A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes

Summary A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7–10 days of culture. The antibody BM8 belonged to the IgG_2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.     

Measurement of the immunoperoxidase staining of macrophages within liver granulomata of mice infected with Mycobacterium tuberculosis.

A quantitative image analysis technique developed for the measurement of the extent of macrophage activation and epithelioid cell differentiation was performed on mice infected experimentally with Mycobacterium tuberculosis. The granulomatous inflammatory response within the liver reached a peak at day 23 and declined by day 33. Animals of strain B10.BR (H-2k) showed an increased granuloma fraction as compared to Balb/k (H-2k) mice, thus confirming the influence of non-H2 genes in the control of granuloma formation in mice. Using a monoclonal antibody against CD11b/CD18 (Mac1;CR3), we observed two subpopulations of macrophages within the granulomata. The small, darkly staining cells at the periphery of granulomata appear to be newly recruited macrophages. Larger, paler staining cells toward the center of granulomata represent activated and mature epithelioid macrophages. Using a semiautomated image analyzer (Quantimet 970), we measured the relative numbers of these macrophage subpopulations. There were more activated macrophages (epithelioid cells) associated with the increased granuloma fraction in the B10.BR mice than in the Balb/k. However, similar numbers of newly recruited peripheral macrophages were found in both Balb/k and B10.BR strains. This technique has shown qualitative as well as quantitative differences in the granulomatous inflammatory response in this murine model of tuberculosis in strains of mice with quite different antibody repertoires to mycobacterial antigens.     

Experimental paracoccidioidomycosis in the syrian hamster: Morphology,ultrastructure and correlation of lesions with presence of specific antigens and serum levels of antibodies

Male hamsters (134) received intratesticular injection of a live cerebriform culture of Paracoccidioides brasiliensis and were sacrificed from 6 hours up to 123 days onwards. Tissues from testis, lymph nodes, lungs, liver, spleen, kidneys and intestines were examined microscopically; presence of specific antigens was saught in lesions of testis, regional lymph nodes and liver by indirect immunofluorescence (IF); inoculation site lesions were studied electron microscopically and circulating specific antibodies measured by complement fixation and IF tests.Up to 24 hours inoculation site lesions showed fungi surrounded by PMNs; 48 hours latter macrophages accumulated forming loose nodules; epithelioid granulomata appeared after 5 days. Fungi, scarce in early lesions, increased in numbers up to the time when epithelioid granulomata dominated the picture; in young granulomata fungi were abundant and small; older granulomata contained rare, vacuolated fungi. Ultrastructurally the space between fungi and host-cells was larger around reproducing forms decreasing in size as the parasites grew larger and being a virtual slit around old degenerated fungi. Immunofluorescence studies revealed that fungal walls were brightly fluoerescent; in early lesions macrophages surrounding fungi or free in the intersticium contained fluorescent antigenic material in the cytoplasm; similar macrophages were observed in draining lymph nodes as early as 18 hours after inoculation, and latter, in macrophage nodules and Kupffer cells in the liver; epithelioid and giant cells appear to block diffusion of antigens, since in epithelioid granulomata fluorescence was limited to fungal walls.Disseminated paracoccidioidomycosis occurred in 100% of animals after day 5 of infection. Besides specific lesions (containing fungi), antigens were identified by immunofluorescence in non specific lesions in the liver (diffuse or nodular Kupffer cell hyperplasia) and in the lymph nodes (histiocytic hyperplasia). Serum antibodies appeared in low titers, up to day 20, increasing onwards. From day 70 on, titers decreased and lesions changed from confluent epithelioid to loose granulomata infiltrated by PMNs; fungi that before were large and quiescent now were small and in active reproduction. Secondary amyloidosis was present in 85% of the animals.In the hamster, Paracoccidioidomycosis develops as a chronic progressive disease and the lesions are related both to fungi and its antigens.     

Ultrastructure of Mycobacterium marinum granuloma in striped bass Morone saxatilis

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close association of lysosomes with mycobacterial phagosomes, as well as the presence of electron-opaque material within phagosomes, suggested phagolysosomal fusion. Development of granulomas involved epithelioid transformation of macrophages, followed by appearance of central necrosis. Desmosomes were present between mature epithelioid cells. The necrotic core region of M. marinum granulomas was separated from overlying epithelioid cells by several layers of flattened, electron-opaque spindle-shaped cells. These cells appeared to be formed by compression of epithelioid cells and, aside from a flattened nucleus, did not possess recognizable organelles. Following the development of well-defined, paucibacillary granulomas, secondary disease was observed. Recrudescence was marked by bacterial replication followed by disruption of granuloma architecture, including loss of epithelioid and spindle cell layers. In advanced recrudescent lesions, normal tissue was replaced by macrophages, fibroblasts, and other inflammatory leukocytes. Large numbers of mycobacteria were observed, both intracellular and suspended in cellular debris.     

Induced inflammatory process in the Antarctic fish Notothenia neglecta

We studied the ability of the Antarctic fish Notothenia neglecta to conduct an induced inflammatory process at 0°C. Indian ink was injected and a cotton suture thread was implanted into muscle of different groups of fish. After 1–2 days of Indian ink injection, the ink was diffused in the perimysium and there was hemorrhage and cellular infiltrate composed mainly of macrophages A (with few and small lysosomes) and neutrophils; after 7–15 days, there were macrophages A and some macrophages B (cytoplasm clear, lamellar cytoplasmic system forming interdigitations); after 30 days, there was Indian ink in the interior of macrophages A. The suture thread process takes place in two phases: the first (up to 7 days) with predominance of macrophages A and few neutrophils, and the second (15–30 days) with predominance of macrophages B. It can be concluded that N. neglecta is responsive to irritant stimulus with inflammatory process indicating adaptation to the antarctic environment. Received: 22 July 1997 / Accepted: 27 April 1998     

Localization of hepatitis A antigen in liver tissue by peroxidase-conjugated antibody method: light and electron microscopic studies.

Hepatitis A antigen (HAAG) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg- positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasm of liver cells obtained from marmosets were infected with HAV contained HAAg.     

Mechanism of vasculitis and aneurysms in Kawasaki disease: role of nitric oxide.

NO in vivo has both beneficial and nonbeneficial effects depending on site and concentration. Peroxynitrite, resulting from the reaction of NO with superoxide radical, causes cellular damage. Nitrotyrosine, end product of NO's toxic effects on cellular proteins, is a stable compound that can be used to detect evidence of harmful quantities of NO. We sought to detect nitrotyrosine in coronary arterioles of DBA/2 mice injected intraperitoneally with Lactobacillus casei cell wall. The inflammatory response induced occurred in perivascular fashion and involved mainly macrophages. It was variable according to time points, being severe on days 10 and 14 and mild to moderate on days 3 and 7. Few basal inflammatory cells appeared in controls injected with phosphate-buffered saline. Western immunoblots of homogenized hearts on days 10 and 14 demonstrated specific nitrated proteins. Immunohistochemistry of frozen sections of diseased hearts showed positive immunoreactivity for nitrotyrosine in coronary arterioles at the same time points. These findings were absent in the controls. We also determined the expression of inducible nitric oxide synthase (iNOS) in controls on days 10 and 14. iNOS colocalized with nitrotyrosine in perivascular macrophages and coronary arterioles of treated mice. Additionally, aneurysms were found on day 10 and intracardiac hemorrhage with consequent death on day 14. These observations supply evidence that NO through its reactive product, peroxynitrite, and its antigen/tissue marker, nitrotyrosine, is directly involved in coronary arteritis and aneurysm development in mice models of Kawasaki disease (KD). This article shows that macrophages are central to this and bolsters the likelihood of L. casei being the cause of KD.     

A method for the induction of blood eosinophilia with simple protein antigens

A new method of immunization is presented in which blood eosinophilia is induced with simple protein antigens in rats. Large inert particles coated with antigen are injected intravenously embolizing the lungs. Eosinophilia is maximal 5 days after a booster injection. It is concluded that the site of tissue localization of antigen is important in blood eosinophilia induction.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Summary Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNAse dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4° C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37° C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsable for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Peritoneal inflammatory cells in plaice, Pleuronectes platessa L.: effects of stress and endotoxin

When plaice were injected intraperitoneally with either oyster glycogen or live Vibrio alginolyticus an acute cellular inflammatory response was observed. The duration of these responses, 7 and 15 days respectively, exceeded the time course of the mammalian cellular inflammatory reaction. Peak leucocyte numbers were found at 2–3 days and neutrophils, which were phagocytic, were more numerous than macrophages. Although the increase in macrophage numbers was less marked, these cells appeared more actively phagocytic than neutrophils. Cortisol injections and environmentally-induced stress caused a significant reduction in the extent of inflammatory cell infiltrates, while endotoxin significantly enhanced the response.     

Method for the preparation of stabile microencapsulated lactic acid bacteria

Summary A method to produce viable and stabile dry microorganisms for food and agricultural purposes was developed. Spray-dried, freeze-dried or liquid culture concentrates of lactic acid-producing bacteria were mixed with various bulking agents to form a homogeneous wet granulation having a water content of 35–60% (w/w). The wet granulation was extruded through a dye onto a spinning plate (350–500 rpm) of a spheronizing device which resulted in the formation of discrete spherical particles. After forming spheres, the aggregate cell particles, both coated and uncoated, were dried to a moisture level of 5–10% using a temperature below the microorganism's optimum growth temperature. The coated and uncoated products were stored at different temperatures and periodically sampled to determine stability. Uncoated cell particles were more stabile at 4°C than at 22°C for 76 days. While both coated (with sodium alginate or carboxymethyl-cellulose) and uncoated particles showed similar stability at 4°C, at higher storage temperatures the applied coating improved the storage stability of the culture particles.     

Experimental pulmonary paracoccidioidomycosis in mice: Morphology and correlation of lesions with humoral and cellular immune response

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6×10⁵ yeast forms ofParacoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay — MIF assay) were investigated since the 1^st to the 360^th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90.The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.     

Endocytosis in cultures of Blastocystis hominis

A study of the function of the electron-dense pits in the vacuolar and granular forms of Blastocystis hominis was undertaken. Immuno-electron microscopy using anti-clathrin antibody and colloidal gold demonstrated clathrin to be associated with all forms of the pits and some cytoplasmic vesicles. Cationized ferritin traced the pathway of endocytosis from the surface of the coated pits through internalization via electron-dense coated vesicles and uncoated vesicles and tubules in the cytoplasm. The cationized ferritin particles accumulated in the central vacuole, suggesting a metabolic or storage role for this structure. Differences in the accumulation of cationized ferritin particles were noted between vacuolar and granular forms. The hydrolytic enzyme acid phosphatase was not detected within the central vacuole suggesting that this structure does not act as a lysosome.     

Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens.

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.     

Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

The role of humoral and cellular mediators in enhanced mammary inflammatory reactions to staphylococcal infection in systemically immunized ewes

Prior systemic immunization with live Staphylococcus aureus vaccine enhances the early recruitment of neutrophils into nonlactating mammary glands infected with staphylococci. The study investigates the role of humoral and cellular mediators in this phenomenon. Intramammary infusion of bacteria suspended in immune sheep serum did not enhance the inflammatory response to infection in nonimmunized ewes despite the presence of complement in the infused serum. Infusion of complement activated by incubation with zymosan evoked a massive neutrophil influx into mammary secretions by 4 hr after infusion. Hemolytic complement activity was not detected in mammary secretions of immunized or nonimmunized ewes. These findings indicate that, despite the inflammatory effect of complement activation, humoral immune factors did not promote neutrophil migration into infected glands. Mammary glands of systemically immunized ewes stimulated 5 days previously with staphylococcal soluble antigens (SSA) supported larger neutrophil influxes during staphylococcal infection than contralateral glands stimulated with endotoxin 5 days prior to infection. Exudates of SSA-stimulated glands had significantly higher cell concentrations, prior to infection, than endotoxin-stimulated glands; however elevated cell concentrations in endotoxin-stimulated glands of nonimmune ewes did not support enhanced inflammatory responses. These findings suggest that qualitative but not quantitative characteristics of mammary leucocytes influence the inflammatory response to infection in systemically immunized ewes.