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N Koch

Publications and source records attributed to N Koch.

At least 19 recordsLinked to original sources
PubMed1991

Rapid intracellular pathway gives rise to cell surface expression of the MHC class II-associated invariant chain (CD74).

In previous investigations, it had been shown that class II and associated invariant polypeptides are sorted to an endocytic route where transport is delayed. Invariant chain (Ii) is degraded in a post-Golgi compartment, presumably an endosomal vesicle, and only class II molecules emerge on the cell surface. By using a mAb against the extracytoplasmic domain of human Ii, we demonstrate, by electron microscopy and by cytofluorometry, surface expression of Ii on lymphoma cells and on human B lymphocytes. We examined surface expression of Ii upon inhibition by brefeldin A of intracellular transport from the endoplasmic reticulum to the Golgi stack. This treatment rapidly depletes the cell surface of Ii. In the subsequent absence of brefeldin A, Ii appears rapidly at the cell surface. Within 5 h, the previous level of surface Ii (sIi) is reconstituted. Chloroquine abrogates depletion of sIi by brefeldin A, apparently by inhibition of internalization of sIi. Because on its route to the cell surface Ii is not proteolytically digested, it was possible that Ii and associated class II molecules are not separated on this pathway. Immunochemical studies reveal that on the cell surface of a B lymphoma cell line a proportion of Ii and class II polypeptides are associated.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1991

Class II major histocompatibility complex molecules of murine dendritic cells: synthesis, sialylation of invariant chain, and antigen processing capacity are down-regulated upon culture.

Dendritic cells (DCs), such as Langerhans cells (LCs) of the epidermis and the DCs of lymphoid organs such as spleen, are potent antigen presenting cells. DCs express high levels of major histocompatibility complex (MHC) class II molecules, but, partly because of the low numbers of primary DCs in any tissue, there has been no detailed study of the biochemistry of their class II molecules. This information may be needed to help explain recent findings that DCs process native protein antigens when freshly isolated from epidermis and spleen. Processing ceases during culture, yet a strong accessory function for activating resting T cells develops. We studied immunoprecipitates of DC class II and invariant chain (Ii) molecules by two-dimensional gel electrophoresis. We found that (i) freshly isolated LCs synthesize large amounts of class II and Ii polypeptides; (ii) Ii molecules that are known to be involved in antigen processing display an unusually large number of sialic acids in fresh LCs; (iii) with culture, class II and Ii synthesis decreases dramatically and has virtually ceased at 3 days; and (iv) the turnover of class II in pulse/chase experiments is slow, being undetectable over a 12- to 32-hr culture period, whereas the turnover of Ii is rapid. We conclude that MHC class II molecules of DCs do not seem to be qualitatively unique. However, the regulation of class II and Ii expression is distinctive in that biosynthesis proceeds vigorously for a short period of time and the newly synthesized class II remains stably on the cell surface, whereas Ii turns over rapidly. This may enable DCs to process and retain antigens in the peripheral tissues such as skin and migrate to the lymphoid organs to activate T cells there.

Animals↗
PubMed1991

Molecules that modify antigen recognition.

MHC class II molecules assemble in the presence of invariant chains. These probably serve not only to protect the peptide-binding site on MHC class II molecules from endogenous peptides, but also to sort MHC class II molecules from the Golgi complex to endosomes and there to retain the class II polypeptides to allow binding of peptides generated from exogenous antigens.

Animals↗
PubMed1991

Correlation between invariant chain expression level and capability to present antigen to MHC class II-restricted T cells.

In this study we investigated the role of the invariant chain (li) in the presentation of hen egg lysozyme (HEL) and measles virus hemagglutinin (HA) antigens to MHC class II-restricted T hybridoma cells. Fibroblastic cells transfected with Ed or Ak genes, and supertransfected or not with the li gene, were used as antigen-presenting cells (APC). For every APC pair analysed, the amount of exogenous antigen needed to obtain a T-cell response was inversely correlated with the level of li expression. Exogenously provided HEL was efficiently presented by li-supertransfected APC at doses of 10 micrograms/ml or below. In contrast, non-li transfected fibroblastic cells, which express a low level of endogenous li, required at least 10 times more HEL to stimulate most of the T hybridoma cells. Analogous results were also obtained using exogenous HA. Finally, two different experiments suggest that basal li expressed in fibroblastic cells is involved in the presentation of exogenous antigen. In the first one, we showed that li/class II ratio was increased in high-density grown fibroblastic cells and that this increase correlates with the ability of the cells to present exogenous antigen. In the second, treating high-density grown cells with an antisense li oligodeoxynucleotide could impair their ability to present exogenous HEL. We also examined the presentation of endogenously-synthesized HEL or HA after introduction of the antigen into the biosynthetic pathway of the APC by transfection of HEL and HA cDNAs. There was no apparent difference in the capability of high density grown fibroblastic cells, transfected or not with li gene, to present endogenous HEL or HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.

Antibodies, Monoclonal↗
PubMed1990

Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element.

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.

Animals↗
PubMed1989

A role of Ia-associated invariant chains in antigen processing and presentation.

Most native antigens require processing in a cellular compartment for efficient presentation to T helper cells. The cellular elements that permit processing are not known. We investigated a possible role of the class II MHC-associated invariant chains in antigen processing. Fibroblast cells that were transfected with class II genes were compared with fibroblasts supertransfected with the invariant chain gene for their capacity to present the fifth component of complement (C5) to C5-specific class II restricted T cell clones or influenza virus protein to a virus-specific T cell clone. Only fibroblasts supertransfected with the invariant chain gene were able to present native antigen, even at very low antigen concentration, whereas both fibroblast types could present cyanogen bromide-fragmented C5 or the virus peptide. Presentation of intact antigen but not of fragmented antigen was totally abrogated by treatment of fibroblasts with chloroquine. The invariant chain gene encodes two polypeptides, li31 and li41. Expression of either li31 or li41 was sufficient to render class II-expressing fibroblasts capable of presenting intact antigen.

Animals↗
PubMed1989

MHC class II invariant chains in antigen processing and presentation.

Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing.

Antigens↗
PubMed1989

[Metabolic disorders in femur head necroses in adulthood].

The discussion paper contains the results of a study about 70 patients with necrosis of femoral head. In 28 patients we found anamnestic hints to large-dose steroid therapy, chronic alcoholism or radiation. As a result of the biochemical blood examination in 29 patients hyperlipoproteinemia and in 25 patients dyslipidemia were determined, accompanied by a carbohydrate metabolic disturbance (15 cases) and a purine metabolic disturbance (13 cases).

Adult↗
PubMed1988

Posttranslational modifications of the Ia-associated invariant protein p41 after gene transfer.

Biochemical analysis of a rat fibroblast cell clone, transfected with the murine Ia associated invariant chain gene, demonstrates the expression of a family of proteins. This indicates that all members of the invariant protein family are derived from the same gene. The proteins p41, Ii, p27, p25, and p10, synthesized in the transfectant cell line, are identical with the invariant proteins previously shown to associate noncovalently with Ia antigens. These proteins are identified by immunoprecipitation and western blotting with a monoclonal antibody against the N-terminus and with an antiserum against the C-terminal part of the invariant chain. One protein, p41, has recently been shown to contain a thyroglobulin repeat (TgR) element. It was suggested that p41 might use the TgR element as a signal sequence which guides its intracellular transport to endosomes or lysosomes. Here, I demonstrate that p41 binds four N-linked carbohydrates and is heavily sialylated. During transport through trans Golgi compartments p41 binds palmitic acid, presumably at the same cytoplasmic cysteine as previously shown for Ii. A consensus sequence surrounding the palmitylated cysteine of the invariant chains (Ii, p41) and the human transferrin receptor was found. The transferrin receptor is known to follow an endocytic pathway, for which its cytoplasmic domain is essential. It is conceivable that the palmitylated domain of the invariant chains is a guiding structure for the membrane fusion process with transport vesicles. A role of the invariant proteins for antigen processing/presentation is discussed.

Animals↗
PubMed1988

Cooperative effect of interferon-gamma and tumor necrosis factor-alpha on the induction of the class II antigen-associated invariant chain expression.

The regulation of the invariant chain (Ii) expression was studied in the human colon carcinoma cell line HT-29 that constitutively expressed neither Ii nor class II antigens. Upon stimulation of HT-29 cells with a combination of human recombinant tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), expression of mRNA and protein of the invariant chain were induced. In contrast, administration of TNF-alpha or IFN-gamma alone had no effect. A delayed induction of Ii mRNA, which was first detected 10-12 h after stimulation, was observed; this suggests an indirect regulatory mechanism. Stimulation with both IFN-gamma and TNF-alpha led to the co-expression of class II antigens with the invariant chain. In order to study the genetic basis for this stimulation the murine invariant chain gene (800 bp 5' flanking sequences and the structural gene) was transfected into HT-29 cells and transfected cells were tested for the ability to respond to IFN-gamma and TNF-alpha. Simultaneous application of both cytokines had a strong effect on the induction of the murine invariant chain. IFN-gamma alone had no effect and TNF-alpha only marginally stimulates murine invariant chain expression. The transfection experiment indicates that the murine invariant chain gene construct contains the structural elements which are responsible for regulation with IFN-gamma and TNF-alpha. We determined whether the cooperative effect of TNF-alpha and IFN-gamma is also found in vivo. Stimulations of mice were performed with TNF-alpha, IFN-gamma and a combination of both. The immunohistological analysis of kidney tissue sections revealed that TNF-alpha had no effect on Ii and Ia expression. Upon IFN-gamma treatment a minor subset of renal tubules showed staining for Ii, and less prominently also for Ia. However, simultaneous application of both cytokines led a strong induction of both Ii and Ia antigens in renal epithelial cells, thus suggesting that this synergistic effect potentially occurs under physiological conditions.

Animals↗
PubMed1987

The production of recombinant HLA-DR beta and invariant chain polypeptides by cDNA expression in E. coli.

In this report we describe the production of recombinant fusion proteins of the HLA-DRw6 beta chain and the murine Ia-associated invariant chain. cDNAs encoding the human HLA-DRw6 beta chain and the murine Ia-associated invariant chain were introduced into bacterial expression plasmids. These plasmids direct the synthesis of the respective molecules as fusion proteins of the bacteriophage MS-2 polymerase by E. coli. Fusion proteins purified from crude E. coli lysates were used to raise antisera in rabbits. These antisera were able to immunoprecipitate biosynthetically labelled class II and invariant chain antigens. Additionally, two anti-DR antisera were raised against single domains of the HLA-DR beta chain thus generating reagents with a defined fine specificity. The anti-murine invariant chain serum was shown to cross-react with the human invariant chain and therefore may be useful for studying invariant chain and Ia antigen expression in different species. The method described here permitted us to produce large quantities of immunologically relevant proteins, for use in the production of polyclonal and monoclonal antibodies. Soluble fragments of the fusion proteins representing certain DR domains may also be useful in functional immunological studies.

Animals↗
PubMed1987

Primary structure of the gene for the murine Ia antigen-associated invariant chains (Ii). An alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin.

The gene for murine Ia-associated invariant (Ii) chains (Ii31 and Ii41) was characterized by sequence analysis. The gene extends over approximately 9 kb and is organized in nine exons. Exon 1 encodes the 5' untranslated region and the cytoplasmic segment, exon 2 the membrane spanning segment and adjacent amino acids and exons 3-8 the extracytoplasmic portion of Ii31. Putative promoter sequences were found upstream of the start of the coding sequence. Between exons 6 and 7 an additional, alternatively spliced exon 6b has been identified. This exon is spliced into the mRNA coding for the Ii-related Ii41 protein. Exon 6b encodes a cysteine-rich domain of 64 amino acids. It shows a remarkably high homology to the repetitive elements in thyroglobulin, a precursor for thyroid hormone. Based on this homology, it is suggested that this domain (TgR) in Tg and in Ii41 may play a role either in hormone formation or as a carrier in the transport of molecules (thyroid hormone or processed antigen respectively) between intracellular compartments.

Amino Acid Sequence↗
PubMed1986

Differential expression of Ia and Ia-associated invariant chain in mouse tissues after in vivo treatment with IFN-gamma.

B10.BR mice were injected i.v. with varying doses of recombinant IFN-gamma on three consecutive days. In tissue sections of 13 organs, the distribution of Ia antigens and Ia-associated invariant chain (Ii) was studied by using an immunoperoxidase technique. In the control animal, Ia and Ii were shown to be co-expressed in most tissues. However, on Kupffer cells, a small number of hepatocytes, and a subset of lymphocytes in lymph nodes and in the splenic red pulp only Ii, and no Ia, was detectable. In contrast, strongly Ia+ interdigitating reticulum cells of T-dependent areas of lymph nodes and spleen were only weakly stained for Ii. IFN-gamma treatment resulted in a dramatic increase of MHC antigen expression throughout the body, with striking differences in the inducibility of certain tissues for Ia and Ii: Bronchial epithelium was clearly induced to express the invariant chain, whereas Ia antigens remained entirely absent. Moreover, in kidney tubules and colon epithelium, Ii was induced more broadly than Ia. In contrast to the induction of Ii on endothelial cells of larger vessels in kidney, heart, and lungs, no de novo expression of Ia or Ii in capillary endothelial cells was observed. The number of detectable Ia+/Ii+ interstitial dendritic cells considerably increased upon exposure to IFN-gamma. Neither neurons nor glial cells were induced to MHC antigen expression. Our data demonstrate that IFN-gamma applied i.v. is a potent inducer or enhancer of Ia antigens and invariant chain in a variety of cell types.

Animals↗
PubMed1986

In vivo induction of H-2K/D antigens by recombinant interferon-gamma.

B10.BR mice received i.v. increasing doses of recombinant interferon-gamma (rIFN-gamma) on three consecutive days. Using an immunoperoxidase technique the distribution of H-2K/D antigens was studied in frozen tissue sections of thirteen organs (kidney, liver, pancreas, esophagus, stomach, small intestine, colon, lungs, heart, brain, thymus, lymph node and spleen). Class I antigens were shown to be induced or enhanced in almost every organ after exposure to IFN-gamma. This effect was particularly conspicuous for renal tubular cells, hepatocytes, bronchiolar epithelial cells, gastric mucous cells, thymic cortical lymphocytes and capillary endothelial cells in heart and kidney. Neurons, glial cells, gastric chief and parietal cells, and pancreas cells were not inducible. The findings show that i.v. application of IFN-gamma leads to strong induction or enhancement of major histocompatibility complex class I antigens in a wide variety of tissues.

Antibodies, Monoclonal↗