Failure to Demonstrate Leukocyte Migration Inhibition in Human Tuberculin Hypersensitivity 1

Abstract

Antigen-specific inhibition of guinea pig peritoneal macrophages by a nondialyzable soluble factor is now well established as an in vitro correlate of experimental delayed hypersensitivity (1, 2). Recently an adaptation of this technique to naturally occurring human hypersensitivity has been reported: antigen-specific inhibition of buffy coat cell migration is stated to correlate excellently with skin sensitivity to whole killed brucella bacilli (3, 4). We have used this buffy coat technique with a different antigen and have been unable to confirm these observations.

Materials and Methods. Forty ml of heparinized venous blood were obtained from subjects known to be either skin test sensitive or insensitive to 0.2 μg of purified protein derivative of tuberculin (PPD). The subjects were either normal volunteers (11 expts.) or patients hospitalized with various rheumatic diseases (13 expts.). None of the latter were taking corticosteroid hormones or other immunosuppressant therapy. Whole blood kept at 37° was allowed to sediment; the buffy coat cells were obtained by centrifugation of the supernatant plasma. The cells were then washed four or more times in Hanks'balanced salt solution (Gibco). Excess erythrocytes when present were lysed by brief exposure to hypotonic saline. The differential leukocyte counts after such treatment averaged 42% lymphocytes (range 8-88%) and viability, assessed by trypan blue exclusion, was usually greater than 95% (range 82-100%). The leukocytes were then suspended at a concentration of 2.5-5.0 × 10⁷ cells/ml in TC 199 (Gibco) and aspirated into capillary tubes which were then centrifuged, scored with a file, fractured, and placed into duplicate Mackaness-type chambers (5) containing 10% fetal calf serum (Gibco) in TC 199, penicillin and streptomycin, and either no antigen or 40 μg/ml of commercial PPD (Merck, Sharpe and Dohme) or preservative-free PPD (generously supplied by Dr. M. W. Fisher, Parke Davis and Company, Detroit, Michigan), both demonstrated to be biologically potent by in vitro and in vivo testing of Freund's adjuvant-immunized guinea pigs.