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Biomedical subjects

M B Widmer

Publications and source records attributed to M B Widmer.

At least 37 records · Page 2Linked to original sources
PubMed1992

The Steel factor.

Steel factor (SLF) is a recently identified growth factor which is the gene product of the murine Steel locus and a ligand for the c-kit tyrosine kinase receptor, the product of the dominant white spotting locus (W). Defects at these genetic loci result in aberrant melanocyte, germ cell, and hematopoietic development. Both the receptor (c-kit) and the ligand (SLF) have been shown to undergo tissue-specific mRNA splicing to produce distinct isoforms which have unique biological functions. As predicted by the phenotype of these mutations, SLF influences the growth and differentiation of melanocytes, primordial germ cells, and a broad spectrum of cell types in the hematopoietic progenitor and stem cell hierarchy. SLF has also been shown to have effects on hematopoietic lineages not predicted by defects seen in the Steel mouse.

Animals↗
PubMed1991

Regulation of alloreactivity in vivo by IL-4 and the soluble IL-4 receptor.

Although numerous in vitro studies have demonstrated that cytokines are involved in the generation of alloreactive effector cells, the role of these regulatory substances in vivo is less well defined. We have recently cloned and expressed cDNAs encoding both membrane bound and soluble forms of the murine IL-4R. The effects of murine rIL-4 and a recombinant, soluble, extracellular portion of the murine IL-4R soluble(s) IL-4R on the generation of alloresponsiveness in vivo were evaluated by measuring the lymphoproliferative response to a localized injection of allogeneic cells and the survival of cardiac allografts. Administration of IL-4 to BALB/c mice resulted in a slight augmentation of the anti-C57BL/6 lymphoproliferative response compared to that occurring in control, mouse serum albumin-(MSA) treated mice. In contrast, the sIL-4R suppressed this response to allogeneic cells in a dose-dependent manner, with a dose of 50 micrograms/kg/day causing nearly complete inhibition of the response as compared to the response observed in controls. The inhibitory effect of sIL-4R was reversed by simultaneous administration of IL-4. A neutralizing antibody against IL-4 (11B11) and another against the IL-4R (M1) were also effective inhibitors of the response when given at 100- to 1000-fold higher concentrations than the amount of sIL-4R required to inhibit the response. In cardiac allograft experiments, BALB/c mice were engrafted with newborn C57BL/6 hearts in the ear pinnae and treated with sIL-4R (50 micrograms/kg) or MSA. Such allografts survived an average of 4 days longer in sIL-4R-treated mice than in MSA-treated controls. In conclusion, neutralization of IL-4 inhibits alloresponsiveness in vivo. These results confirm a regulatory role for this pleiotropic cytokine in allograft rejection and suggest a therapeutic value for IL-4 antagonists alone or in combination with other immunosuppressive regimens.

Animals↗
PubMed1991

Characterization and pharmacokinetic parameters of recombinant soluble interleukin-4 receptor.

The interleukin-4 receptor (IL-4R) is expressed as a 140-Kd membrane glycoprotein that binds IL-4 with high affinity. Recently, cDNA clones for the murine IL-4R have been isolated. One clone encodes an integral membrane protein, while another encodes a protein in which translation is terminated before the transmembrane region, thus producing a soluble form of the IL-4R (sIL-4R). HeLa cell clones overexpressing sIL-4R were isolated using a novel filter-overlay and 125I-IL-4 ligand binding technique. Quantitative analysis demonstrated that the kinetics and affinity of IL-4 binding to the recombinant sIL-4R were similar to the native membrane-bound IL-4R. As low doses of sIL-4R specifically inhibited IL-4-induced proliferative responses in vitro, sIL-4R biodistribution and elimination parameters were evaluated to assess the pharmacokinetic potential of sIL-4R as a therapeutic agent. Pharmacokinetic studies demonstrated that radiolabeled sIL-4R had a distribution half-life of 9 minutes and an elimination half-life of 2.3 hours following intravenous (IV) administration. When administered by intraperitoneal or subcutaneous (SC) injection, the elimination half-lives were prolonged to 4.2 hours and 6.2 hours, respectively. Although the initial blood level of sIL-4R was reduced if administered by SC injection, the bioavailability was comparable with IV administration. The main sites of sIL-4R elimination were the liver and kidney.

Animals↗
PubMed1991

Interleukin 2 stimulates serine phosphorylation of CD45 in CTLL-2.4 cells.

Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.

Animals↗
PubMed1991

Inhibition of cytokine function: potential in autoimmune disease.

Multiple lines of evidence implicate cytokines in the pathogenesis of autoimmune and inflammatory diseases. Neutralization of the biological activities of cytokines by interfering with ligand binding to specific cell-surface cytokine receptors thus represents an approach to controlling such diseases. Several agents, including novel, receptor-based antagonists, are being developed and tested in disease models.

Animals↗
PubMed1990

Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.

The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.

Antigens, CD↗
PubMed1990

Regulation of alloreactivity in vivo by a soluble form of the interleukin-1 receptor.

In vitro studies have shown that cytokines are involved in the regulation of the immune response, but their role in vivo is less well defined. Specific cytokine antagonists enable the identification of particular cytokines involved in the response and offer a means for modifying it. Systemic administration of a soluble, extracellular portion of the receptor for interleukin-1 (sIL-1R) had profound inhibitory effects on the development of in vivo alloreactivity. Survival of heterotopic heart allografts was prolonged from 12 days in controls to 17 days in mice treated with sIL-1R. Lymph node hyperplasia in response to a localized injection of allogeneic cells was completely blocked by sIL-1R treatment. The inhibition was overcome by simultaneous administration of interleukin-1 (IL-1); thus, sIL-1R acts by neutralizing IL-1. These results implicate IL-1 as a regulator of allograft rejection and demonstrate the in vivo biological efficacy of a soluble cytokine receptor.

Animals↗
PubMed1990

Regulation of T cell proliferation by IL-7.

The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.

Animals↗
PubMed1990

Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

Amino Acid Sequence↗
PubMed1990

IL-1 and its receptor are translocated to the nucleus.

The internalization and intracellular transport of IL-1 and its receptor were examined in the murine T cell line EL-4. For 4 h after internalization intracellular 125I-IL-1 alpha remains bound to its receptor without degradation. Electron microscope autoradiography demonstrates that internalized IL-1 accumulates in purified nuclei. The IL-1 extracted from these nuclei is still bound to receptor. As no receptors for IL-1 were detected in untreated nuclei, these results suggest IL-1 driven translocation of the cell surface IL-1R complex to the nucleus. IL-1R internalization was correlated with IL-1 signal transduction events required to induce growth factor production from several subclones of EL-4 cells. The subsequent transport of the internalized IL-1R complex to the nucleus suggests the possibility for a nuclear site for IL-1R signaling.

Amino Acid Sequence↗
PubMed1990

Lymphopoiesis and IL-7.

Interleukin 7 (IL-7) is a cytokine initially referred to as pre-B cell growth factor because of its growth-promoting activity for cells early in the B lymphocyte lineage. Subsequent to IL-7 gene cloning and expression, IL-7 was also found to influence the activity of cells in the T lineage. Some of the effects of IL-7 on B and T lymphocyte behavior, both in vitro and in vivo, are discussed here. The growth of pre-B cells in Whitlock-Witte long-term bone marrow cultures [1] provided the basis for the cloning of IL-7 [2]. Such cultures consist of at least two cellular elements: 1) a stromal cell layer and 2) lymphoid cells growing in clusters around the stromal cells. A cDNA encoding IL-7 was cloned utilizing a direct expression cloning strategy. A transformed stromal cell line was used as a source of RNA from which a cDNA library was constructed, inserted into a mammalian expression vector and expressed in COS 7 cells. The COS cells were then screened for secretion of IL-7 into the culture medium as defined by the ability to support stromal cell-independent growth of the lymphoid elements from long-term Whitlock-Witte cultures. In addition to its effects on cells from long-term marrow cultures, IL-7 stimulates growth of cells in cultures of fresh bone marrow [3]. Cells from whole bone marrow plated in semisolid agar form colonies in the presence of IL-7. If the marrow is first enriched for cells expressing the B220 antigen before culture in IL-7, the plating efficiency is increased dramatically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interleukin 7 stimulates growth of fetal thymic precursors of cytolytic cells: induction of effector function by interleukin 2 and inhibition by interleukin 4.

Interleukin 7 (IL-7) and interleukin 2 (IL-2) stimulate the outgrowth of distinct populations of thymocytes in lobe submersion cultures (LSC) established with day 12-14 murine fetal thymus. Analysis of the expression of cell surface markers in previous studies showed that IL-7 favors the expansion of a more immature population. In the experiments reported here, populations grown in IL-7 and IL-2 were found to differ functionally as assessed by the expression of cytolytic activity. Whereas cells derived from IL-2-supplemented LSC were highly cytolytic for a broad panel of targets, cells that emerged in IL-7-supplemented cultures exhibited little or no such activity, even in the presence of facilitating lectin. However, there were cells with cytolytic potential present in IL-7-grown populations, as demonstrated by the abrupt appearance of effector function 3 days after their exposure to IL-2. Limiting dilution analysis showed that the absolute number of cells in the cytolytic lineage in fetal thymic lobes increased during culture in LSC. Interestingly, identical increases occurred in IL-7-supplemented and IL-2-supplemented LSC, despite the fact that only the latter population exhibited appreciable lytic activity. Collectively, these results suggest that IL-7 stimulates outgrowth of cell populations which contain functionally inactive cytolytic precursors whose activity is inducible by IL-2. In contrast to IL-2, IL-4 failed to stimulate the appearance of a lytic population from IL-7-supplemented LSC. Furthermore, IL-4 interfered with cell proliferation and acquisition of lytic activity normally induced by IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Precursor frequency and lytic specificity of interleukin 2 and interleukin 4-responsive murine lymphocytes.

The response to IL-2 and IL-4 of several functionally defined lymphoid cell types was assessed at the individual responding cell level in limiting dilution experiments in which exogenous IL-2 or IL-4 was used to promote cellular activation and expansion. Splenic precursors that give rise to alloreactive CTL in LDA supplemented with IL-2 were approximately 3-fold more frequent than those detected in IL-4. Of special interest was the observation that cytolytic cultures arising in primary allogeneic LDA supplemented with IL-4 exhibited a greater degree of lytic specificity than those arising in IL-2. This difference may be due to an inherently broader specificity of CTL cultured in IL-2 or to a concomitant activation of CTL and lymphokine-activated killer(s) (LAK) in individual IL-2 supplemented microcultures. In this regard, estimation of LAK precursor(s) (LAK-P) frequencies in LDA cultures established without an overt antigenic stimulus revealed that IL-2 activates a 20-fold higher frequency of LAK-P than does IL-4. Finally, an examination of the proliferative response to IL-2 and IL-4 of several CTL and PTL clones demonstrated that IL-4 responsive cells comprise a subset of cells that respond to IL-2, irrespective of their functional phenotype.

Animals↗
PubMed1989

Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vivo or in vitro.

In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.

Antigens, CD↗
PubMed1989

Improved survival and leukocyte reconstitution without detrimental effects on engraftment in murine recipients of human recombinant granulocyte colony-stimulating factor after transplantation of T-cell-depleted histoincompatible bone marrow.

In vivo administration of human recombinant granulocyte colony-stimulating factor (rG-CSF) was evaluated for effects on survival, hematologic recovery, and engraftment in an allogeneic murine bone marrow transplantation (BMT) model involving T-cell depletion. Post-BMT recipients of continuous subcutaneous infusions of rG-CSF (n = 62) for 14 days had a significant survival advantage compared with post-BMT controls (n = 60) that received phosphate-buffered saline (PBS) infusions. Moreover, recipients of rG-CSF had significantly increased numbers of circulating leukocytes on days 7 and 14 post-BMT. Engraftment was not adversely affected. Administration of rG-CSF after transplantation of T-cell-depleted histoincompatible bone marrow benefits survival and leukocyte recovery without compromising engraftment.

Animals↗
PubMed1989

Effect of IL-7 on the growth of fetal thymocytes in culture.

The effects of IL-7 on the in vitro growth and differentiation of day 12 to 14 murine fetal thymocytes were examined in three culture systems. In single cell suspension cultures, IL-7 and IL-2 induced a DNA synthetic response in a short term (1 day) assay, but neither cytokine supported continued cell growth. In conventional fetal thymus organ cultures, the addition of exogenous IL-7 resulted in a twofold increase in cell number over that which normally develops in unsupplemented fetal thymus organ cultures during a 7-day period. The most striking effects of IL-7 were noted in lobe submersion cultures (LSC), a system in which thymocyte growth was totally dependent on the addition of exogenous cytokine. Cells proliferated for a period of approximately 2 wk in IL-7, and cell viability could be maintained even longer. A high percentage of cells recovered after 7 to 14 days from IL-7-supplemented LSC resembled the earliest detectable fetal thymocytes with regard to cell surface markers: they expressed Pgp-1, lacked CD4, CD8, and CD3 and many expressed the IL-2R. These results suggest that IL-7 promotes the growth of cells that occur early in the T cell lineage. Cell populations recovered from LSC supplemented with IL-7 and IL-2 exhibited differential expression of some surface markers, particularly CD3 and NK1.1. In addition, cells from LSC supplemented with IL-7 were found to proliferate upon subsequent exposure to IL-2, but cells from LSC containing exogenous IL-2 were no longer responsive to IL-7. These results imply that IL-7 and IL-2 may act at different stages of thymocyte differentiation. Together with previous observations of IL-7-specific mRNA expression in the thymus, this study provides evidence highly suggestive of a pivotal role for IL-7 in T cell development.

Animals↗