Biomedical subjects

M P Beckmann

Publications and source records attributed to M P Beckmann.

At least 37 records · Page 2Linked to original sources

PubMed1991

Molecular characterization of the interleukin-8 receptor.

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.

Amino Acid Sequence↗

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

The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors.

Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.

Animals↗

PubMed1991

Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130.

Leukemia inhibitory factor (LIF) is a cytokine with a broad range of activities that in many cases parallel those of interleukin-6 (IL-6) although LIF and IL-6 appear to be structurally unrelated. A cDNA clone encoding the human LIF receptor was isolated by expression screening of a human placental cDNA library. The LIF receptor is related to the gp130 'signal-transducing' component of the IL-6 receptor and to the G-CSF receptor, with the transmembrane and cytoplasmic regions of the LIF receptor and gp130 being most closely related. This relationship suggests a common signal transduction pathway for the two receptors and may help to explain similar biological effects of the two ligands. Murine cDNAs encoding soluble LIF receptors were isolated by cross-hybridization and share 70% amino acid sequence identity to the human sequence.

Amino Acid Sequence↗

PubMed1991

Expression of receptors for interleukin 4 and interleukin 7 on human T cells.

Human recombinant interleukin 4 (IL-4) and interleukin 7 (IL-7) have been modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 and IL-7 receptors (R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA) or immobilized CD3 monoclonal antibody the intensity of biotinylated IL-4 staining increased approximately twofold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused a considerable increase in IL-4 receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R although it was able to cause a significant increase in receptor expression on T cells co-cultured with PHA or CD3. Freshly isolated T cells expressed high levels of IL-7R, as determined by biotinylated IL-7 binding and flow cytometry, which did not change significantly with culture in medium alone. Stimulation with PHA, Concanavalin A (Con A) or CD3 had little effect on the intensity of staining. In contrast, activation with phorbol ester resulted in a decrease in IL-7R expression. Similarly, in the presence of IL-4 or IL-7, but not IL-2, the intensity of staining with biotinylated IL-7 was lowered. Analysis of purified T-cell populations showed that IL-7R were present, and IL-4R could be induced, on both CD4+ and CD8+ populations. Analysis of IL-4 receptor expression by this flow cytometric technique was supported by results from 125I-labeled IL-4 binding and by Northern blot analysis of mRNA levels. Taken together, the results of these studies show that the use of biotinylated cytokines and flow cytometry provides a very sensitive method with which to study the expression and regulation of cytokine receptors.

Animals↗

PubMed1991

Assays for lymphokines, cytokines and their receptors.

There have been several recent developments in the detection of cytokines, their receptors, and the genes that encode them. New methods for characterizing cytokines and lymphokines include the use of molecular hybridization techniques, bioassays and antibody-mediated detection systems. The most common techniques used for identifying and characterizing cytokine receptors rely upon the use of radiolabeled, fluoresceinated or biotinylated ligands.

Animals↗

PubMed1991

Regulation of murine in vivo IgG and IgE responses by a monoclonal anti-IL-4 receptor antibody.

Although the cytokine interleukin 4 (IL-4) stimulates LPS-activated mouse B lymphocytes to secrete both IgG1 and IgE, an anti-IL-4 antibody completely inhibits IgE responses but has little or no effect on several in vivo IgG responses. IL-4 might, therefore, have a restricted role in the generation of in vivo humoral immune responses. Alternatively, IgG1 responses might be stimulated by IL-4 secreted by T cells that are interacting directly with B cells, so that anti-IL-4 antibody cannot neutralize IL-4 before it binds to a B cell IL-4 receptor. In contrast, an antibody that blocks the IL-4 receptor (IL-4R) should equally inhibit responses to IL-4 produced proximal to or distant from a B cell. This reasoning led us to determine the ability of an anti-IL-4R mAb to affect antibody production in mice injected with a goat antibody to mouse IgD (GaM delta) or inoculated with the nematode parasite Heligmosomoides polygyrus. Anti-IL-4R mAb, like anti-IL-4 mAb, blocked IgE responses by greater than 95% and enhanced IgG2a responses to a variable extent. Anti-IL-4R mAb, however, had only a modest and variable inhibitory effect on the induction of IgG1 responses, although it caused these responses to terminate more rapidly. A combination of anti-IL-4 and anti-IL-4R mAbs totally blocked goat anti-mouse IgD antibody (GaM delta)-induced IgE production but had no additive inhibitory effect on IgG1 production. These observations are most consistent with the view that IL-4 is required for a primary IgE response, but has relatively little role in the induction of IgG1 responses in the in vivo systems studied.

Animals↗

PubMed1990

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.

Animals↗

PubMed1990

A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins.

Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.

Amino Acid Sequence↗

PubMed1990

Cytokine receptors and B cell functions. I. Recombinant soluble receptors specifically inhibit IL-1- and IL-4-induced B cell activities in vitro.

IL-1 and IL-4 are important mediators of B cell growth and differentiation. The cell-surface receptors for these cytokines have recently been cloned and recombinant soluble receptors have been produced that bind their respective ligand. The ability of soluble forms of the murine IL-1R (sIL-1R) and IL-4R (sIL-4R) to inhibit B cell functions in vitro was examined. Proliferation of B cells treated with anti-Ig plus IL-1 or IL-4 was inhibited by the appropriate soluble receptor. sIL-4R also inhibited IL-4-dependent B cell differentiation as measured by: induction of IgG1 and IgE secretion by LPS blasts, down-regulation of IgG3 secretion by LPS blasts, increased Ia expression, and increased Fc epsilon R (CD23) expression. The inhibitory effects of the soluble receptors were found to be highly specific in that sIL-4R had no effect on IL-1-induced B cell activity and sIL-1R had no effect on IL-4 activity, further demonstrating the existence of two independent pathways of B cell activation directed by IL-1 and IL-4.

Animals↗

PubMed1990

Induction of B cell activities by interleukin 4 is inhibited by a receptor-specific monoclonal antibody in vitro.

The effects of interleukin (IL) 4 on B cell growth and differentiation are mediated through binding of IL 4 to a specific cell surface receptor. The murine T cell IL 4 receptor (IL 4R) has recently been cloned and monoclonal antibodies (mAb) which bind specifically to the IL 4R have been developed. The ability of two of these anti-IL 4R mAb (M1 and M2) to inhibit IL 4-induced B cell functions in vitro was examined. The M1 mAb inhibited the ability of IL 4 to induce B cell proliferation in a dose-related fashion. The inhibition was specific for proliferation induced by IL 4 in that the antibody did not affect induction of proliferation by IL 1. Similarly, M1 inhibited IL 4-dependent B cell differentiation as measured by induction of IgG1 and IgE secretion, decreased IgG3 secretion, increased Ia expression, and increased Fc epsilon R (CD23) expression. In contrast, the anti-IL 4R-specific mAb M2 had no effect upon any of these activities. The ability of M1 but not M2 to inhibit IL 4-induced B cell growth and differentiation correlated with the inhibition of binding of radiolabeled IL 4 by M1. These reagents should be valuable tools with which to analyze the involvement of IL 4 in immune responses.

Animals↗

PubMed1990

A new cytokine receptor superfamily.

The amino acid sequences of several, recently cloned cytokine receptors show significant homologies, primarily in their extracellular, ligand-binding domains. With one exception, their cognate cytokines mediate biological activities on a variety of hematopoietic cell types; thus we have designated the receptors as the hematopoietic receptor superfamily.

Amino Acid Sequence↗

PubMed1990

A soluble form of the interleukin 4 receptor in biological fluids.

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.

Animals↗

PubMed1990

Regulation of interleukin 4 receptors on human T cells.

Human recombinant interleukin 4 (IL-4) was modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 receptors (IL-4R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA), or immobilized CD3 mAb the intensity of biotinylated IL-4 staining increased approximately 2-fold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused an increase in receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R, although it was able to induce a significant increase in receptor expression on T cells co-cultured with PHA or CD3 mAb. Flow cytometric analysis of purified T cell subsets confirmed that the up-regulation of IL-4R occurred on both CD4+ and CD8+ subpopulations. Two-color staining of T cells activated with PHA and IL-7 revealed that this increase in IL-4R expression occurred almost exclusively on cells expressing the p55 IL-2Ra subunit, although a significant number of cells expressing p55 do not express IL-4R. Analysis of IL-4R expression by this flow cytometric technique was substantiated by 125I-labeled IL-4 binding data and Northern blot analysis of IL-4R mRNA levels, suggesting that use of biotinylated human IL-4 for ligand binding and its detection by flow cytometry provides a very sensitive method for the study of IL-4R regulation.

Biotin↗

PubMed1990

Lymphokine control of in vivo immunoglobulin isotype selection.

Several specific conclusions can be drawn from these studies: 1. IL-4 is required for the generation of both primary polyclonal and secondary antigen-specific IgE responses in vivo. 2. IL-4 is required to maintain established, ongoing, antigen-specific and polyclonal IgE responses. 3. Most, but not all, polyclonal IgE production during a secondary immune response is IL-4-dependent. Memory B cells that have already switched to IgE at the DNA level may no longer require stimulation with IL-4 to be induced to secrete IgE. 4. The generation of a secondary IgE response is not dependent upon the presence of IL-4 during primary immunization. However, if IL-4 is not present during primary immunization, it is required during secondary immunization for the generation of an IgE response. 5. IL-4 does not appear to be required for the generation of in vivo IgG1 responses, and in at least some instances, does not contribute significantly to the generation of IgG1 responses in vivo. 6. A late-acting form of T-cell help other than IL-4 appears to be required for the generation of an IgE, but not an IgG1 response. 7. An antibody that inhibits IL-4 binding to IL-4 receptors affects Ig isotype selection in the same way as an antibody that neutralizes IL-4. 8. IFN-gamma can act in both spontaneous and induced immune responses to suppress IgE production. 9. IFN-gamma can also suppress IgG1 production and stimulate IgG2a production. However, IFN-gamma appears to suppress polyclonal IgG1 responses more than antigen-specific IgG1 responses, and it enhances, but is not required for, the generation of IgG2a responses. 10. IFN-alpha appears to resemble IFN-gamma in its ability to inhibit IgE and enhance IgG2a responses in GaM delta-injected mice, but it requires the presence of IFN-gamma to suppress IgG1 production in these mice. 11. Both IFN-alpha and IFN-gamma appear to be able to decrease IgE production in some human patients. 12. There is no direct evidence that IL-5 contributes to the generation of in vivo antibody responses. Two general conclusions may also be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms.

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.

Amino Acid Sequence↗

PubMed1988

Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains.

Human platelet-derived growth factor (PDGF) consists of two distinct but related polypeptide chains designated PDGF-A and PDGF-B. The gene encoding PDGF-B has given rise to the v-sis oncogene. In the present study the transforming activities of PDGF-A and PDGF-B genes are compared. The PDGF-A chain gene is markedly less efficient in inducing transformation than the PDGF-B gene under the influence of the same promoter. There are significant differences in the secretory and growth stimulating properties of the two chains. These properties appear to account for the much more potent transforming ability of the PDGF-B gene. These findings provide insights into biologic properties of a growth factor responsible for potent autocrine stimulation of abnormal cell proliferation.

Animals↗