3-dependent hematopoietic cell line Ba/F3 transduce the mSCF-dependent growth signal, indicating that established cell clone will provide a unique cellular system for the study of SCF/c-kit signal transduction mechanism.Abbreviations SCF stem cell factor - IL interleukin - CSF colony stimulating factor - IMDM Iscove's Modified Dulbecco's Medium - DME Dulbecco's Modified Eagle's Medium - FCS fetal calf serum - PCR polymerase chain reaction - EDTA ethylenediaminetetra-acetic acid; sodium dodecyl sulfate     

Expression of murine interleukin-2 cDNA in E. coli and biological activities of recombinant murine interleukin-2

Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E. coli. Recombinant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.     

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.     

Molecular cloning of 114/A10, a cell surface antigen containing highly conserved repeated elements, which is expressed by murine hemopoietic progenitor cells and interleukin-3-dependent cell lines

Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.     

An Epstein-Barr virus-transformed B cell line produces autoregulatory interleukin-1 that regulates bone remodeling

In this study, we demonstrate that an Epstein-Barr virus-transformed B cell line, A-11, produced interleukin-1 (IL-1), a cytokine that regulates bone remodeling. A-11 cells produce IL-1 in a cell dose- and culture time-related manner. The IL-1 activity was neutralized by recombinant human IL-1 (rhIL-1) alpha antiserum, but not by rhIL-1 beta antiserum. The IL-1 was semi-purified by (NH4)2SO4 precipitation, Superose prep 12 gel filtration, and anion-exchange chromatography strongly stimulated in vitro bone resorption. The stimulatory effect of the purified IL-1 on bone resorption was prostaglandin independent. Purified IL-1 inhibited DNA and collagen synthesis in the osteoblastic cell line MC3T3-E1. However, it enhanced significantly the cellular activity of alkaline phosphatase (EC 3.1.3.1), a marker enzyme for differentiation of osteoblasts. On the other hand, A-11 cell proliferation was inhibited by addition of rhIL-1 alpha antiserum, but not by rhIL-1 beta antiserum. And cell proliferation was stimulated by exogenous rhIL-1 alpha and -beta.     

Interleukin-1 binding, internalization, and processing in a murine osteoblastic cell line, MC3T3.E1.

We have investigated the interaction of IL-1 and its receptor on a murine osteoblastic cell line, MC3T3.E1, with regard to binding, internalization, and the fate of the receptor-ligand complex following internalization. Binding experiments indicated that this cell line possesses a high affinity receptor (Kd 1.02 x 10(-10) M) that binds both IL-1 alpha and IL-1 beta, and has approximately 6500 receptors per cell. Cross-linking experiments indicated that the receptor has a molecular weight of 100,000 daltons. Binding of IL-1 to the receptor is inhibited by the Interleukin Receptor Antagonist Protein (IRAP). These characteristics suggest that the murine osteoblastic receptor resembles that found on T lymphocytes and fibroblasts. Internalization experiments showed that this process is fairly rapid and results in degradation of the ligand and subsequent loss of degraded IL-1 from the cell. In this respect, processing of the receptor-ligand complex mimics that observed with IL-1 receptors on murine bone marrow cells, pre-B cells, and macrophages. Although the reasons for these differences are unclear, it may be that, unlike fibroblasts, osteoblasts may function as an effector cell which rapidly removes IL-1 from the immediate environment via ligand degradation while at the same time initiating bone resorption via stimulation of osteopontin biosynthesis.     

Suppression of prostaglandin E(2)-mediated c-fos mRNA induction by interleukin-4 in murine macrophages

When murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentration-dependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E(2) from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE(2) brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE(2) and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE(2), which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE(2), and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGE(2)-mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3.     

Chemo-immunotherapy of murine tumors using interleukin-2 (IL-2) and cyclophosphamide

Summary The antitumor effect of interleukin-2 (IL-2), alone and in combination with cyclophosphamide was assessed in mice with established sarcoma (MCA 105, H-2^b), carcinoma (M109, H-2^d) and T lymphoma (PIR-2, H-2^b). Whereas administration of IL-2 alone (5×10⁴–10×10⁴ U, i.p. twice daily, for 4–8 consecutive days) prolonged the survival of mice with the solid neoplasms, it enhanced tumor growth and decreased survival of mice with the lymphoma. In the PIR-2 lymphoma, no IL-2 receptor (TAC) could be detected, nor could we demonstrate IL-2 tumor growth stimulation in vitro. A synergistic therapeutic effect was achieved in mice with the solid tumors, but not in mice with the lymphoma, only when IL-2 was given 1–4 days after cyclophosphamide (100–200 mg/kg). Conversely, administration of IL-2 1–4 days prior to cyclophosphamide resulted, in all three tumor systems, in enhanced tumor growth and in decreased survival as compared with mice receiving cyclophosphamide alone. Similarly, treatment with IL-2 both before and after cyclophosphamide was less efficacious than a single course of IL-2 given after-wards. It is concluded that for maximal therapeutic efficacy, IL-2 should be administered following chemotherapy, and that certain tumors may respond adversely to IL-2 treatment.     

Prostaglandin E(2) production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E(2) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE(2) production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC(50) of 1.0 microM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE(2) production by activated macrophages.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by interleukin-3 and calcium ionophores

An immortalized interleukin-3 (IL-3)-dependent progenitor cell line, BAF-3, undergoes programmed cell death (apoptosis) when deprived of IL-3. This program is characterized by an early degradation of DNA into oligonucleosome-length fragments that precedes by several hours the loss of cell viability. In the absence of IL-3, DNA fragmentation and cell death can be prevented by the calcium ionophores A23187 (1 microM) and ionomycin (0.5 microM). This addition of calcium ionophore maintains cell viability while reversibly arresting the cell cycle. Apoptosis by growth factor deprivation is also a mechanism of cell elimination in bone marrow cells removed from the stromal micro-environment, as DNA fragmentation and cell death was shown to take place in primary cultures of IL-3-responsive bone marrow cells after IL-3 removal.     

Induction of IL 2 responsiveness in a murine IL 3-dependent cell line

A mouse IL 3-dependent cell line, FD.C/1, can be induced to IL 2 growth responsiveness by culture in IL 2-conditioned culture medium. The IL 2-dependent cell lines derived by this procedure have been designated FD.C/2 cells. Once established, the FD.C/2 cells respond to human, rat, and mouse IL 2. When cultured with murine IL 3, FD.C/2 cells did not proliferate, appearing to accumulate in the G1 phase of the cell cycle. Other human and mouse lymphokines failed to stimulate FD.C/2 cell growth. The growth dependence of FD.C/2 cells on IL 2 could not be reversed to IL 3. Cell lines derived by these procedures could provide in vitro models for hemopoietic differentiative events.     

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.     

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.     

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.