Clara Cell Secretory Protein Is Reduced in Equine Recurrent Airway Obstruction

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

Influence of Pilin Glycosylation on Pseudomonas aeruginosa 1244 Pilus Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.

Schistosoma mansoni phosphofructokinase: immunolocalization in the tegument and immunogenicity

Schistosoma mansoni depends for its survival on glycolysis. Two glycolytic enzymes, glyceraldehyde-3P-dehydrogenase and triose-phosphate dehydrogenase, found in both the adult and schistosomular tegument, have been reported to confer partial protection against cercarial infection. This paper describes the immunogenic properties of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis, and its localization in the tegument and adjacent tissues. Recombinant schistosome PFK was used as antigen. A polyclonal antibody against purified PFK from Fasciola hepatica was affinity purified using recombinant PFK and used in combination with immunogold labelling to identify PFK by transmission electron microscopy in cryosections. In both adult worms and in schistosomula most immunogold label localized in the cytoplasmic syncytial region with less being found in the tegument. There was no significant PFK localization within or external to the outer membrane. Sera from mice immunized with recombinant S. mansoni PFK with Freund's adjuvant or alum plus rIL-12 demonstrated high titres of anti-PFK IgG, but no protection against cercarial infection. Sera from mice that were acutely or chronically infected or multiply exposed to irradiated cercariae did not recognize recombinant schistosome PFK in either Western blotting or ELISA. Similarly, sera from humans infected with S. mansoni did not recognize PFK. We conclude that in spite of the high immunogenicity of rPFK in mice, it is not a significant immunogen during the course of infection and does not confer protection from schistosomiasis. One main difference between PFK and the other 2 glycolytic enzymes seems to be the inaccessibility of PFK to the outside surface of the tegument.

Segregation of cell adhesion molecules into membrane microdomains facilitates leukocyte-endothelial interactions

Cells interact with their extracellular environments by means of a variety of cellular adhesion molecules (CAM) and surface ligands. In many instances, CAMs interact in a sequential temporal fashion which suggests that these adhesion molecules may occupy or be polarized to various membrane microdomains on the cell surface. Detection of CAMs can be accomplished by a variety of methods including immunofluorescent microscopy and flow cytometry, and by the use of immunocytochemical markers (i.e. colloidal gold) in electron microscopy. The development of high resolution field emission SEM in the mid 1980's and the Autrata modification of the YAG detector for backscatter electron detection at low voltage has greatly facilitated the recognition of colloidal gold probes for detection of surface CAMs. Low voltage FESEM with Bse imaging provides increased resolution of cell surface topography (~3nm at 3-4 keV) which can be observed in 3-dimensions, and simultaneously permits detection/high spatial resolution of immunogold label by atomic number contrast.

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells.

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double-immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.

Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture.

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.

Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils.

Previous studies have suggested that the leukocyte adhesion proteins Mac-1 and p150,95 are stored in a latent intracellular pool in neutrophils, and cellular fractionation studies have shown that Mac-1 is localized primarily in the peroxidase-negative specific granules. To determine the subcellular location of leukocyte adhesion receptors (LAR), we used immunocytochemical techniques on frozen thin sections of human blood leukocytes that had been incubated for peroxidase to mark the peroxidase-positive azurophil granules. To enhance the sensitivity of detection, polyclonal antibodies against immunoaffinity-purified p150,95 were raised in rabbits and absorbed with leukocytes from a patient deficient in this protein. The antiserum reacted with p150,95 and two other antigens with the same beta subunit, Mac-1 and lymphocyte function-associated antigen 1 (LFA-1). In neutrophils, we observed immunogold label for LAR predominantly on the membranes of peroxidase-negative granules, and in smaller amounts on the plasma and perinuclear membranes. In double-label experiments, there was colocalization of LAR with lactoferrin in some of the peroxidase-negative granules. We conclude that the latent pool of LAR resides in the membranes of peroxidase-negative granules. A significant increase in label on the plasma membrane of neutrophils stimulated with PMA is consistent with secretion of LAR to the exterior of the cell during degranulation. While LFA-1 appears very early in neutrophil maturation, it is becoming clear that Mac-1 and p150,95 are upregulated from an intracellular storage pool of peroxidase-negative granules that appear during the myelocyte stage of differentiation. Further studies are indicated to determine the significance of these proteins on the plasma membrane of two other granulocytes, eosinophils and basophils.

A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.