apyrase and Pseudomonas-Infections

Airway defenses are regulated by a complex purinergic signaling network located on the epithelial surfaces, where ATP stimulates the clearance of mucin and pathogens. The present study shows that the obstructive disease cystic fibrosis (CF) affects the activity, expression, and tissue distribution of two ectonucleotidases found critical for the regulation of ATP on airway surfaces: NTPDase1 and NTPDase3. Functional polarities and mRNA expression levels were determined on primary cultures of human bronchial epithelial (HBE) cells from healthy donors and CF patients. The in vitro model of the disease was completed by exposing CF HBE cultures for 4 days to supernatant of the mucopurulent material (SMM) collected from the airways of CF patients. We report that NTPDase1 and NTPDase3 are coexpressed on HBE cultures, where they regulate physiological and excess nucleotide concentrations, respectively. In aseptic conditions, CF epithelia exhibit >50% lower NTPDase1 activity, protein, and mRNA levels than normal epithelia, whereas these parameters are threefold higher for NTPDase3. Exposure to SMM induced opposite polarity shifts of the two NTPDases on both normal and CF epithelia, apical NTPDase1 being mobilized to basolateral surfaces and bilateral NTPDase3 to the apical surface. Their immunolocalization in human tissue revealed that NTPDase1 is expressed in epithelial, inflammatory, and endothelial cells, whereas NTPDase3 is restricted to epithelial cells. Furthermore, the SMM-exposed CF HBE cultures reproduced the impact of the disease on their in vivo distribution. This study provides evidence that an extensive remodeling of the enzymatic network regulating clearance occurs in the airways of CF patients.

Topics: Adenosine Triphosphatases; Adult; Airway Remodeling; Antigens, CD; Apyrase; Azides; Cystic Fibrosis; Down-Regulation; Epithelial Cells; Humans; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Signal Transduction; Staphylococcal Infections; Up-Regulation

Previous experimental studies have shown that extended-wear contact lens usage results in a centripetal migration of Langerhans cells from the conjunctiva into the central cornea. To test the consequences of this, Langerhans cells were induced into the cornea before Pseudomonas aeruginosa infection in BALB/c mice that are normally resistant (the cornea heals) and in C57BL/6 mice that are susceptible (the cornea perforates) to bacterial challenge.. Mean clinical scores, slit lamp examination, adenosine diphosphatase (ADPase), and acid phosphatase staining as well as immunostaining with DEC-205, B7-1, CD4, and interleukin-2 receptor (IL-2R) antibodies and histopathologic, RT-PCR, and delayed-type hypersensitivity (DTH) analyses were used to examine the effects on bacterial disease after polystyrene bead induction of Langerhans cells into the cornea before bacterial challenge.. No difference in disease response was observed in bead- versus sham-treated C57BL/6 mice after bacterial infection; however, significant differences leading to corneal perforation were seen in BALB/c mice that included an increased number of Langerhans cells in the central cornea at 1 and 6 days after infection, an increased number of B7-1+ (mature) Langerhans cells at 6 days after infection, CD4+ and IL-2R+ T cells at 5 days after infection, enhanced DTH, and increased mRNA levels for IFN-gamma in cornea and cervical lymph nodes. Alternately, levels of IL-4 were significantly higher in the cornea and cervical lymph nodes of sham- versus bead-treated animals.. These data provide evidence that Langerhans cells are critical in the innate immune response to P. aeruginosa and provide new information regarding the mechanisms governing resistance versus susceptibility to bacterial infection with this opportunistic pathogen.

Topics: Acid Phosphatase; Animals; Antigens, CD; Apyrase; B7-1 Antigen; CD4-Positive T-Lymphocytes; Cornea; Eye Infections, Bacterial; Female; Hypersensitivity, Delayed; Immunoenzyme Techniques; Interferon-gamma; Interleukin-4; Keratitis; Langerhans Cells; Lectins, C-Type; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Minor Histocompatibility Antigens; Models, Animal; Pseudomonas Infections; Receptors, Cell Surface; Receptors, Interleukin-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Apyrase; B7-1 Antigen; B7-2 Antigen; CD28 Antigens; Cell Movement; Corneal Diseases; Female; Genetic Predisposition to Disease; Injections, Intraperitoneal; Langerhans Cells; Lymph Nodes; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; Species Specificity; Staining and Labeling

The morphology, distribution and quantitation of dendritic (Langerhans) cells (LC) was determined by analysis of ADPase stained epithelial flat mounts from 6-8 week young adult (resistant) and 24 month old (susceptible) aged mice before and after experimental infection with P. aeruginosa topically applied to the scarified cornea. The contralateral eye (controls) was also scarified and phosphate buffered saline applied similarly. This study has examined the changes in ADPase positive cell populations of the conjunctival limbal epithelium and corneal epithelium of naturally resistant mice (Swiss-Webster and CD2F1) following corneal infection with Pseudomonas aeruginosa at two different ages, young adult (8 week old) and aged (24 month old). The young adult mice recover from their infection and restore corneal clarity while the aged mice have extensive ocular destruction and corneal scarring. Conjunctival limbal dendritic cell numbers in young adult mice were found to be significantly increased at day seven post infection and then returned to baseline levels. In contrast, conjunctival limbal dendritic cell numbers in aged mice were found to increase slowly and to peak at fourteen days after infection. Other differences between the two ages (young adult and aged) included an initial increase in dendritic cells five hours post infection in the young adult groups and an initial decrease at five hours in the aged groups of mice.

Topics: Aging; Animals; Apyrase; Cornea; Corneal Diseases; Female; Langerhans Cells; Mice; Phosphoric Monoester Hydrolases; Pseudomonas Infections