STUDIES ON IVIECHANISIVIS OF CELLULAR IMMUNITY
The relationship between microorganism and host cell during development of an infectious process has never been clearly defined. In order to fully understand the effect of a microbial pathogen on a mammalian cell, it is important to know how each affects the enzymatic machinery of the other. Recently there have been some investigations of the biochemical aspects of host-parasite interactions (Berk and Nelson, 1960; Berk et al, 1960a, b; Kun and Miller, 1948; Puziss and Wright, 1959; Suter, 1956). Studies in our laboratory with a mouse monocyte-Pseudomonas aeruginosa system have indicated that host cell metabolism can be disturbed radically during contact with an infectious agent. For example, succinoxidase activity of in vivo infected mouse phagocytes was depressed, while lactate production from hexoses was markedly stimulated (Berk et al, 1960a, b). It is the purpose of this paper to describe 3 hydrolases in mouse mononuclear cells and to report the effect of P. aeruginosa on the activity of these enzymes.
METHODS
The procedures for obtaining exudates and maintaining the streptomycin-resistant strain of P. aeruginosa were essentially those previously reported (Berk and Nelson, 1960; Nelson and Becker, 1959). Warm mineral oil (0.5 ml per mouse) was used to induce peritoneal exudates in Received for publication May 13, 1961. * This investigation was supported by research grant E-2298 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. 8 to 10-week-old female mice (Webster strain) 18 hours before harvesting of cells. The pooled monocytes from 40 mice were washed twice with saline by centrifuging for 10 minutes at 500 ref. Hemocytometer counts indicated that 1 ml contained approximately 5 X 108 packed cells. Most experiments employed intact cells, but when disrupted cells were required homogenates of equivalent numbers were prepared by sonic treatment in a 9 kc per second Raytheon magnetostrictive apparatus or by disruption at 20,000 psi in a French pressure cell (Aminco). Cellular debris was centrifuged off in the cold at 600 ref and the resultant supernatant was recentrifuged at 30,000 to 35,000 ref for 20 to 30 minutes. In some cases a minute amount of light yellow-green sediment was obtained at this speed. In experiments with infected mice, 109 to 10ro washed P. aeruginosa were administered intraperitoneally 15 to 60 minutes before autopsy. Monocytes were washed 2 to 3 times with heparinized saline containing 1000 to 4000 units of polymyxin per ml to remove and destroy extracellular bacteria. In some experiments 1000 units of polymyxin per ml were also incorporated into reaction mixtures. Similar procedures were used when monocytes were allowed to ingest dead microorganisms in vivo. In experiments employing toxins or bacteria in vitro, monocytes were pre-incubated with these supplements for 2 minutes prior to substrate addition. The pH of all supplements was adjusted to the optimal pH for each enzyme. Particles were obtained by disrupting the bacteria in a French pressure cell at 20,000 psi, centrifuging at 2000 ref to remove cellular debris, and concentrating at 35,000 ref. Assay.-Beta glucuronidase was assayed by the method of Dodgson et al (1953) with phenolphthalein glucuronide as substrate. Into test tubes were pipetted 20 to 50 ,aM of substrate, 2 ml of 0.1 M acetate buffer, pH 3.9, and 0.5 ml of a monocyte suspension in acetate buffer. The heparin concentration of the reaction mixtures was 20 units per mI. The reaction mixtures were incubated at 37 C for 30 to 60 minutes. At the end of the incubation period, 1 ml of 10% trichloroacetic acid was added and the resultant super innatant was assayed after centrifugation. The amount of phenolphthalein liberated was determined by its color intensity at pH 10.5 and 540 m«.