Synthesis and expression of MHC class II molecules in the absence of attached invariant chains by recombinant-interferon-gamma-activated bone-marrow-derived macrophages.

Pure populations of in vitro propagated bone marrow-derived macrophages are constitutively Ia negative. Co-culturing of these cells with recombinant interferon-gamma (rIFN-gamma) resulted in the appearance of high amounts of Ia antigens at the cell surface of essentially all cells. The continuous presence of the stimulus was a prerequisite for sustained Ia expression because removal of the stimulus resulted in rapid decline of surface Ia. Two-dimensional (2D) gel analysis (1D isoelectric focusing, 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of class II molecules synthesized by rIFN-gamma-stimulated bone marrow macrophages (BMM phi) revealed that, in contrast to class II co…

Increased Level of Intracellular MHC Class II Molecules in Murine Langerhans Cells Following In Vivo and In Vitro Administration of Contact Allergens

Treatment of murine Langerhans cells (LC) with contact allergens results in increased internalization of cell membrane constituents and therefore in depressed cell-surface expression of major histocompatibility complex (MHC) class II molecules during the first hours after haptenization. In this presentation we show that this downregulation of cell-surface-expressed Ia-antigens is accompanied by an augmentation of the intracellular pool of MHC class II molecules. Rat MoAb 2G9 was developed, which recognizes IA and IE molecules of the d-haplotype. This MoAb competes with the murine MoAb MK-D6 for binding sites to IAd-molecules. After blocking the cell-surface-expressed molecules with 2G9 and …

Modulation of accessory cell function of immortalized bone marrow-derived macrophages by granulocyte/macrophage colony-stimulating factor.

To generate cloned macrophage populations with sensitivity towards granulocyte/macrophage colony-stimulating factor (GM-CSF), bone marrow-derived macrophages (BMM phi) were immortalized by transformation with SV40. A panel of transformed clones was established. The majority of clones represented independently derived transformants, as evidenced by restriction fragment length polymorphism using genomic DNA digested with EcoRI and TaqI and the 5.2 kb SV40 DNA for hybridization analysis. The cells belong to the macrophage lineage according to several criteria, e.g. the presence of nonspecific esterase, their phagocytic capacity and their morphology. Many clones were potent antigen-presenting c…

Analysis of Invariant Chain Processing in 3 Day Cultured Rat Langerhans Cells

MHC class II molecules, critical peptide binding elements involved in the presentation of exogenous antigen to T helper cells, are expressed constitutively by Langerhans cells (LC) within their epidermal microenvironment. Several studies in mouse and man demonstrated, that short term in vitro culture of LC entails remarkable functional and penotypic alterations, including a profound increase of class II elements exposed at the LC’s surface1. Biosynthetic analysis revealed a downregulation of class II synthesis during the culture period2,3. In recent work on rat LC we described the uncoupling of the coordinately regulated biosynthesis of class II and invariant chain proteins in the course of…

Recombinant GM-CSF Induces in Vitro Differentiation of Dendritic Cells from Mouse Bone Marrow

The unprecedented functional capacity of dendritic cells (DC) in sensitizing resting T cells and their role in triggering T dependent immune responses attract increasing interest in this unique accessory cell population. Like macrophages (Mph) DC have been described to originate in the bone marrow (BM) (1). While the cytokine-promoted in vitro differentiation of Mph from BM-cells is well established, a convincing in vitro culture system for propagating mouse DC from BM-cells has not yet been reported. This work demonstrates the differentiation of DC from mouse bone marrow cells by a short term in vitro culture system supplemented with rGM-CSF.

Uptake of microparticle-adsorbed protein antigen by bone marrow-derived dendritic cells results in up-regulation of interleukin-1α and interleukin-12 p40/p35 and triggers prolonged, efficient antigen presentation

Dendritic cells synthesize and express major histocompatibility complex (MHC) class II peptide-binding elements constitutively and, therefore, belong to the category of professional antigen-presenting cells. Unlike other cells that show constitutive class II expression, such as B cells and certain T cell clones, dendritic cells possess the unique capacity to activate naive T cells. Using dendritic cells generated in vitro by culture of mouse bone marrow in the presence of low doses of recombinant mouse granulocyte/macrophage colony-stimulating factor, we found that discrete maturation stages of these cells can be distinguished which were correlated with defined functional capabilities. The …

An MHC class II-expressing T cell clone presenting conventional antigen lacks the ability to present bacterial superantigen.

We have analyzed the response of rat T cells to myelin basic protein (MBP) and the bacterial superantigen, staphylococcal enterotoxin E (SEE). Rat T cells reactive with MBP can respond to SEE presented by spleen cells but not to SEE presented by LOA, a rat T cell clone that expresses both I-A and I-E MHC class II molecules, even though LOA is much more efficient than splenic APC in the presentation of MBP. The inability of LOA to present superantigen is not due to a structural difference in MHC II molecules between LOA and the splenic APC or to differential expression of major accessory/adhesion molecules, including CD2, CD5, CD4 and CD44, on LOA. The non-responsiveness of SEE/LOA-induced T…

Complete cDNA sequence coding for the MHC class II RT1.B alpha chain of the Lewis rat.

Sequence of rat cDNA clone pLR 112 coding for the RT1.D 1 chain

Two different subtypes of antimitochondrial antibodies are associated with primary biliary cirrhosis: identification and characterization by radioimmunoassay and immunoblotting.

Antimitochondrial antibodies from patients with primary biliary cirrhosis react with different mitochondrial polypeptides as demonstrated by Western blots. The IgG fractions of a patient with primary biliary cirrhosis Stage I reacting exclusively with a pair of polypeptides at 48,000 daltons (p 48) on Western blot and from a patient with Stage III primary biliary cirrhosis reacting exclusively with a single 62,000 dalton polypeptide (p 62) were labeled with 125I; two radio-immunoassays were established detecting antimitochondrial antibodies against p 62 and p 48, respectively. Autologous sera blocked the assay, but the two reference sera did not block each other. Fourteen of 40 patients wit…

Development of Rat DC by in Vitro Culture of Bone Marrow Cells

Dendritic cells (DC) represent a subpopulation of leukocytes of bone marrow (BM) origin, involved in crucial immunological reactions. DC play a fundamental role in the primary immune response by stimulating quiescent T cells. In this study we describe an in vitro culture system to raise DC from unfractionated bone marrow (BM) cells of LEWIS rats in the presence of low doses of mouse recombinant GM-CSF, that was successfully used in previous work to culture mouse DC1,2,3.

Contact Allergens Modulate the Expression of MHC Class II Molecules on Murine Epidermal Langerhans Cells by Endocytotic Mechanisms

MHC class II molecules play an important role during the sensitization phase of allergic contact dermatitis. To study the influence of contact allergens on the expression of these molecules by murine epidermal Langerhans cells (LC), we performed a flow-cytofluorometric analysis of the Ia-antigen expression after in vivo application of contact allergens. A distinct decrease in the Ia-antigen expression of the entire LC population was noticed 3 h after in vivo application of the contact allergen 2,4-dinitrofluorobenzene (DNFB). This decrease was transient and balanced 24 h after in vivo application of DNFB. A downregulation was also detectable after in vivo application of the contact allergen…

Biochemical analysis of class II antigens. Identification of a two- and a three-polypeptide chain complex of I-A locus equivalent molecules in the rat.

The polypeptide chain composition of class II antigens from LEW rat spleen cells was studied utilizing cross-reactive mouse alloantiserum A. TH anti-A.TL (specificity anti-Iak) and the monoclonal antibodies MRC-OX6 and MRC-OX3 for immunoprecipitation. Two-dimensional gel mapping of A. TH anti-A. TL immunoprecipitates revealed that, as in the mouse, two groups of class II antigens exist corresponding to I-A and I-E locus equivalent structures. In the absence of reducing agents three monomeric chains α, 36 kDa (p36); γ, 33 kDa (p33); and β, 23 kDa (p23), were detected for I-A equivalent antigens, whereas I-E equivalent molecules separated into five monomeric chains: α, 37 kDa (p37); γ, 33 kDa…

Immunochemical characterization of anti-acetylcholine receptor antibodies in primary biliary cirrhosis

Although the presence of anti-mitochondrial antibodies is the main characteristic of primary biliary cirrhosis (PBC), other autoantibodies have been described in this disease. This study employs immunoblot methods to test whether the sera of PBC patients also contain antibodies directed against nicotinic acetylcholine receptors (AChR). We show that the majority of patients' sera indeed react with AChR just as sera of myasthenic patients do. In contrast, however, these anti-AChR antibodies do not lead to significant clinical symptoms of myasthenia. In all cases studied, PBC sera recognized a protein with the molecular weight of the alpha-chain of acetylcholine receptor (40 kDa). In addition,…

Nucleotide sequence of rat invariant γ chain cDNA clone pLRγ34.3

Granulocyte-macrophage colony-stimulating factor-cultured bone marrow-derived macrophages reveal accessory cell function and synthesis of MHC class II determinants in the absence of external stimuli.

The antigen-mediated activation of a number of T cell clones by bone marrow (BM) cells cultivated in the presence of various colony-stimulating factor (CSF) preparations was investigated. BM macrophages (BMM phi) grown in L929 cell supernatant as a crude source of macrophage colony-stimulating factor (M-CSF) as well as BM cells propagated in the presence of recombinant M-CSF exhibited transient antigen presentation potential to some T cell clones, being maximal on day 7 and having declined to a low level by day 19 of in vitro culture. Treatment of these long-term-cultivated BMM phi populations with recombinant interferon-gamma (IFN-gamma) resulted in predominant antigen presentation capacit…

Processing requirements for the recognition of insulin fragments by murine T cells.

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A…

Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD and Edman degradation

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at −80 °C to prevent peptide losses.

Full length cDNA of rat RT1.DMa and RT1.DMb and expression of RT1.DM genes in dendritic and Langerhans cells.

MHC encoded DM heterodimers and classical MHC class II complexes meet in an endosomal/lysosomal compartment where DM heterodimers support peptide loading of MHC class II. Studies on peptide loading of rat class II and on peptide persistence in cells of the dendritic lineage prompted us to establish full length cDNA clones coding for the subunits alpha and beta of rat DM molecules as well as a mAb directed against the luminal moiety of the beta subunit. Here we describe the establishment of the first full length cDNA clones of rat RT1.DMa and RT1.DMb. The mode of expression of RT1.DM at the transcript level in bone marrow culture-derived dendritic cells, in Langerhans cells and in a number o…

Uptake of Bead-Adsorbed Versus Soluble Antigen by Bone Marrow Derived Dendritic Cells Triggers Their Activation and Increases Their Antigen Presentation Capacity

The property to internalize particles has for long time been ascribed primarily to macrophages. DC in contrast were considered generally as phagocytosis negative. Fully mature DC which can be isolated from various tissues of the body do indeed not take up particulate material; however immature DC which arise in differentiating bone marrow cultures do exhibit phagocytic capacity1. Consistent with their immature phenotype epidermal Langerhans cells were also described to possess phagocytic potential2.

Modulation of MHC Class II Determinants on Rat Langerhans Cells During Short Term Culture

Epidermal Langerhans cells (LC) are regarded as the most peripheral outpost of the immune system. They play a pivotal role during the onset of an immune response in the skin. One of the principal functions of LC is reflected in their extraordinary potency to present antigen to high activation requiring naive T cells.

Biochemical properties of MHC class II molecules endogenously synthesized and expressed by mouse Langerhans cells

The cell surface expression and biosynthesis of Langerhans cells (LC)-derived major histocompatibility complex (MHC) class II molecules from epidermal cells (EC) prepared freshly and cultured for up to 3 days was investigated. Based on the constitutive expression of MHC class II determinants by LC, a panning and magnetic bead selection procedure was employed, yielding 65% and 86% of I-A+ cells, respectively. Phenotypical and cytochemical examinations revealed that the two LC preparations were free of contaminating macrophages as well as B and T cells. Freshly prepared enriched LC were highly efficient in the stimulation of protein antigen-specific T cell clones, while LC purified from short…

Induction of anamnestic T cell proliferation by antigen-pulsed, bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMM phi) were grown in a liquid culture system in the presence of L cell-conditioned medium as a source of colony-stimulating factor. After a 4-h pulse with antigen, cultured irradiated BMM phi were capable of presenting the antigen to primed T cells as assessed in a T cell proliferation assay. Proliferation was optimal when BMM phi were used between days 5 and 8 of bone marrow cell culture. T cells of Lyt1 and Lyt123 phenotype had to be present at the start of the culture period to yield an optimal response. Conventional antisera and monoclonal antibodies directed against the H-2 I region and the I-A subregion, respectively, proved inhibitory in this system…

Relationship between the target antigen of liver-kidney microsomal (LKM) autoantibodies and rat isoenzymes of cytochrome P-450

Chronic active hepatitis (CAH) is a clinical syndrome of different etiologies. Liver-kidney microsomal (LKM) autoantibodies characterize a subgroup of HBsAg negative CAH, which is considered to be an autoimmune liver disease. By immunoblotting analysis (IB) LKM positive sera have been shown to react strongly with a poly-peptide band at 50 kD. Therefore we investigated various rat microsomal enzymes with a molecular weight around 50 kD as potential candidate target antigens. These included epoxide hydrolase, cytochrome P-450 reductase, and phenobarbital-inducible isoenzymes of cytochrome P-450 (PB1, PB2, PB3a, PB3b). By radioimmunoassay (RIA) and IB LKM positive sera were shown to react with…

Identification of transcripts of the T cell antigen receptor beta chain gene and major histocompatibility complex class II genes in antigen-presenting cloned BK-BI-2.6.C6 cells.

The cloned murine cell line BK-BI-2.6.C6 has previously been shown to exhibit T cell characteristics, to synthesize and express MHC class II molecules, and to present protein antigens to antigen-dependent T cell clones. As a more definitive proof of the T-cell nature of these cells, transcripts of the rearranged T cell antigen receptor (TcR) beta gene were assessed by Northern blot analysis. BK-BI-2.6.C6 cells constitutively transcribe mRNA for the light chain of TcR and express the disulphide-linked alpha, beta TcR heterodimer at the cell surface. In addition mRNA for the polymorphic MHC class II subunits A alpha and A beta as well as for the invariant gamma chain were detected. BK-BI-2.6.…

Synthesis and cell surface display of class II determinants by long-term propagated rat T line cells

We have investigated the capacity of the encephalitogenic BS rat T cell line bs 83 and its variant clone bs 83.III.C6 to synthesize and express RT1.B-specific class II molecule subsets defined by monoclonal antibodies (mAb) MRC-OX6 and MRC-OX3. Earlier studies had indicated that mAb MRC-OX6 recognizes three distinct molecular species: an immature oligomeric polypeptide chain complex comprised of the polymorphic subunits alpha, beta and the invariant proteins of the gamma group; a biosynthetic intermediate composed of post-translationally modified alpha, beta and gamma chain (denoted p35) and a fully glycosylated alpha, beta two-chain complex derived from the plasma membrane. MRC-OX3 was sho…

The TH1 Lymphokine Interferon-γ is a Potent Upregulator of Dendritic Cells with Phagocytic Capacity in GM-CSF Supplemented Bone Marrow Cultures

Myeloid dendritic cells (DC), macrophages and granulocytes are descendants of a hematopoietic progenitor cell that originates in the bone marrow1. Thus, bone marrow derived cells distributed in tissue culture in the presence of GM-CSF give rise to the three leukocyte populations which under various in vitro culture conditions proceed in differentiation and phenotypic maturation2–7.

Complete coding nucleotide sequence of cDNA for the class II RT1.B beta I chain of the Lewis rat.

We have established the first full length cDNA clone for the beta light chain of the MHC class II alpha, beta heterodimer (isotype RT1.B) of the rat. Clone pLR beta 118 was obtained from a self-primed lambda gt10 cDNA library of IFN-tau treated bone marrow-derived macrophages of the Lewis rat. Subcloning of pLR beta 118 into a transcription vector with subsequent in vitro transcription and translation using the reticulocyte lysate system in the presence of microsomes followed by immunoprecipitation with mAb OX6 and two-dimensional gel electrophoresis revealed the intact RT1.B beta I-chain.

Cell surface display of rat invariant γ chain: detection by monoclonal antibodies directed against a C-terminal γ chain segment

A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant gamma chain (amino acid 142-216) was generated using distinct fusion proteins that contain this gamma segment for immunization and hybridoma screening. Additional fusion protein were prepared carrying discrete regions of the gamma chain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native gamma chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenoc…

The invariant chains of mouse class II antigens: biochemical properties and molecular relationship

The proteins p40 (Mr = 40 000), p32 (Mr = 32 000), p28 (Mr = 28 000), p20 (Mr = 20 000) and p10 (Mr = 10 000) are described which occur in noncovalent association with the polymorphic alpha, beta heterodimer of class II antigens. They were investigated with respect to their molecular characteristics and their mutual structural relationship. p32, the predominant species of this group corresponds to the invariant chain gamma (Ii). In contrast to the polymorphic subunits alpha and beta, proteins p40, p28, p20 and p10 migrated like gamma in electrophoretically constant positions, when class II molecules of different subregions and different alleles were assessed by two-dimensional gel electroph…