In vitro inhibition of lymphocyte proliferation by Pseudomonas aeruginosa phenazine pigments

Abstract

Human lymphocyte proliferation is inhibited in vitro in the presence of killed Pseudomonas aeruginosa or cell-free P. aeruginosa culture supernatants. A comparison of culture supernatants obtained under similar conditions from Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Pseudomonas cepacia strains demonstrated that all P. aeruginosa supernatants were strongly inhibitory, whereas supernatants from other bacteria were mildly inhibitory or not inhibitory at all. These P. aeruginosa inhibitors prevent proliferative responses of resting cells upon mitogen activation and decrease [3H]thymidine uptake when added to human lymphocytes undergoing active proliferation in culture. The inhibitory effect is reversible and not due to cytotoxicity. Most of the inhibitory activity present in crude supernatants was detected in ultrafiltrates of molecular weights below 2,000. Purified P. aeruginosa pyocyanine, a low-molecular-weight phenazine pigment present in culture supernatant, was strongly inhibitory for lymphocyte proliferation. Extraction of pyocyanine and phenazine pigments from inhibitory P. aeruginosa supernatants eliminated their inhibitory activity. Inhibitors were recovered from reverse-phase chromatographic cartridges by both chloroform and methanol elution, indicating that pyocyanine and other phenazine pigments present in P. aeruginosa supernatants are responsible for the inhibition of lymphocyte proliferation. In addition to the identification of phenazine pigments as lymphocyte proliferation inhibitors, several criteria ruled out major contributions of P. aeruginosa polysaccharide, exotoxin A, and proteases to this phenomenon. P. aeruginosa strains selected for very low protease production or for very low exotoxin A production produced supernatants as inhibitory for lymphocyte proliferation as supernatants obtained from clinical P. aeruginosa isolates. Purified P. aeruginosa lipopolysaccharide and protease preparations failed to induce reversible lymphocyte proliferation inhibition. Finally, heat inactivation of P. aeruginosa supernatants at 100 degrees C for 60 min inactivates exotoxin A and proteases but produced only a moderate decrease of the inhibitory activity for lymphocyte proliferation.

Small Cytoplasmic Antigens from Pseudomonas aeruginosa Stimulate γδ T Lymphocytes

“…exotoxin A, a heat-labile protein, that was found to be cytotoxic in vitro for human peripheral blood macrophages and depressed lymphocyte proliferation and cytokine synthesis [22]. Furthermore, P. aeruginosa phenazine pigments and proteases have been described as inhibitors of lymphocyte activation and proliferation [23,24]. Other antigenic preparations have also demonstrated a stimulatory effect on gd T lymphocytes after heat treatment [25,26].…”

Cystic fibrosis: A review of pulmonary infections and interventions

“…175*176 Lymphocyte functions may be blocked by bacterial products. 145 body, 1?7.156,157 correlated with disease progression.…”

“…16' ) Most recently, Sorenson has demonstrated inhibition of lymphocyte proliferation in a nonspecific, reversible manner by P. aeruginosa pigments. 145 Other studies have revealed abnormalities in lymphocyte subpopulations. I7O During acute exacerbations, decreases in B lymphocytes and theophylline-resistant helper cells occur, while theophylline-sensitive suppressor cells are increased.…”

Inactivation of human gamma interferon by Pseudomonas aeruginosa proteases: elastase augments the effects of alkaline protease despite the presence of alpha 2-macroglobulin

“…A number of studies have shown that the opportunistic pathogen Pseudomonas aeruginosa can nonspecifically inhibit T-lymphocyte-mediated immunity in vitro (14,15,26,32,36) and in vivo (5,14,30). Patients infected with P. aeruginosa often show evidence of impaired cell-mediated immunity (28,33,34).…”