Mary Jane Thomassen

Inhibitory effect of cystic fibrosis serum on pseudomonas phagocytosis by rabbit and human alveolar macrophages.

Summary: This report presents experimental observations indicating the presence of an inhibitory activity in cystic fibrosis (CF) serum which impairs phagocytosis of Pseudomonas aeruginosa by rabbit as well as human alveolar macrophages. Of the 49 patient serum samples studied, 40 consistently showed ≥ 60% inhibition, 3 showed no inhibition and 6 were in the range of 20–60% inhibition of Pseudomonas phagocytosis. In parallel studies, the phagocytosis of S. aureus and S. marcescens was found not to be inhibited by CF serum. Mixing of CF serum with normal serum could not overcome the inhibitory effect, indicating the presence of an inhibitory factor rather than the lack of a necessary component. The inhibitory activity is not lost upon exposure of serum to glass, upon freezing the serum once, or upon heating at 56 C for 30 minutes.Speculation: The serum of cystic fibrosis patients selectively inhibits alveolar macrophage function in vitro; the expression of this inhibitory activity in vivo may compromise effective host control of infection. Investigation of the origin, nature and pathophysiologic role of the activity may suggest new approaches to the control of Pseudomonas pulmonary infection.Pultionary infection is a major factor in the morbidity and mortality associated with cystic fibrosis (CF) (6). Pseudomonas, a uhrquitous organism in the environment, is cystic fibrosis (CF) (6). Pseudomonas, a uhrquitous organism in the environment, is usually not pathogenic for healthy individuals. However, individuals with the chronic lung disease of CF are particularly susceptible to opportunistic Pseudomonas aeruginosa infections. The frequency of this organism in CF pulmonary infections is inadequately explained. It is well known that most CF patients have elevated levels of Pseudomonas antibodies in their sera and pulmonary secretions (l2,14). While recently there has been an indication that a lymphocyte unresponsiveness to Pseudomonas may be acquired as the infection progresses (18,19), no other immunologic abnormality has been consistendy observed (5.10). Extrapulmonary infection is extremely rare and sepsis is almost never, seen after the first months of life (22). This unusual susceptibility to Pseudomonas despite apparently normal systemic humoral and cellular immunity. suggests that local pulmonary host defense mechanisms are defective in CF. Several recent studies have indicated that lung defenses can, to a certain extent, function independently of systemic humoral and cell mediated immune systems (9,15,20,21).Lung defenses include mucociliary transport as well as phagocytic cells, lymphocytes, and immunoglobulin secretion. Since mucociliary transport in some CF patients is compromised (5). clearing of the bacteria becomes more dependent on the efficient action of the phagocytic cells. Previous studies in our laboratory (2) and by Biggar, et al. (I) have shown that CF serum impairs phagocytosis of Pseudomonas by rabbit alveolar macrophages. This report presents experimental observations indicating the presence of an inhibitory activity in CF serum which impairs phagocytosis of Pseudomonas by human as well as rabbit alveolar macrophages.

Ultrastructure and function of alveolar macrophages from cystic fibrosis patients

Summary: Alveolar macrophages were isolated from three cystic fibrosis patients, and the structure and function of these cells were compared to that of normal alveolar macrophages. The cystic fibrosis (CF) and normal alveolar macrophages were able to phagocytize Pseudomonas in the presence of normal serum, but cells from both sources had decreased phagocytosis of Pseudomonas in the presence of CF seram. Phagocytosis of Staphylococcus was not inhibited. Ultrastructural studies showed CF macrophages to be morphologically normal, however, in contrast to CF polymorphonuclear cells, they had not been heavily engaged in phagocytosis. The similarities between CF and normal macrophages suggest that the chronic pulmonary infection of CF may be due to an extrinsic factor in an altered lung environment rather than to any intrinsic cellular defect of the alveolar macrophage.Speculation: Functional and morphologic observations indicate that cystic fibrosis alveolar macrophages are not providing an adequate phagocytic defense against Pseudomonas. This defective phagocytosis does not appear to arise from an intrinsic problem with the macrophages, but rather appears to be due to extrinsic factors, i.e.. an altered lung environment together with a substance(s) present in cystic fibrosis serum which selectively inhibit Pseudomonas phagocytosis.

Effect of mucoid property on antibiotic susceptibility of Pseudomonas aeruginosa.

Clinical isolates ofPseudomonas aeruginosa from patients with cystic fibrosis (CF) were examined for susceptibility to the antibiotics carbenicillin, ticarcillin, tobramycin, gentamicin, and tetracycline. Minimal inhibitory concentrations of the antibiotics were determined for mucoid and nonmucoid isolates from the same patient by a single-colony replica plating method. This method allows the rapid determination of antibiotic susceptibility of a single cell’s progeny and the individual screening of each colony against all antibiotics. Twenty of 34 (58%) cystic fibrosis patients had a mucoid isolate which was more susceptible to antibiotics than their nonmucoid isolate of the same serotype. Nonmucoid revertant segregants of mucoid strains isolated from 50% of the patients demonstrated greater resistance to at least one antibiotic than the original mucoid strain. Multiple isolates from 25 patients were serotyped by Difco (Liu) or Homma antiserum; only 2 patients harbored multiple strains with no common serotyping antigens. Serotypes of the nonmucoid revertants were the same as the original mucoid isolate even if the susceptibilities of the two strains were not similar.

Phagocytic inhibitory activity in serum of cats immunized withPseudomonas aeruginosa lipopolysaccharide

Sequentially collected sera from cats chronically immunized withPseudomonas aeruginosa lipopolysaccharide (LPS) serotype 5 were assessed for their effect on phagocytosis by alveolar macrophages from nonimmunized cats. Phagocytosis was measured by incubating macrophage monolayers for 20 min in the presence of3H-labeled bacteria and 5% serum from control or immunized animals. Sustained phagocytic inhibitory activity developed in the sera of eight of 11 immunized cats (mean inhibition ranged from 30% to 73%) after 13 weekly immunizations. The activity was specific because phagocytosis ofStaphylococcus aureus andP. aeruginosa of another serotype (type 6) was unimpaired. An enzyme-linked immunosorbent assay showed an increase in the amount of LPS-specific IgG in postimmunization sera from the eight cats with inhibitory activity. The IgG appeared to be serotype specific because significantly higher titers were obtained against LPS serotype 5 than LPS serotype 6. The results suggest that phagocytic inhibitory activity in sera from LPS-immunized cats may be due to anti-LPS IgG.

Function of human alveolar macrophages from normal volunteers and cigarette smokers: effect of cystic fibrosis serum.

Previous studies demonstrated that serum from cystic fibrosis (CF) patients specifically inhibited Pseudomonas phagocytosis by both normal and CF alveolar macrophages. In the present study, inhibition of Pseudomonas aeruginosa phagocytosis by CF serum was significantly less on macrophages from heavy smokers than on cells from normal volunteers (P < 0.01). Normal volunteer cells cultured for 10 days were also less affected by CF serum as compared to cells cultured for 24 hours from the same individual (P < 0.01). Altered morphology (increased size and spreading on glass surfaces) and increased intracellular glycosidases of these cells were suggestive of a difference in the state of activation compared to normal cells. Macrophages from heavy smokers and 10‐day cultures from normal volunteers were inhibited by heated CF serum, suggesting that complement‐mediated opsonization was responsible for attachment or ingestion of P. aeruginosa in CF serum by these macrophages.

Phagocytosis ofPseudomonas aeruginosa by feline alveolar macrophages: Strain differences in nonimmune serum

Phagocytosis of three strains ofPseudomonas aeruginosa by cat alveolar macrophages was compared by applying Michaelis-Menten kinetics to analyze the phagocytic process. The rate constant derived from such analyses showed marked differences in both the Vmax and Km. Variant bacterial properties among the three strains may be responsible for the observed differences in the rate constants.