asialo-gm1-ganglioside and Respiratory-Tract-Infections

The purpose of the present study was to determine the impact of NK cells on the development of protective adaptive immunity in response to nasal-pulmonary immunization against mycoplasma. Depletion of NK cells before nasal-pulmonary immunization enhanced resistance to mycoplasma respiratory infection. The effect of NK cells on the generation of protective immunity in lungs was dependent on lymphoid cells, as immunization of either SCID mice or immunocompetent mice depleted of CD4(+) T cells did not demonstrate any increased resistance in the presence or absence of NK cells. The presence of NK cells at the time of nasal-pulmonary immunization modulated mycoplasma-specific cytokine responses in lungs and lower respiratory nodes. In particular, NK cells skewed the mycoplasma-specific T cell cytokine responses in the draining lymph nodes to higher IL-4, IL-13, and IL-17 while lowering IFN-gamma responses. Adoptive transfer of total lung lymphocytes isolated from immunized mice into naive mice led to a significant reduction in the mycoplasma numbers in lungs, and the resistance was greater if cells were obtained from immunized mice that were depleted of NK cells. Similar results were obtained if purified B cells, T cells, or CD4(+) T cells were used. Interestingly, this is the first time that a favorable role of functional CD4(+) T cells in mediating protection in mycoplasma respiratory disease was demonstrated. Thus, NK cells can influence the responses of multiple lymphocyte populations capable of mediating resistance to mycoplasma infection.

Topics: Administration, Inhalation; Animals; Antigens, Bacterial; Cells, Cultured; Female; G(M1) Ganglioside; Immunity, Innate; Killer Cells, Natural; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, SCID; Mice, Transgenic; Mycoplasma Infections; Mycoplasma pulmonis; Respiratory Tract Infections; Vaccination

Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

Topics: Adenosine Triphosphate; Amiloride; Animals; Bronchi; Butadienes; Calcium Signaling; Cells, Cultured; Cystic Fibrosis; Egtazic Acid; Epithelial Cells; Epithelial Sodium Channels; Estrenes; Flagellin; G(M1) Ganglioside; Humans; Ion Transport; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Nitriles; Patch-Clamp Techniques; Phosphatidylinositol 4,5-Diphosphate; Pseudomonas aeruginosa; Pseudomonas Infections; Pyrrolidinones; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Respiratory Tract Infections; Sodium; Sodium Channels; Trachea

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. 125I-labeled flagellin bound to the glycolipids GM1 and GD1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla+ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

Topics: Animals; Bacterial Adhesion; Bacterial Proteins; CHO Cells; Cricetinae; DNA-Binding Proteins; Fimbriae Proteins; Flagella; Flagellin; Flow Cytometry; G(M1) Ganglioside; Gangliosides; Inflammation; Macrophages; Mice; Mice, Inbred BALB C; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Tumor Cells, Cultured; Virulence

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.

Topics: Adolescent; Adult; Animals; Bacterial Adhesion; Cells, Cultured; Child; Cystic Fibrosis; Epithelium; Female; G(M1) Ganglioside; Humans; Male; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Opportunistic Infections; Pseudomonas aeruginosa; Pseudomonas Infections; Rabbits; Regeneration; Respiratory Physiological Phenomena; Respiratory System; Respiratory Tract Infections

The effect of recombinant human interleukin-2 (rIL-2) on the course of experimental chronic respiratory tract infection caused by Klebsiella pneumoniae in mice was examined. rIL-2 was administered subcutaneously once a day for 7 or 14 days, starting 2 weeks after the mice were infected. Administration of 2 or 20 micrograms of rIL-2 per mouse daily for 7 days reduced bacterial counts in the lungs dose dependently. At a dose of 0.2 microgram per day, proliferation of bacteria in the lungs was suppressed after 14 days of administration. Agglutinin titers in serum were not affected by rIL-2 treatment. Monocyte and lymphocyte counts in peripheral blood were increased by administration of 20 micrograms of rIL-2 daily for 14 days but not by treatment for 7 days. In addition, clearance of bacteria from the lungs after aerosol exposure was enhanced by treatment for 7 days before infection. Thus, rIL-2 acted therapeutically or prophylactically in the presence or absence, respectively, of a specific antigen. These effects were not abolished by anti-asialo GM1 antibody. This suggests that activation of natural killer cells does not play a critical role in the therapeutic and prophylactic effects of rIL-2.

Topics: Aerosols; Agglutination Tests; Animals; Antibodies; Chronic Disease; Female; G(M1) Ganglioside; Glycosphingolipids; Interleukin-2; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Leukocyte Count; Lung Diseases; Mice; Mice, Inbred CBA; Recombinant Proteins; Respiratory Tract Infections