Immunology, 1975, 28, 1051.
Serum Factors Capable of Opsonizing Shigella for Phagocytosis by Polymorphonuclear Neutrophils W. P. REED Department of Medicine, Veterans Administration Hospital, and University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A.
(Received 19th March 1974; acceptedfor publication 16th November 1974)
Summary. Twenty-five Shigella strains were tested for their susceptibility to phagocytosis and killing by polymorphonuclear neutrophils (PMN). The studies identified several serum factors that could participate in opsonization. The strains varied remarkably in their susceptibility to killing when heat-stable opsonins were employed, but all strains were killed when exposed to heat-labile opsonins and PMN. The heat-stable opsonin was shown to be IgG, whereas IgM was ineffective in the absence of complement, and 11S IgA was never effective. Heatlabile opsonization required immunoglobulin as well as complement, but IgM was the only immunoglobulin demonstrated to participate in this reaction. The alternative C3 activating pathway is required for efficient heat-labile opsonization of Shigella, but some opsonization also appeared to occur through the Cl-C4-C2 pathway of C3 activation. INTRODUCTION Many strains of Shigella are susceptible to killing by a combination of immunoglobulins, components from either the early classical or alternative complement pathways, and components of the terminal complement sequence (Reed and Albright, 1974). However, many strains are resistant to killing by serum alone. This study examines the role of polymorphonuclear neutrophils (PMN) in killing Shigella strains which are either sensitive or resistant to killing by serum, and also investigates the serum factors capable of opsonizing Shigella for ingestion by the PMN. MATERIALS AND METHODS
Bacteria and sera All strains ofShigella were isolated from patients in New Mexico in the 3 years preceding the study, and were preserved by serial subculture on nutrient agar, and by lyophilization. Grouping and typing was by slide agglutination with Bacto-Shigella antisera (Difco, Detroit, Michigan). The strains used belonged to serogroups B2, B3, C4, C6 and D. Serum from healthy donors separated after clotting at 40 for 30-60 minutes was stored at -80° in small aliquot samples. After thawing, these sera still had normal haemolytic complement levels. Sera were tested for antibody activity against Shigella Correspondence: Dr William P. Reed, Veterans Administration Hospital, 2100 Ridgecrest Drive, SE, Albuquerque, New Mexico 87108, U.S.A.
1052 W. P. Reed by microtitre indirect haemagglutination (IHA) of human 0 Rh-negative erythrocytes coated with bacterial antigen (Reed and Albright, 1974).
Removal of antibody Antibody to Shigella was removed from serum by overnight absorptions with heatkilled organisms at 00 in the presence of 0-01 M ethylene diamine tetraacetic acid (EDTA, Sigma, St Louis, Missouri). The volume of packed organisms was double the serum volume, since smaller quantities of organisms sometimes failed to remove all the opsonic capacity. The resulting absorbed serum had no detectable haemagglutinating or complement-fixing antibody to the strain of Shigella used for the absorptions, but C3 proactivator (C3PA) remained detectable by immunoelectrophoresis. EDTA was removed by dialysis against continuing changes of phosphate-buffered saline at 00 for 30 minutes in a hollow cellulose fibre b/HFO 1/20 beaker dialyser (Bio Rad, Richmond, California), and the resulting serum still contained 42 CH50 units of haemolytic complement (normal >80 units). Immunoglobulins Two lots of colostral IlS IgA were prepared by the method of Tomasi, Tan, Solomon and Prendergast (1965). When concentrated to 4 mg of IgA/ml, the llS fraction contained neither IgG nor IgM by radial immunodiffusion (limits of detection: 0-01 mg of IgG/ml, 0 03 mg of IgM/ml). However, after one preparation appeared to promote complement-mediated opsonization, it was passed through a solid immunoabsorbent column with Sepharose-bound anti-IgM (goat) (Hyland, Costa Mesa, California) according to the method of Cautrecasas (1970). Immunoglobulin M was isolated by passing the 19S serum fraction from a 10-40 per cent sucrose density gradient through a Sephadex G-200 gel filtration column and collecting only the IgM-containing fractions. When concentrated to 0-8 mg IgM/ml, there was no detectable IgA or IgG by radial immunodiffusion (limit of detection: 0f02 mg IgA/ml using 1 iS IgA standard). Two 7S fractions of human serum, one from a sucrose density gradient and the other from a Sephadex G-200 gel filtration column served as the source for IgG. At 12 mg of IgG/ml a trace of IgA was detectable, but there was no IgM by radial immunodiffusion. All the immunoglobulin-containing preparations exhibited IHA activity to the strains for which their opsonizing capacity was determined.
Inactivation of complement and its components The heat-labile components of complement and the alternative complement activating pathway were destroyed by heating sera at 560 for 30 minutes. C3 was also removed by Sepharose-bound antiserum to C3 (goat, Hyland) at 40 in the presence of 0-01 M EDTA. The effluent from the column was concentrated to the original serum volume and EDTA was removed by negative pressure dialysis against continuing changes of phosphatebuffered saline at 40 for 2 hours in a hollow cellulose fibre beaker dialyser. There was no remaining detectable haemolytic complement activity unless C3 (10 mg/ml, Cordis, Miami, Florida) was added. The alternative C3 activating pathway of serum was inactivated in the presence of 0-01 M EDTA by Sepharose-bound anti-C3 activator (Behring, Woodbury, New York) which also reacts with C3PA. After concentration to the original volume and removal of EDTA, the resulting serum had no detectable C3A or C3PA by Ouchterlony analysis.
Opsonization of Shigella for Phagocytosis 1053 However, the six different sera treated this way still contained 70-84 CH50 units of haemolytic complement, thus showing the classical or Cl-C4-C2 pathway to be intact. The classical C3 activating pathway was inactivated by chelating Ca2 + with 0 01 M ethyleneglycol-bis (B-aminoethyl ether) NNV,N'-tetraacetic acid (EGTA, Sigma, St Louis, Missouri) as used by Fine, Marney, Colley and Sergent (1972), and Johnston, Newman and Struth (1973). Since the function of the alternative pathway depends upon Mg2 +, 0-01 M magnesium chloride was also added.
Phagocytic-killing test The phagocytic bactericidal test of Maaloe (1946) as modified by Hirsch and Strauss (1964) was employed. Briefly, plastic tubes with 1 ml of an incubation mixture in Hanks's solution contained approximately 2 x 106 washed viable organisms from an 18-hour broth culture, an equal number of washed normal human PMN obtained by sedimentation of blood in 6 per cent dextran 75 (Travenol Laboratories, Morton Grove, Illinois), and 0-1 ml of serum or other material being tested as an opsonin source. Thus, the highest concentration of serum or other opsonin in the tube was 1 :10, and in some tests further doubling dilutions were made. All tests included control tubes with PMN omitted, and tubes with the opsonin source omitted. After thorough mixing, a sample was removed for bacterial counting with a calibrated 0`001-ml platinum wire loop. The tubes were then tightly stoppered and incubated at 370 with end over end rotation at 12 rev/min. Samples for bacterial counting were again removed at 30, 60 and 120 minutes of incubation. Each sample was diluted in 1 ml of distilled water to disrupt the PMN and spread unkilled micro-organisms, and 0 1 ml was pipetted into a Petri dish and mixed with 10 ml of warm (450) Pennassay (Difco) agar. Colonies were counted electronically with a Biotron Automated Colony Counter Model Cl 11 (New Brunswick Scientific, New Brunswick, New Jersey) after incubation for approximately 40 hours. Counts were expressed as a percentage of the initial counts, and plots of survival were made on semilog paper. RESULTS FRESH AND HEATED SERA AS A SOURCE OF OPSONINS
Sixteen strains known to be susceptible to killing by incubation with fresh unheated serum alone ( 75 per cent survival in 2 hours) were tested as above, and also with fresh serum from the same donor as a source of opsonins. Heated serum promoted PMN phagocytosis and killing of some strains but other strains actually increased in numbers when the only opsonins present were heat stable. There was no apparent relationship between the serogroups of organisms and their susceptibility to heat stable opsonins of the test serum. Five additional heated sera were each employed as the source of opsonins with one of the strains that showed growth,
W. P. Reed