Biomedical subjects

K Eichmann

Publications and source records attributed to K Eichmann.

At least 19 recordsLinked to original sources

PubMed1995

Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages.

The present study addresses the regulatory mechanisms involved in the arginine metabolism of macrophages by arginase and nitric oxide synthase. Induction of both enzymes with LPS or by mixed lymphocyte reaction has been reported. Here, we demonstrate that these enzymes can be independently induced in murine bone-marrow-derived macrophages with the appropriate agonists. Arginase expression is specifically triggered by IL-4, IL-10, PGE2 as well as non-toxic or detoxified LPS. Conversely, IFN gamma induces only NO synthesis in these cells. The results demonstrate that the metabolism of arginine in macrophages is controlled by TH1/TH2-dependent cytokines and suggest a regulatory role of arginase on the NO synthesis by intracellular substrate depletion.

Amino Acid Oxidoreductases↗

PubMed1995

Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines.

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively.

Amino Acid Oxidoreductases↗

PubMed1995

Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules.

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

Amino Acid Sequence↗

PubMed1994

Streptavidin-tricolor is a reliable marker for nonviable cells subjected to permeabilization or fixation.

Cell death is normally accompanied by loss of integrity of the cell membrane. Concomitant loss of osmotic pressure and permeability for DNA binding dyes like propidium iodide make it possible to distinguish viable from nonviable cells. However, after permeabilization for intracellular staining or after fixation of the cells, we find that propidium iodide leaks out of nonviable cells and is transferred to formerly viable cells. Cell size cannot be used for examining viability in permeabilized or heterogeneous cell populations. Here we show that streptavidin-tricolor enters specifically and irreversibly into dead cells, and is not transferred to formerly viable cells after fixation or permeabilization. Therefore, streptavidin-tricolor can be a useful dead-cell marker in experimental situations where conventional methods fail to distinguish between viable and nonviable cells.

Animals↗

PubMed1993

Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

Animals↗

PubMed1992

Autoreactivity of low but not of high CD4 variants of an antigen-specific, I-A-restricted mouse T cell clone.

Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.

Animals↗

PubMed1992

Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes.

The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.

Animals↗

PubMed1992

In vivo depletion of interferon-gamma leads to susceptibility of A/J mice to mouse hepatitis virus 3 infection.

The possible role of interferon-gamma (IFN-gamma) in the resistance of A/J mice to MHV3 infection was investigated. Monoclonal antibodies specific for IFN-gamma, CD4 and CD8 molecules were administered in vivo to deplete selectively the IFN-gamma synthesized or the appropriate subset of T cells. The animals were then infected with MHV3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the IFN-gamma synthesis in sera and peritoneal exudates. After MHV3 infection, a full resistance of control A/J mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. The IFN-gamma synthesis in sera and peritoneal exudates of depleted mice, after MHV3 infection, drastically decreased when compared to that detected in control mice. The data presented are consistent with the hypothesis that IFN-gamma plays an essential role in the resistance of A/J mice to MHV3 infection.

Animals↗

PubMed1992

Expression of murine soluble CD4 protein in baculovirus infected insect cells.

The expression of murine soluble CD4 (L3T4) protein (sCD4) by baculovirus-infected insect cells was characterized. The yield of sCD4 reached 2 mg/l culture supernatant late in infection. Nevertheless, a large amount of sCD4 remained cell-associated, presumably in the endoplasmic reticulum or an early golgi compartment, as indicated by the endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitivity of its carbohydrate chains. The secreted form of sCD4 is modified with both endo-beta-N-acetyl-D-glucosaminidase D (endo-D) and endo-H-sensitive oligosaccharides. It was possible that the incomplete secretion indicated faulty glycosylation or improper folding of the sCD4 protein. However, inhibitor studies showed that complete carbohydrate processing is not required for secretion of sCD4 by insect cells. Moreover, maintained reactivity with a panel of monoclonal Ab as well as phase partitioning experiments suggested that secretion is apparently not caused by misfolding of the sCD4 protein. Similar results were obtained with biologically active murine interleukin-4 produced by insect cells. This indicates that an inefficient secretory pathway may be a general problem of baculovirus-infected insect cells and is not a consequence of incorrect molecular conformation.

Animals↗

PubMed1992

Human peripheral blood gamma delta T cells respond to antigens of Plasmodium falciparum.

Peripheral blood lymphocytes from donors previously unexposed to malaria parasites proliferate in vitro when stimulated with whole parasitized red blood cells of several different strains of Plasmodium falciparum. Here we show that both cells enriched for both memory (CD45R0+) and naïve (CD45R0-) phenotype can respond. Cells involved in these responses occur at frequencies similar to those observed for recall antigens such as tetanus toxoid but at lower frequencies than observed for the superantigens staphylococcal enterotoxin B or the mitogenic lectin phytohemagglutinin (PHA). Proliferation is inhibited by antibodies to class II MHC and to CD3 molecules. Stimulation of purified CD45R0- T cells by whole parasitized red blood cells for 6 days results in the generation of a large proportion of gamma delta T cell blasts of V gamma 9V delta 2 TCR phenotype and in the acquisition of the CD45R0 molecule within the blast cell population. The rapid generation of a vigorous primary in vitro gamma delta T cell response by malarial parasites may reflect the situation during primary malarial infection.

Animals↗

PubMed1991

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.

Amino Acid Sequence↗

PubMed1991

Affinity enhancement and transmembrane signaling are associated with distinct epitopes on the CD8 alpha beta heterodimer.

CD8 is a heterodimeric membrane glycoprotein on MHC class I-restricted T lymphocytes that cooperates with the alpha beta CD3 TCR in the recognition of MHC class I molecules presenting antigenic peptides. Co-operation has two components: enhancement of the affinity of MHC/peptide-TCR interaction, and signal transduction through the T cell membrane. The cytolytic function of CTL is primarily dependent on the affinity-enhancement component of CD8-TCR cooperation whereas activation of resting CD8+ T cells is primarily dependent on transmembrane signaling. Using a panel of mAb, two to the alpha-chain and three to the beta-chain of CD8, we investigated the relationships between epitopes and functional regions of the CD8 molecule. Two of the antibodies, one to the alpha-chain and one to the beta-chain of CD8, inhibit the cytolytic function of CTL but not the generation of CTL from resting T cells. Another two antibodies, also one to the alpha- and one to the beta-chain, inhibited the generation of CTL while enhancing the cytolytic function of CTL. These results suggest that both the alpha- and beta-chain of CD8 possess two distinct regions, one involved in affinity enhancement and the other in transmembrane signaling. The former may be the MHC class I-binding region whereas the latter may associate with the alpha beta CD3 TCR. The data can explain the apparent functional equivalence of CD8 alpha alpha homodimers and alpha beta heterodimers.

Animals↗

PubMed1991

Biased T cell receptor V alpha region repertoire in the synovial fluid of rheumatoid arthritis patients.

Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of synovial fluid T cells from two RA patients. TcR alpha chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the alpha chain constant (C alpha) gene segment, and the nucleotide sequences of 51 functionally rearranged cDNA clones were determined. Twenty different V alpha genes and 26 different J alpha gene segments were utilized in these cDNA clones. Three of the V alpha gene segments which are frequently (8%-17% total) expressed in synovial fluid T cells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals. The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V alpha genes is biased.

Antibody Diversity↗

PubMed1991

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

Animals↗

PubMed1991

The immune system: cells and molecules for the integration of self and non-self.

In a reductionist view, the immune system consists of 4 cell types, 3 diverse receptor systems and 10 to 15 soluble mediators including lymphokines and antibody molecules. Using these elements, the immune system generates 3 different effector mechanisms: Activated macrophages for the killing of intracellular microorganisms, cytotoxic T cells for the killing of virus-infected cells, and antibodies for the neutralization of soluble and cell-associated antigens of various types. While it is already very difficult to derive reliable results form reductionists experiments, such as studying immune responses in culture systems, it is infinitely more complicated to study the influence of physical exercise on the immune system.

B-Lymphocyte Subsets↗

PubMed1990

Production of murine interleukin-4 and interleukin-5 by recombinant baculovirus.

The cDNAs coding for murine interleukin-4 and -5 have been expressed using the baculovirus AcNPV as a vector in insect cells. Interleukins are secreted into the culture medium of virus-infected insect cells at high levels. Recombinant baculoviruses were isolated using a simple and fast selection scheme based on assays for interleukin activity. The cloning strategy described should be generally applicable to the production of any interleukin or other proteins with biological activity in the baculovirus system.

Animals↗

PubMed1990

Deviations in thymocyte development induced by divalent and monovalent ligation of the alpha/beta T cell receptor and by its cross-linking to CD8.

Fetal thymic organ cultures (FTOC) were tested as a model system to induce, in a polyclonal fashion, negative and positive thymic selection events. By flow cytometry, thymocytes developed in FTOC differed in several parameters from their in vivo differentiated counterparts. In particular, no clear distinction was possible between CD4+CD8+ immature cells with low TCR expression and mature CD4+ or CD8+ cells with high TCR expression. Thymocyte development in FTOC was manipulated with three different antibody reagents: anti-V beta 8 (F23.1), anti-Lyt-2.2 (19/178) and the quadroma derived bifunctional antibody HPHT-2, carrying one binding site of each. This antibody served also as a monovalent anti-V beta 8 reagent in FTOC from Lyt-2.1 mouse strains. Antibody 19/178 suppressed the development of single positive CD8+ cells, but only at very high concentrations. F23.1 and HPHT-2 suppressed the development of CD4+V beta 8+ and CD8+V beta 8+ thymocytes at relatively low concentrations giving rise to V beta 8 occupancies from about 2% upwards. Suppression was equally pronounced in cells with low and high TCR densities. Moreover, V beta 8 suppression occurred upon divalent and monovalent V beta 8 binding and was not significantly influenced by V beta 8-CD8 cross-linking. This suggests that ligation of the TCR alone is sufficient for clonal deletion. The data do not exclude a role for CD8 as an accessory adhesion molecule but suggest that exogenous cross-linking of CD8 to the TCR is not essential in transmembrane signaling for clonal deletion. At lower antibody concentrations giving rise to V beta 8 occupancies below detection, V beta 8-CD8 cross-linking by HPHT-2, but no divalent and monovalent V beta 8 ligation, induced an increase of CD8+V beta 8+ cells at the expense of CD4+ V beta 8+ cells with no change in the proportion of total V beta 8+ thymocytes. The latter effect was quantitatively of borderline significance but reproducible. These latter results are compatible with the hypothesis that cross-linking of the alpha beta TCR and CD8 on the thymocyte surface provides a maturation signal resulting in loss of CD4 from CD4+ CD8+ double positive immature thymocytes.

Animals↗

PubMed1990

Anti-CD4 modifies the kinetics but not the magnitude of anti-CD3-induced interleukin 2 production in peripheral T cells.

We have studied the effect of T cell receptor (TcR) cross-linking and TcR-CD4 cross-linking on the induction of interleukin (IL) 2 and IL 2 receptor mRNA expression in peripheral T cells. We confirm our previous results using bioassays, suggesting that CD4 is involved in T cell activation by up-regulating the expression of IL 2 receptors. Furthermore, we show that this also concerns other functional T cell genes such as IL 4 and interferon-gamma. In contrast, similar levels of IL 1 mRNA are found in T cells activated with anti-CD3 with and without anti-CD4 antibody, suggesting that the induction of the IL 2 gene is regulated by TcR cross-linking alone and not influences by CD4. However, anti-CD4 appears to accelerate the induction of IL 2 mRNA in the T cell populations, presumably by a more rapid adhesion of the cells to the antibody-coated surface.

Animals↗