Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer.

The Z39Ig protein (complement receptor for C3b and iC3b) is expressed on resident tissue macrophages in various tissues. This study was undertaken to examine the distribution of Z39Ig+cells and their phenotypic features in rheumatoid arthritis (RA) synovium, in comparison with those of osteoarthritis (OA) and psoriatic arthritis (PsA) synovium. Monoclonal anti-Z39Ig antibody was produced by immunizing Z39Ig transfected murine pre B cells and used for the identification of Z39Ig+cells. Z39Ig+cells were further stained with antibodies to macrophages, fibroblast-like synoviocytes, complement receptors and dendritic cells by using the double immunostaining method in normal, RA, OA and PsA synovium. RA synovial mononuclear cells were double-stained using anti-Z39Ig and anti-CD11c antibodies and sorted into Z39Ig+CD11c+cells and Z39Ig+CD11c-cells. These cell populations were then analysed by electron microscopy. The expression of the Z39Ig protein was limited to intimal macrophages in normal, RA, OA and PsA synovium. The numbers of Z39Ig+CD11c+cells and the ratios of Z39Ig+CD11c+cells to Z39Ig+cells were increased in the synovial lining layer of RA as compared with those of OA and PsA. The ultrastructural analysis of Z39Ig+CD11c+cells showed the character of macrophages with many secondary lysosomes and swelling of mitochondria. Z39Ig+ cells appeared to be useful for identification of resident tissue macrophages in normal synovium and the corresponding macrophages in the synovial lining layer of inflammatory arthritis. Expansion of Z39Ig+CD11c+cells was characteristic of RA synovial lining layer.

The intron 5-inserted form of rat erythropoietin receptor is expressed as a membrane-bound form.

The cDNA encoding an intron 5-inserted form of the erythropoietin receptor (I5Epo-R) has been cloned from rat. DNA sequence analysis reveals that the insertion of intron 5, which consists of 79 bp, causes a shift in reading frame and results in termination in the region of exon 7. The deduced amino acid sequence is composed of 316 amino acid residues, which is a molecular weight of 34220. To study the function of rat I5Epo-R, we established a Chinese hamster ovary cell line expressing rat I5Epo-R. Western blot analysis and binding studies with 125I-recombinant human erythropoietin showed that the transfected cells expressed rat I5Epo-R with a molecular size of 36 kDa as a membrane-bound form, but not as a soluble form, and had a single class of binding sites with a Kd of 700 pM.

ADP-ribosylated actin as part of the actin monomer pool in rat brain.

Mono-ADP-ribosylation in mammals is poorly understood. In this study, we found mono-ADP-ribosylated actin in rat brains. Mono-ADP-ribosylated actin by ADP-ribosyltransferase or nonenzymatic reaction was shown at a different position from the unmodified actin in the isoelectrical focusing. High-pressure liquid chromatography utilizing a reverse phase (ODS) column separated ADP-ribosylated actin from unmodified actin. In the two-dimensional gel electrophoreses and high-pressure liquid chromatography, the endogenously ADP-ribosylated actin was detected in the supernatant fraction from the rat brain extract, where a nonpolymerizing actin was present after removal of the polymerizing actin. The concentration of NAD and ADP-ribose, after microwave irradiation, was 220 nmol and 150 nmol/g of rat brain tissue. Actin ADP-ribosylated by purified ADP-ribosyltransferase failed to form actin filaments after the addition of Mg2+. Actin ADP-ribosylated by the nonenzymatic reaction could polymerize with the addition of Mg2+. The enzymatically modified actin could form actin filaments after treatment with ADP-ribosylhydrolase but not after treatment with phosphodiesterase. These results suggest that ADP-ribosylated actin by enzymatic or nonenzymatic reaction is one of the sequestering factors in actin-actin binding and is a part of the actin pool in the rat brain.

ADP-ribosylation in adrenal glands: purification and characterization of mono-ADP-ribosyltransferases and ADP-ribosylhydrolase affecting cytoskeletal actin.

Mono-ADP-ribosylation in mammals is poorly understood. In this study, we purified four mono-ADP-ribosyltransferases and one ADP-ribosylhydrolase from rat adrenal medulla. The four purified mono-ADP-ribosyltransferases had molecular weights of 69,000 by gel filtration, pH optima of 8.0, and Kms for their action on NAD of about 20 microM. The four enzymes ADP-ribosylated to the alpha-subunit of heteromeric GTP-binding proteins. After tryptic digestion of alkylated actin mono-ADP-ribosylated by the purified mono-ADP-ribosyltransferases or botulinum C2 toxin, the two radioactive peptide patterns were identical. The purified ADP-ribosylhydrolase with mono-ADP-ribosylated actin as the substrate had a molecular weight of 61,000 on gel filtration, a pH optimum of 7.5, and a Km for mono-ADP-ribosylated actin of about 7 microM. The enzyme released ADP-ribose from ADP-ribosylated actin and the alpha-subunit of hetromeric GTP-binding proteins. Actin monomers mono-ADP-ribosylated by the four mono-ADP-ribosyltransferases did not form actin filaments after the addition of Mg2+. After release of ADP-ribose by ADP-ribosylhydrolase, actin filaments formed on the addition of Mg2+, suggesting that the polymerization and depolymerization of cytoplasmic actin the adrenal chromaffin cells may be regulated by mono-ADP-ribosylation.

Effects of mono-ADP-ribosylation on cytoskeletal actin in chromaffin cells and their release of catecholamine.

To better understand the physiological role of mono-ADP-ribosylation in animals, we examined its role in chromaffin cells. Monoclonal antibodies against rat brain ADP-ribosylhydrolase were prepared, one of which (9E7) completely inhibited the enzyme's activity with ADP-ribosylated actin as the substrate. After actin monomers were polymerized by the addition of Mg2+, mono-ADP-ribosylation induced actin depolymerization. After mono-ADP-ribosylation, the actin monomers did not polymerize by the addition of Mg2+. Polymerized actin cosedimented with chromaffin granules but mono-ADP-ribosylated actin did not. After ADP-ribosylhydrolase on the membrane of chromaffin granules was incubated with 9E7, mono-ADP-ribosylated actin did not cosediment with chromaffin granules. When chromaffin cells permeabilized with saponin were incubated with NAD and 9E7, actin and rho protein was mono-ADP-ribosylated and stimulated catecholamine release from the cells. In histochemical experiments, catecholamine and actin filaments disappeared when the permeabilized chromaffin cells were treated with NAD and 9E7. These findings indicate that mono-ADP-ribosylation breaks the actin barrier in order to move granules during exocytosis, and ADP-ribosylactin hydrolase may keep the granules within the actin barrier.

Characterization of a novel monoclonal antibody that senses nitric oxide-dependent activation of soluble guanylate cyclase.

Two monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the alpha- and beta-subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the beta-subunit and its affinity did not change with NO. Neither mAb cross-reacted with particulate GC. Cultured Purkinje cells from rats were treated with S-nitroso-N-acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased.

Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena gracilis.

In Euglena gracilis, the activity of ADP-ribosyl cyclase, which produces cyclic ADP-ribose, oscillated during the cell cycle in a synchronous culture induced by a light-dark cycle, and a marked increase in the activity was observed in the G2 phase. Similarly, the ADP-ribosyl cyclase activity rose extremely immediately before cell division started, when synchronous cell division was induced by adding cobalamin (which is an essential growth factor and participates in DNA synthesis in this organism) to its deficient culture. Further, cADPR in these cells showed a maximum level immediately before cell division started. A dose-dependent Ca2+ release was observed when microsomes were incubated with cADPR.

Ultrastructural immunocytochemical studies of blood group substances in human eccrine glands.

We investigated the ultrastructure of blood group antigens A, B, and H in human eccrine glands by means of the immunogold labeling technique. Blood group antigens A, B, and H were found in the Golgi apparatus, secretory granules, and over the apical and basolateral cell membranes of dark cells of eccrine glands depending on the blood group phenotype of the donors. Both A and B antigens were found in the dark cells of AB donors. The labeling pattern of the Golgi stacks seemed to have a polarity whereby the anti-blood group A antibody labeled all the stacks, whereas anti-blood groups B and H bound to the trans side of the Golgi complex. These observations suggest that the blood group substances are secreted into the lumen after being processed through the Golgi apparatus and the immature and mature granules in the dark cells of human eccrine glands.

Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules.

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II. (J Histochem Cytochem 47:481-488, 1999)

Postembedding staining of Brunner's gland with lectin-ferritin conjugates.

Postembedding staining of intracellular carbohydrates of rat Brunner's gland cells embedded in Epon and acrylamide was carried out with Ricinus communis agglutinin-ferritin, concanavalin A-ferritin, and wheat germ agglutinin-ferritin conjugates. Th Golgi vacuoles and mucous granules were stained with these conjugates. In each staining, the tissues embedded in acrylamide were stained more strongly than those embedded in Epon. The staining intensity of wheat germ agglutinin-ferritin was the strongest among the three conjugates and the staining intensity of Ricinus communis agglutinin-ferritin was stronger than that of concanavalin A-ferritin in both embedding methods. Free ferritin showed almost no binding to these structures and staining with the conjugates was inhibited by the addition of appropriate competitive sugars to the staining solutions. Osmium-postfixed tissues were not stained well with the conjugates. Washing of the sections with bovine serum albumin solution after staining was an essential step in the present method to reduce the nonspecific adsorption of the conjugates. The present method was very simple and had good reproducibility.

Sulfated glycosaminoglycans in guinea pig neutrophils studied by use of cationic colloidal gold.

Using a high electron resolution staining method, cationic colloidal gold (CCG, pH 1.0) staining, we studied the fine structural localization of sulfated glycosaminoglycans (GAGs) in various maturational stages of guinea pig neutrophils. Azurophil and specific granules of neutrophils reacted positively to CCG, with variety in labeling according to maturation. All immature azurophil and specific granules were labeled selectively. Mature granules lost their affinity with CCG. CCG-positive labeling was also observed in the trans to trans-most Golgi apparatus of promyelocytes and myelocytes. Prior absorption with poly-l-lysine prevented CCG labeling of tissue sections. Mild methylation of ultrathin sections at 37C did not alter CCG labeling, whereas CCG labeling disappeared after active methylation at 60C. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG labeling. These findings suggest the existence of sulfated GAGs not only in immature azurophil but also in immature specific granules of neutrophils. Sulfation of GAGs occurs in the trans- to trans-most Golgi apparatus of neutrophil granulocytes. A possible correlation between accumulation of sulfated GAGs and maturation of specific granules in neutrophils is also discussed.

Distribution of glycoconjugates in the kidney studied by use of labeled lectins.

Distribution of glycoconjugates in different areas of the rat kidney was studied by light and electron microscopy using six different horseradish peroxidase-labeled lectins. Glomeruli and brush borders of the proximal tubules reacted differently to these lectins, which indicated differences in the carbohydrate compositions of those regions. The ascending limb of Henle's loop (ALH) had strong binding sites for peanut agglutinin (PNA) and soybean agglutinin (SBA). Dolichos biflorus agglutinin (DBA) did not stain the cells of ALH but did stain those of distal convoluted tubules (DCT). DBA is a good marker for distinguishing ALH from DCT. DBA, PNA, and SBA were also good markers of the collecting duct. Ricinus communis agglutinin (RCA-1) and wheat germ agglutinin (WGA) diffusely stained the various components of different parts of the kidney.

Novel method to investigate kinetics of rat skin cells by means of an occlusive dressing method using bromodeoxyuridine.

We developed a novel technique to detect S-phase skin cells by applying bromodeoxyuridine (BrdU) epicutaneously using an occlusive dressing (OD) method. BrdU was scarcely absorbed from the skin with a simple epicutaneous application, whereas the incorporation of BrdU was very well promoted with the use of our OD method. We applied BrdU on the backs of rats using this method and investigated the conditions required for an optimal response, with a special focus on the period of application, the concentration of BrdU used and vehicles suitable for the immunocytochemical staining of this agent. From these experiments, we were able to determine that an application time of at least 60 min was necessary to liable S-phase cells, a 2% concentration of BrdU was needed to obtain consistent labeling and aqueous vehicles are satisfactory solvents for BrdU preparations. Epidermal keratinocytes and S-phase cells in the upper portion of dermis were clearly labeled after either intraperitoneal injection of BrdU or after administration by means of the OD method. To ascertain whether this latter method could provide an effective alternative to intraperitoneal injection, we compared the labeling patterns of both methods with respect to the speed of migration of BrdU-labeled basal cells from the basal layer to the horny layer of epidermis. Using either of these two methods, basal keratinocytes were labeled immediately after administration. Three days after the first administration, BrdU-labeled cells were detected in the middle layer of the epidermis, but after 8 days, they were no longer evident in epidermal tissue. As another means of comparing both methods, we used antibody to proliferating cell nuclear antigen (PCNA) and compared the ratio of PCNA-positive basal cells to BrdU-labeled basal cells. The number of PCNA-positive cells was about 4.6 times greater than the number of BrdU-labeled basal cells by both methods. We concluded that the OD method could be used as a substitute for intraperitoneal injection in order to observe cell kinetics using bromodeoxyuridine.

Purification of bovine soluble guanylate cyclase and ADP-ribosylation on its small subunit by bacterial toxins.

Soluble guanylate cyclase (sGC) consisting of two different subunits (alpha: Mr = 74,000, beta: Mr = 69,000) was purified more than 12,000-fold in terms of specific activity from the supernatant of bovine lung homogenates and characterized. The heme content determined with the pyridine hemochromogen method and Bradford's protein assay was 0.8 heme per dimer. Cholera, pertussis, and botulinum C3 toxins modified exclusively the beta-subunit of sGC, yielding the ADP-ribose-bound compound with 1:1 stoichiometry, and Vmax for the cyclase reaction was increased 10 times by this modification. When the ADP-ribosylation of sGC was performed simultaneously with two or three bacterial toxins which have distinct amino acid specificities, the resultant enzyme had only one ADP-ribose, and the activity was the same as that of the enzyme modified with one toxin. When NO was incorporated into the reaction mixture containing the ADP-ribosylated sGC, the cyclase activity noticeably increased by approximately the same amount as that seen for the unmodified enzyme. Such effects were not seen with CO. When ADP-ribosylated sGC was incubated with Mn2+, the enzyme activity was synergistically increased. The heme-deleted sGC was also ADP-ribosylated by bacterial toxins and its activity was raised. These findings suggest that sGC has an ADP-ribosylation site near the GTP binding site, like other GTP-binding proteins, and that the beta-subunit regulates the activity.

Purification and characterization of arginine:mono-ADP-ribosylhydrolase from Euglena gracilis Z.

Arginine:mono-ADP-ribosylhydrolase was purified from a protozoan, Euglena gracilis Z, using [32P]mono-ADP-ribosylated actin as a substrate. The enzyme showed molecular mass of 33 kDa both in SDS PAGE and gel filtration, indicating it to be a monomeric protein. It was strongly inhibited by ADP and ADP-ribose and activated by Mg2+, DTT, and 2-mercaptoethanol. These results suggest that it recognizes the ADP-ribose moiety of the modified protein. Since the enzyme activity increased in S phase and late G0 phase in a synchronous dividing culture, the enzyme may function in the regulation of the cell cycle.

Purification and characterization of ADP-ribosyl cyclase from Euglena gracilis.

ADP-ribosyl cyclase, which catalyzes the conversion from NAD+ to cyclic adenosine diphosphoribose (cADPR), is proposed to participate in cell cycle regulation in Euglena gracilis. This enzyme, which was found as a membrane-bound protein, was purified almost the homogeneity after solubilization with deoxycholate, and found to be a monomeric protein with a molecular mass of 40 kDa. Its Km value for NAD+ was estimated to be 0.4 mM, and cADPR, a product of the enzyme, inhibited the enzyme competitively with respect to NAD+ whereas another product, nicotinamide, showed noncompetitive (mixed-type) inhibition. In contrast to mammalian CD38 and BST-1, Euglena ADP-ribosyl cyclase lacked cADPR hydrolase activity.

Proliferation and migration kinetics of stem cells in the rat fundic gland.

The proliferation and migration of stem cells in the developing and adult rat fundic gland have been studied using BrdU immunohistochemistry and BrdU-GSA II (Griffonia-simplicifolia agglutinin-II) double staining. In the developing rat fundic gland, stem cells were first scattered throughout all levels of the epithelia and then concentrated in the depth of the pits. With the elongation and maturation of the fundic glands, stem cells left the gland base and moved upward. By 4 weeks after birth, the development of the fundic gland was completed and stem cells were confined to a narrow proliferative zone in the isthmus, reaching the adult distribution pattern. In the adult rat fundic gland, stem cells in the isthmus differentiated and migrated upward and downward, replacing the surface mucous cells and glandular cells respectively. For upward migration, it took about one week for stem cells to migrate from the isthmus to the surface. For downward migration, it took about two weeks for stem cells to migrate from the isthmus to the neck, and it took 30-36 weeks to reach the gland unit's blind end. Finally stem cells were lost at the deepest level of the glands. The results obtained by simple topographical distribution in the present experiment agreed well with those obtained by quantitative analysis, suggesting the usefulness of BrdU immunohistochemistry for cell kinetic studies.

Fabry disease: ultrastructural lectin histochemical analyses of lysosomal deposits.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal alpha-galactosidase activity. Globotriaosylceramide accumulates predominantly in lysosomes of various tissues. Former studies have clarified the nature of this disease, and the accumulated materials in the lysosomes have been analyzed using biochemical techniques. In the present study, transmission electron microscopy was used to reveal the fine structure of these lysosomal deposits, and sugar residues in the lysosomal deposits in Fabry disease were examined by lectin histochemistry combined with enzyme digestion. This is the first report to describe the lysosomal sugar residues in Fabry disease analyzed using lectin histochemistry at the ultrastructural level. With these techniques, we were able to detect alpha-galactosyl, beta-galactosyl and glucosyl sugar residues in the lysosomal deposits. The experimental procedures used in this study have considerable potential for use in investigations of glycolipid and glycoprotein storage diseases without the need for complex methodology and expensive materials.

Immunohistochemical study of cell proliferation and differentiation in epidermis of mice after administration of cholera toxin.

Cholera toxin causes reversible epidermal hyperplasia. We observed maximal thickness of the epidermis on the fourth day after treatment and a return to pretreatment values by day 7. The increase in thickness occurred in the basal and intermediate layers, with these layers becoming two to three times thicker than those of normal epidermis. The time sequence of epidermal proliferation was studied using bromodeoxyuridine (BrdU) labelling. We observed a maximum number of labelled basal cells within the first 24 h. Only a few cells were labelled 7 days after toxin injection. Griffonia simplicifolia-IB4 (GSA-IB4), Ulex europaeus-I (UEA-I) and Griffonia simplicifolia-II (GSA-II) lectins were used for the analysis of epidermal cell differentiation in the tissue sections. To study keratinocyte differentiation, further immunological staining was performed using two anticytokeratin antibodies, PKK2 and PKK3 mouse monoclonal antibodies. From the immunocytochemical results, we conclude that synchronous differentiation of the epidermis occurs after cholera toxin administration.

Inositol 1,4,5-trisphosphate and cyclic ADP-ribose mobilize Ca2+ in a protist, Euglena gracilis.

Inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) released Ca2+ from microsome fraction prepared from Euglena gracilis in dose-dependent manners. Caffeine, which also induced Ca2+ release from the microsomes, caused desensitization of the Ca2+ response to cADPR, although the Ca2+ response to InsP3 was not affected by caffeine. Further, ruthenium red inhibited the Ca2+ release induced by cADPR, but not by InsP3. These results suggest that cADPR functions as an endogenous messenger to activate a caffeine-sensitive, Ca(2+)-release mechanism, whereas InsP3 induces Ca2+ release by a distinct mechanism in E. gracilis.