Effects of pneumolysin on human polymorphonuclear leukocytes and platelets

M K Johnson, D Boese-Marrazzo, W A Pierce
1981 Infection and Immunity  
Pneumolysin was bound by human polymorphonuclear leukocytes in a reaction which occurred very rapidly at 0°C. Low concentrations of pneumolysin were found to stimulate leukocyte migration and lysosomal enzyme secretion. At increasing lysin levels, inhibition of spontaneous migration and chemotaxis, cell death, and lysis were observed. Pneumolysin was also found to lyse platelets and to activate serum to become chemotactic. In a study of the mechanism of pathogenesis involved in ocular
more » ... in ocular infections with Streptococcus pneumoniae, it was observed (6) that a highly purified pneumolysin preparation (homogeneous upon examination with acrylamide gel electrophoresis) produced an acute inflammatory reaction in rabbit eyes. Large amounts of discharge containing polymorphonuclear leukocytes (PMNs) accumulated within a short time after the conjunctival instillation of this agent. Intracorneal injection of lysin was also followed by PMN infiltration. We therefore surmised that interaction between lysin and PMNs may be an important feature of ocular infections with this organism. The present work represents an in vitro study of the effects of pneumolysin on various properties of PMNs. MATERIALS AND METHODS Purification of pneumolysin. The methods for production and purification of pneumolysin, for determination of its purity, and for titer with rabbit erythrocytes have been described previously (8) . The specific activity of the preparations used in this study was approximately 106 hemolytic units (HU) per mg of protein. Chemicals and media. Zymosan, formylmethionylleucylphenylalanine, and human serum albumin were obtained from Sigma Chemical Co. RPMI 1640, minimal essential Eagle medium with Earle salts (MEM), Hanks balanced salt solution, and medium 199 with modified Earle salts were obtained from GIBCO Laboratories and contained 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.4. A stock solution of cytochalasin B (Aldrich Chemical Co.) was prepared by dissolving 0.5 mg per ml of dimethyl sulfoxide. Preparation of PMNs and platelets. Fifty milliliters of blood was drawn from healthy volunteers, using ethylenediaminetetraacetate as an anticoagulant. After addition of 10 ml of 6% Macrodex (Pharmacia Fine Chemicals, Inc.), the blood was allowed to stand at room temperature for 1 h. The leukocyte-rich upper layer was removed, combined with RPMI 1640 for a total volume of 50 ml, and centnfuged for 8 min at 115 x g. The resulting platelet-rich supernatant layer was removed and, when desired, reserved for platelet isolation. The leukocyte pellet was resuspended in 50 ml of RPMI 1640, recentrifuged, suspended in 10 ml of RPMI 1640, and layered over 10 ml of lymphocyte separation medium (Bionetics Laboratory Products). After centrifugation for 40 min at 500 x g, the supernatant layers (including the material at the interface) were decanted, and the pellet was washed once in 50 ml of the medium that was to be used for the final cell suspension. Contaminating erythrocytes were removed by hypotonic lysis, and the PMNs were suspended in medium to give either 1 x 107 or 2.5 x 107 per ml, as needed. Viability of the cells (determined with 0.04% trypan blue) was at least 99%, and the differential count revealed that over 95% of the cells were PMNs. When we needed to isolate platelets, the plateletrich supernatant fraction was subjected to another centnifugation at 900 x g for 10 min. After the removal of contaminating erythrocytes, the platelets were suspended in Hanks balanced salt solution with 0.1% Pentex bovine serum albumin (BSA) to an optical density of 1.0. Binding experiments. Pneumolysin (258 HU) diluted in Hanks balanced salt solution-BSA was combined with 107 PMNs suspended in the same medium (the total volume was 3.1 ml). After various periods of incubation on ice, the PMNs were removed by filtration (Swinnex, 0.45-pm filter), and the unbound lysin concentration was assayed in the filtrate. Chemotaxis. Measurements of chemotaxis under agarose were made essentially as described by Nelson et al. (10). Agarose (SeaKem) was used at a concentration of 0.018 g/ml, and L-glutamine was used at a concentration of 4 mM. Plates were made in quadruplicate; each plate contained six pairs of wells, the outer containing chemoattractant and the inner containing the PMNs. The plates were incubated for 3 h, after which spontaneous migration and chemotactic response were differentiated as described by Nelson et 171 on May 9, 2020 by guest http://iai.asm.org/ Downloaded from
doi:10.1128/iai.34.1.171-176.1981 fatcat:dflf5g2igvbe3nvkgy3rhtzlfa