Biomedical subjects

A Gessner

Publications and source records attributed to A Gessner.

At least 37 records · Page 2Linked to original sources

PubMed1998

Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase.

The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet been identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys-->Ser mutants (C57S MIF, C60S MIF, and C57S/C60S MIF) of human MIF (wtMIF), and alkylated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. In this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60. Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. In conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism.

Animals↗

PubMed1998

The Th1/Th2 paradigm and experimental murine leishmaniasis.

In recent years, the Th1/Th2 concept has become of prime importance in the understanding of heterogeneous responses of the immune system and implications thereof for infectious and autoimmune diseases. Originally established on the basis of different cytokines produced by T cell clones, it is now known that the Th1/Th2 concept really defines totally different immune pathways that affect most if not all cells of the immune system. Murine experimental leishmaniasis was the first model to confirm the relevance of the Th1/Th2 concept in vivo. Herein, we summarize the current knowledge on the characteristics and generation of Th1 and Th2 cells, as well as on recent advances of the application of this concept to murine cutaneous leishmaniasis.

Animals↗

PubMed1998

Experimental murine leishmaniasis and the Th1/Th2 cell concept.

Herein, the current knowledge about the immune response during murine cutaneous leishmaniasis is summarized. Special regard is given to the characteristics and generation of Th1 and Th2 cells during this disease. Originally established on the basis of different cytokines produced by T cell clones, it is now known that the Th1/Th2 concept really defines totally different immune pathways that affect most, if not all cells of the immune system. Murine experimental leishmaniasis was the first model to confirm the relevance of the Th1/Th2 concept in vivo. In particular, data from this laboratory will be presented on the role of different IL-4 receptor allotypes, on the role of the transcription factor interferon-regulatory-factor-1 (IRF-1) and the importance of the enzyme inducible NO synthase (iNOS). This work was supported by the SFB 263 and the Fonds der Chemischen Industrie.

Animals↗

PubMed1997

Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes.

The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.

Alleles↗

PubMed1997

Structure activity studies of the cytokine macrophage migration inhibitory factor (MIF) reveal a critical role for its carboxy terminus.

Carboxy-truncated mutants of human MIF (MIF(1-104) and MIF(1-109)) were used in structure activity studies. CD spectroscopy revealed an overall structural similarity between the mutants and MIF. Denaturant-induced unfolding demonstrated that the C-terminus contributed significantly to the conformational stability of MIF. This appears to be due to the formation of two C-terminal beta-strands. The mutants were enzymatically active, exhibiting half of the enzymatic redox activity of MIF. However, immunological analysis showed that deletion of both 5 and 10 C-terminal residues resulted in loss of the macrophage activating properties of MIF, providing functional evidence that the C-terminus is important for immunological activity and trimer formation. A more detailed study of the C-terminus may assist in identifying the molecular basis for the immunological and enzymatic activities of MIF.

Amino Acid Sequence↗

PubMed1997

A dominant mechanism coordinately suppresses the expression of Th2 lymphokines.

This study, we present evidence for a negatively acting control mechanism that coordinately suppresses the synthesis of the Th2 lymphokines IL-4, IL-5 and IL-6. This control mechanism operates in the murine thymoma cell line BW 5147. When cells of this line were fused to four independently established, well-defined Th2 cell clones, all resulting 74 lymphokine-secreting hybridomas secreted IL-2 which was not secreted by any of the parental Th2 cell clones. Most interestingly, however, none of the 74 hybridomas retained the capacity of the parental Th2 cells to express IL-4. Likewise, the secretion of IL-5 and IL-6 was also suppressed. Obviously, BW 5147 cells dominated the pattern of lymphokines produced, although the lymphokine pattern of Th2 cells was previously considered to be irreversibly fixed due to terminal differentiation of these cells. Suppression of IL-4 production was also observed at the mRNA level, as tested in Northern blot assays. Putative DNA target sequences for suppression of IL-4 gene transcription were not part of the proximal IL-4 promotor regions. Remote DNA control sequences may exist which coordinately regulate the proper, stage-specific expression of the Th2 lymphokines IL-4, IL-5 and IL-6.

Animals↗

PubMed1996

Intracellular murine IFN-gamma mediates virus resistance, expression of oligoadenylate synthetase, and activation of STAT transcription factors.

IFN-gamma is a pleiotropic cytokine that plays a major role in anti-infectious immune responses. The physiologic effects of IFN-gamma are thought to be mediated by the binding of extracellular IFN-gamma to its receptor at the cell surface, thereby triggering an intracellular signaling cascade. In this work, we present evidence for a completely intracellular mechanism for IFN-gamma to induce virus protection. Murine fibroblasts were transfected with the cDNA for murine IFN-gamma, and although no detectable amounts of IFN-gamma were released, these cells were resistant to lysis by the cytolytic vesicular stomatitis virus. In contrast to exogenously added IFN-gamma, the effect of the endogenously produced IFN-gamma was not abolished by treatment with neutralizing Abs. To test whether intracellular signal transduction occurs, an IFN-gamma variant was constructed with the carboxyl-terminal endoplasmic reticulum retention signal Lys-Asp-Glu-Leu (KDEL). Transfection of fibroblasts with this mutant IFN-gamma, anchored in the endoplasmic reticulum, led to virus resistance, thus demonstrating that biologic effects of this protein do not necessarily require binding to the receptor at the cell surface. However, the antiviral state induced by transfection with IFN-gamma-KDEL was strictly dependent on the presence of the IFN-gammaR, since fibroblasts derived from IFN-gammaR-deficient mice (IFN-gammaR -/-) were not rendered virus resistant. The virus resistance induced was accompanied by enhanced expression of 2'-5' oligoadenylate synthetase and constitutive activation of STAT1 (signal transducers and activators of transcription). Hence, autocrinous effects of IFN-gamma in cells naturally producing this cytokine might occur even in the absence of its secretion. The mechanisms involved in signaling appear to be identical with or closely related to those occurring after binding of IFN-gamma to its receptor at the cell surface.

2',5'-Oligoadenylate Synthetase↗

PubMed1996

Two distinct stimulus-dependent pathways lead to production of soluble murine interleukin-4 receptor.

The IL-4R exists in two forms, either membrane bound or as a soluble (s) molecule. Since the sIL-4R binds to its ligand with high affinity, thereby acting as an immunoregulatory molecule, we were interested in the processes leading to its release. First, the release of sIL-4R in the model of murine leishmaniosis was analyzed. Infection of mice with Leishmania major resulted in up-regulation of sIL-4R production by Ag-stimulated CD4+ T cells, with a maximum around 7 days after infection. To clarify the mechanisms underlying sIL-4R release, in vitro studies were performed. After stimulation of naive lymphoid cells with IL-4, sIL-4R release was dependent on up-regulation of spliced IL-4R mRNA, as shown by inhibition with specific antisense oligonucleotides. In contrast to this, no increase in the spliced IL-4R mRNA and no inhibitory influence of antisense oligonucleotides were observed after stimulation of T cells from IL-4-deficient mice with anti-CD3 mAb. Thus, TCR stimulation can lead to IL-4-independent sIL-4R production. Under these conditions proteolytic shedding of membrane-bound IL-4R appears to be the principal mechanism of release, since in contrast to stimulation with IL-4, iodinated sIL-4R could only be immunoprecipitated after cell surface labeling and subsequent TCR stimulation. The common gamma-chain, a component of the IL-4R complex, did not appear to be involved in the pathways leading to sIL-4R expression. This analysis suggests the existence of two differentially regulated pathways of sIL-4R release, possibly having different consequences for the regulation of IL-4 bioactivity.

Alternative Splicing↗

PubMed1996

Expression and co-cytokine function of murine thioredoxin/adult T cell leukaemia-derived factor (ADF).

Human ADF (adult T cell leukaemia-derived factor), an isoform of thioredoxin, promotes proliferation of certain human lymphoid cell lines and is involved in many thiol-dependent reducing reactions. To study functional aspects of the murine homologue, we established inducible overexpression of murine ADF in E. coli and a purification method which led to an apparently homogeneous 14 kDa protein. This recombinant ADF was tested in proliferation assays with murine Th2 cells (D10.G4.1) and CTLL-2 cells. In synergy with IL-2, IL-4, IL-7 and IL-9 ADF displayed co-cytokine activity. These proliferative effects were neutralized by an affinity-purified polyclonal rabbit anti-ADF antiserum. The effects of ADF were critically dependent on the presence of 2-mercaptoethanol. Bacterial thioredoxin had similar effects on the proliferation of murine T cells. Thus, the thiol-related reducing capacity of these proteins is essential for their growth promoting activity. As investigated at the levels of mRNA and protein in several murine cell clones and lines as well as in mouse tissues ADF is expressed ubiquitously. Finally it could be demonstrated by competitive PCR that in contrast to cytokine mRNAs (e.g. IL-4 and IL-13) the expression of ADF mRNA in murine Th2 clones and spleen cells is not influenced by stimulation of these cells through the T cell receptor complex. Murine ADF therefore represents a protein constitutively expressed in a wide variety of cells with the capacity to enhance the proliferative effect of several cytokines on murine T cells.

Animals↗

PubMed1996

Invasion, control and persistence of Leishmania parasites.

Significant advances in research on the immunopathogenesis of leishmaniasis include the discovery of novel putative evasion and survival strategies of Leishmania parasites, a more detailed understanding of the function and regulation of interleukin-12, definition of molecules involved in cognate interaction between macrophages and T cells and new ideas concerning the mechanisms of host resistance and susceptibility. The use of transgenic mice for (re)probing certain immunological aspects of leishmaniasis has yielded not only predictable and confirmatory but also unexpected and pioneering results which require critical appreciation.

Animals↗

PubMed1995

Stable transfection of cloned murine T helper cells.

Here we describe a protocol for the stable transfection of murine T helper (Th) cells and long term culture of the resulting transfectants. The electroporation protocol was established for the murine Th2 clone L1/1 by testing different parameters determining the electric field (capacitance, voltage, single or twin pulse) as well as the activation status of the cells. The transfected T cells were genetically altered by stable integration of the neomycin resistance gene, encoded in the vector pM5neo, into the genome. For selection and long term culture of stable transfectants a scheme combining selection with the antibiotic neomycin (G-418, Geneticin) and repeated stimulation with antigen presenting cells (APC) and antigen was established. This protocol should also be applicable to other antigen reactive T cells. The resistance of the T cells to neomycin correlated directly with expression of the transferred neomycin resistance gene as demonstrated by mRNA analysis. Applying periodic reselection with neomycin the transfected Th2 cells were found to be stable for more than 18 months in culture and displayed an unaltered antigen recognition and lymphokine production pattern as compared with the untransfected L1/1 Th2 cells.

Animals↗

PubMed1995

Fibroblasts as efficient antigen-presenting cells in lymphoid organs.

Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

Animals↗

PubMed1995

Neutralization of endogenous IL-6 suppresses induction of IL-1 receptor antagonist.

IL-1 is a potent cytokine that promotes host defense and inflammation. These processes may be modulated by an IL-1 receptor antagonist (IL-1Ra) that binds to and blocks IL-1 receptors. The objective of this study was to define the cellular origin and regulation of IL-1Ra production during bacterial infection. Oral infection of mice with Yersinia enterocolitica resulted in expression of IL-1Ra mRNA and synthesis of IL-1Ra in Peyer's patches (PP), the local site of infection, as well as in noninfected organs such as spleens. By immunostaining, recruited circulating neutrophils were identified to be the primary source of IL-1Ra in tissues. Only approximately 20% of the IL-1Ra-staining cells were accounted for by inflammatory macrophages. Strikingly, neutralization of IL-6 by anti-IL-6 antiserum caused a suppression of both IL-1Ra mRNA in PP and synthesis of IL-1Ra in circulating neutrophils. Confirmatory evidence that IL-6 participates in the generation of IL-1Ra was obtained when rIL-6 induced, and anti-IL-6 antiserum blocked, IL-1Ra expression in cultures of macrophage and polymorphonuclear leukocytes (PMN). These findings suggest that IL-6 induced induction of IL-1Ra may provide a negative feedback loop, facilitating resolution of the inflammatory response locally and presumably at remote sites of infection.

Animals↗

PubMed1995

Stimulation of B-cell lymphopoiesis by interleukin-7 leads to aggravation of murine leishmaniasis.

The effect of recombinant interleukin-7 (IL-7) on the clinical course of murine leishmaniasis and the development of the accompanying immune response was investigated. Previously, IL-7 has been shown to possess stimulatory capacity for different cell types of the immune and haematopoietic system critically involved in the defence against Leishmania major (L. major), such as macrophages which are activated for the elimination of the parasite by IL-7. In contrast to these in vitro data, the present study indicates that treatment of genetically susceptible BALB/c mice with IL-7 at the onset of the infection leads to enhanced lesion development and a significantly accelerated death of the animals. This was correlated with a 40-fold increased parasite burden in spleens and lymph nodes. While the specific antibody response against L. major was not altered and lymphocytes of IL-7-treated mice produced comparable amounts of the T-helper type-2 (Th2) cytokines IL-4 and IL-10, less interferon-gamma (IFN-gamma) was measurable after antigenic stimulation of lymph node and spleen cells in vitro. One of the major changes appearing by the first week after infection in IL-7-treated mice was the increase of the total cell number in spleen and lymph nodes draining the local infection. Analysis of the cellular composition revealed that the enhanced cellularity was predominantly due to a rise in the B-cell compartment. Since antigen presentation by B cells has been implicated in the development of Th2 cells, the disease-aggravating activity of IL-7 is thought to be primarily due to augmentation of B lymphopoiesis.

Animals↗

PubMed1995

Effect of IL-7 treatment on Leishmania major-infected BALB.Xid mice: enhanced lymphopoiesis with sustained lack of B1 cells and clinical aggravation of disease.

The Xid immunodeficiency was characterized by a total lack of B1 cells and reduced numbers and functions of B2 cells. In BALB.Xid mice, this defect results in an reduced susceptibility against infections with parasites such as Trypanosoma cruzi and Leishmania major. Since IL-7 acts on the B cell compartment by stimulation of pre-B cell proliferation, we analyzed the effect of recombinant IL-7 on L. major infection in BALB.Xid mice. After application of a single dose of IL-7 simultaneously with the infection, the clinical course in BALB.Xid mice was markedly aggravated, resembling that of normal BALB/c mice. IL-7-induced disease promotion was accompanied by an up to 100-fold higher parasite load in several tissues of these mice. When cytokine production of purified, L. major-specific CD4+ T cells from lesion-draining lymph nodes was examined, the IFN-gamma production seen in untreated BALB.Xid mice was suppressed in IL-7-treated animals. One of the major effects of IL-7 treatment in the lymphoid organs of BALB.Xid mice was the increase of the total number of B220, sIgM and MHC II-positive cells. These cells belonged to the B2 subset, since cells expressing surface molecules characteristic for B1 cells (Mac-1 and Ly-1) remained absent in spleens, lymph nodes and the peritoneum. In conclusion, selective up-regulation of B2 cells by IL-7 in the absence of B1 cells is associated with disease aggravation in L. major-infected BALB.Xid mice.

Animals↗

PubMed1994

Interleukin-10 inhibits antimicrobial activity against Leishmania major in murine macrophages.

The stimulation of macrophages is of importance to the defense against intracellularly replicating microorganisms such as Leishmania. In this study the direct effect of recombinant interleukin-10 (IL-10) on the leishmanicidal effector functions of murine peritoneal or bone marrow derived macrophages was investigated. IL-10 almost completely inhibited the killing of intracellular leishmania at concentrations above 10 ng/ml. This inhibitory effect was independent of the stimulus used as the activation of macrophages by IFN-gamma and IL-7, recently shown to possess macrophage activating properties, were suppressed by IL-10. Kinetic experiments revealed that IL-10 must be present during the process of macrophage activation and that the leishmanicidal effector function of fully activated macrophages was not influenced. Furthermore, in the absence of exogenously added IL-10, the addition of neutralizing antibodies against IL-10 or IL-10-specific antisense phosphorothioate DNA-oligonucleotide led to an enhanced killing of parasites after stimulation with either IFN-gamma or IL-7. In accordance with this, IL-10 mRNA was readily detectable in murine macrophages by PCR with reverse transcribed mRNA. These results indicate that IL-10, which is endogenously produced by macrophages, acts as an autocrine deactivating factor supporting the survival of the parasite.

Animals↗

PubMed1994

Recombinant soluble interleukin-4 (IL-4) receptor acts as an antagonist of IL-4 in murine cutaneous Leishmaniasis.

This study was performed to evaluate the soluble interleukin-4 receptor (sIL-4R) as a potential antagonist of interleukin-4 (IL-4) in an infectious disease. It is shown that antigen-triggered proliferation and cytokine secretion of Leishmania major-specific, cloned Th2 cells in vitro can be inhibited dose dependently by recombinant murine, but not control human, sIL-4R. In vivo, we found that endogenous synthesis of IL-4 mRNA is upregulated during the first week of infection, while an increase of IL-4R mRNA occurred later after infection of BALB/c mice with L. major. To interfere successfully with the IL-4 ligand-receptor interaction, we therefore chose to treat infected BALB/c mice with recombinant sIL-4R during the onset (e.g., days 0 to 7) of the immune response. Treatment with murine, but not with human, sIL-4R during the first week of infection rendered BALB/c mice clinically resistant to L. major, led to a 7- to 12-fold reduction of the parasite load in spleen and lymph nodes at 7 weeks of infection, shifted the pattern of cytokines towards a Th1 type, and provided durable resistance against reinfection. Thus, it could be demonstrated that the balance among sIL-4R, membrane-bound IL-4R, and their ligand IL-4 can be modulated in vivo, thereby modifying the antiparasitic immune response. These results suggest a therapeutic value of sIL-4R in diseases in which neutralization of IL-4 is desirable.

Animals↗

PubMed1994

Homodimeric murine interleukin-3 agonists indicate that ligand dimerization is important for high-affinity receptor complex formation.

Homodimeric murine interleukin 3 (mIL-3) agonists were generated by intermolecular cystine-bonding. Steady-state binding assays and association kinetics performed at 4 degrees C using these agonists revealed specific binding to both the high- and low-affinity receptor. DSS-mediated crosslinking studies performed at 4 degrees C with agonist concentrations compatible with high-affinity receptor complex formation allowed to detect protein complexes of the alpha chain, the beta chain(s) and the high-affinity receptor complex migrating with apparent molecular weights of 90 kDa, 140 kDa, and above 180 kDa, respectively. In contrast, monomeric mIL-3 was crosslinked to the alpha chain receptor only unless high concentrations were used. Binding studies performed at 4 degrees C revealed a positive cooperative interaction of monomeric mIL-3 with the low-affinity receptor. Proliferation studies and association kinetics performed at 37 degrees C showed that under physiological conditions these agonists were at least 2- to 3-fold more potent than monomeric mIL-3. We therefore propose that dimerization of mIL-3 may be involved in high-affinity receptor complex formation.

Animals↗