Effect of Pseudomonas aeruginosa cytotoxin on thymidine incorporation by murine splenocytes.

The interaction of highly purified Pseudomonas aeruginosa cytotoxin (PAC) with murine splenocytes was examined. Added at culture initiation, PAC (0.1 to 0.5 microgram/ml) inhibited subsequent [3H]deoxythymidine incorporation measured between 42 to 48 h. Incorporation of [3H]deoxythymidine was inhibited 50% in lipopolysaccharide-, phytohemagglutinin-, and concanavalin A-stimulated cultures by 0.20, 0.32, and 0.39 microgram of PAC per ml, respectively. It is concluded that PAC exhibits a narrow inhibitory concentration response range of 0.1 to 0.5 microgram/ml which, secondarily, is affected by the presence of mitogens. Antitoxin added at splenocyte culture initiation, directly after PAC, yielded greater than or equal to 86% protection against PAC inhibition of [3H]deoxythymidine incorporation. Addition of antitoxin to cultures at different times after PAC demonstrated a time-dependent loss of antitoxin protective effect over a 12-h period, indicating that PAC became cell associated and refractory to antitoxin within this time period. PAC preincubated with splenocytes at 4 degrees C for less than or equal to 1 h could not be removed by washing of cells and was fully inhibitory to [3H]deoxythymidine incorporation when these cells were cultured at 37 degrees C. This finding was confirmed by demonstrating that 125I-labeled PAC bound immediately to cells. It is concluded that PAC action on splenocytes is dose- and time-dependent and consists of a two-phase process: (i) a very rapid binding of PAC to the cell surface available to antitoxin, and (ii) a slower toxicity development phase of ca. 12 h, during which PAC becomes refractory to antitoxin.