Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells

Summary Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP antibody producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin ( or chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin antibody-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.     

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. I. Endocytosis and intracellular translocation of proteoglycan-gold conjugates

Proteoglycans (Mr approximately 200 000) isolated from bovine arterial tissue were decorated with 17 nm diameter gold particles for tracing in electron microscopic thin sections and surface replicas. Lysine and arginine residues of their proteoglycan protein core are assumed to be essential for gold conjugation. The resulting proteoglycan-gold conjugates, which appear as pearl string-like gold strands of about 170 nm in length were used to visualize binding, endocytosis and intracellular translocation of proteoglycans by homologous cultured arterial smooth muscle cells. The proteoglycan-gold conjugates bind to coated as well as to non-coated cell surface membrane areas at 4 degrees C. This is followed by the formation of membrane invaginations. Postincubation at 37 degrees C leads to a time-dependent uptake of proteoglycan-gold conjugates via non-coated and coated vesicles which after fusion are translocated to multivesicular bodies and to large sized vesicles within 1 h. After conversion of these vesicles to lysosomal compartments the gold particles are uncoupled from the proteoglycans and are concentrated within residual vacuoles. From these vacuoles the gold particles are extruded. In contrast to the surface-bound proteoglycan-gold conjugates the released gold particles are condensed to bulky aggregates. The results, which include competition, inhibition and pulse chase experiments, extend biochemical data on endocytosis and degradation of proteoglycans.     

Golgi complex is brefeldin A resistant in multidrug resistant cells.

The multidrug resistance (MDR) is one of the main reasons for chemotherapeutic failures in cancer patients. The overexpression of mdr1 gene product, P-glycoprotein (Pgp), leads to the appearance of resistant tumor cells. In the previous paper (Erokhina, 1997) we have demonstrated that the first stages of Pgp-mediated MDR are accompanied by the reorganization of cytoskeleton elements and the vacuolar system. These data were true for two independently isolated sublines of Syrian hamster embryo fibroblasts transformed by Raus sarcoma virus. In this study, we continued the investigation of the properties of the vacuolar system in Pgp-expressing cells. Brefeldin A (BFA), which is not a Pgp substrate, affects different elements of the vacuolar system and blocks vesicular transport. Our data demonstrate that BFA has different effects on parental and resistant cells. In parental cells, the Golgi apparatus and vesicular transport are sensitive to BFA, while in resistant sublines, BFA affects the vesicular transport but not the Golgi apparatus structure. We discuss the existence of similar and different BFA targets in parental and resistant cells and their role in the evolution of multidrug resistance mechanisms.     

A freeze-fracture study on the differentiation of Golgi and plasma membranes in plant cells

Dictyosomes, Golgi vesicles, and plasma membranes were investigated after freeze-fracture in cells from growing root tips of cress (Lepidium sativum L.), that are distinguishable by different cellulose content of the cell wall, into (i) meristematic cells during early formation of the cell plate, (ii) statocytes of the root cap, and (iii) cortex cells of the differentiation zone. The results of this study show that the number of intramembrane particles (imps) is high in dictyosome cisternae, but low in membranes of budding or dictyosome-derived vesicles. Imps are disperse in the vesicle membranes of meristematic cells (i), but are often grouped into clusters in vesicle membranes of statocytes (ii), and of cortex cells (iii). For the number of particle aggregates in vesicle membranes, the following relation holds: (i) < (ii) < (iii). The number of particles on both fracture faces (PF and EF) of the plasma membrane differs widely between the cell types investigated. There are approximately 250, 1400, and 3100 imps microns-2 on the PF and 50, 500, and 300 on the EF of (i), (ii), and (iii), respectively. The structural complexity of the plasma membrane as judged by the degree of particle aggregations on the PF and the number of cellulose microfibrils in the cell wall show the same relationship: (i) < (ii) < (iii). Thus, the strong correlation between the distribution of imps in vesicle membranes, the structural complexity of the plasma membrane, and the content of cellulose microfibrils indicate that selection of imps during vesicle formation at dictyosome cisternae is an integral component of biogenesis and structural differentiation of plant plasma membranes.     

Expression and targeting of intracellular antibodies in mammalian cells.

Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.     

Golgi detection in mitotic and interphase cells by antibodies to secreted galactosyltransferase

Secreted galactosyltransferase from bovine milk was used to induce antibodies cross-reacting with corresponding intracellular enzymes in a variety of cell lines and tissues. In contrast to the original antigen, the reactive intracellular galactosyltransferase appears as individual species (apparent MW approx. 42000-46000) in SDS-polyacrylamide gel electrophoresis. In indirect immunofluorescence microscopy affinity-purified IgGs locate the galactosyltransferase in a distinct perinuclear and juxtanuclear position indicative for the Golgi region. The rearrangement of labelled structures upon colcemid or monensin treatment--drugs known to influence Golgi morphology and function--is further proof for a Golgi association. The fate and distribution of Golgi elements during mitosis is described at the light microscopical level using galactosyltransferase as easily identifiable marker. In addition we evaluate the utilization of wheat germ agglutinin (WGA) binding for Golgi identification on tissue culture cells and show that WGA is not a reliable marker for certain cell types such as MDCK.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.     

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.     

Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes

Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.     

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.     

Chromogranin A in the human gastrointestinal tract: an immunocytochemical study with region-specific antibodies.

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411-424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 17-38 and 176-195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.     

Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of na?ve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.     

Endocytosis mediated by the mannose receptor in liver endothelial cells. An immunocytochemical study

Immunocytochemical labeling of ultrathin cryosections from rat liver showed that mannose-terminated glycoproteins are removed rapidly from the blood stream mainly by the sinusoidal endothelial cells. The mannose-terminated glycoprotein ovalbumin was injected intravenously into rats 1 min, 6 min, and 24 min before perfusion fixation of the liver. Several minor and at least three major subcellular compartments were shown to be involved in the endocytic process. One minute after injection, ovalbumin was found at the cell surface, in coated pits, in coated vesicles, in tubular structures, and bound to the membrane of large early endosomes of which some showed a cisternal structure. After 6 min, ovalbumin was found in the lumen of large electron-lucent late endosomes and after 24 min in electron-dense structures, presumably lysosomes. The early endosomes have an ultrastructure which, together with the labeling pattern, indicates that this compartment has the same function as the CURL identified in parenchymal liver cells. The results are in accordance with recent biochemical findings indicating that ovalbumin endocytosed by endothelial cells is found sequentially in three different subcellular fractions depending on the time between injection and cooling for fractionation (G. M. Kindberg, T. Berg: Intracellular transport of endocytosed mannose terminated glycoproteins in rat liver endothelial cells. In: E. Wisse, D. L. Knook, K. Decker (eds.): Cells of the Hepatic Sinusoid. Vol. 2. pp. 120-124. Kupffer Cell Foundation. Rijswijk The Netherlands 1989).     

Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin

In the accompanying paper (Griffiths, G., P. Quinn, and G. Warren, 1983, J. Cell Biol., 96:835-850), we suggested that the Golgi stack could be divided into functionally distinct cis, medial, and trans compartments, each comprising one or two adjacent cisternae. These compartments were identified using Baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV) and treated with monensin. This drug blocked intracellular transport but not synthesis of the viral membrane proteins that were shown to accumulate in the medial cisternae. In consequence, these cisternae bound nucleocapsids. Here we show that this binding markedly increased the density of the medial cisternae and allowed us to separate them from cis and trans Golgi cisternae. A number of criteria were used to show that the intracellular capsid-binding membranes (ICBMs) observed in vivo were the same as those membranes sedimenting to a higher density in sucrose gradients in vitro, and this separation of cisternae was then used to investigate the distribution, within the Golgi stack, of some specific Golgi functions. After labeling for 2.5 min with [3H]palmitate, most of the fatty acid attached to viral membrane proteins was found in the ICBM fraction. Because the viral membrane proteins appear to move from cis to trans, this suggests that fatty acylation occurs in the cis or medial Golgi cisternae. In contrast, the distribution of alpha 1-2- mannosidase, an enzyme involved in trimming high-mannose oligosaccharides, and of galactosyl transferase, which is involved in the construction of complex oligosaccharides, was not affected by monensin treatment. Together with data in the accompanying paper, this would restrict these two Golgi functions to the trans cisternae. Our data strongly support the view that Golgi functions have specific and discrete locations within the Golgi stack.     

Binding and activation of hemolytic complement by IgG antibodies: cooperativity between antibodies of different hapten specificity

The ability of IgG antibodies with different hapten specificities to fix C1 and activate C as a function of hapten density on a red cell surface was investigated. Rabbit anti-methotrexate and anti-folinic acid IgG antibodies in a mixture were highly efficient in fixing C1 and activating C when cells carried simultaneously high levels of both haptens. We wished to find out whether in a C-activating IgG complex both IgG molecules had to be in a form that could activate C1. By reducing hapten density of one of the haptens on a double labeled cell, complexes were generated where only one in a pair of IgG molecules was in the activating form; such a pair had the same activating efficiency as a pair in which both IgG molecules were in the activating form. It was concluded that cooperative activation of C in C1-binding IgG complexes required only one IgG in the complex to be in the activating form.     

Malignant melanoma in fine needle aspirates and effusions. An immunocytochemical study using monoclonal antibodies

The value of monoclonal antibodies (MAbs) for the immunodetection of keratin, vimentin and two melanoma-associated antigens recognized by NKI/C3 and NKI/Bteb for the diagnosis of malignant melanoma has been previously established on histologic preparations. In the present study, cytologic preparations from 20 fine needle aspirates and effusions from patients with malignant melanoma were evaluated using these antibodies. Twenty of 20 smears were negative for keratin, and 20 of 20 smears were positive for vimentin. Positivity for NKI/C3 was seen in 12 of 12 cases studied, and for NKI/Bteb in 12 of 13 cases. These results indicate that a panel of MAbs consisting of anti-keratin, anti-vimentin, NKI/C3 and NKI/Bteb is useful for a more accurate diagnosis of malignant melanomas on cytologic preparations. The expression of these antigens in melanoma cells in cytologic smears can be a valuable aid in the detection of primary (noncutaneous) and metastatic melanomas by fine needle aspiration.