Journal o f Immunological Methods, 26 ( 1 9 7 9 ) 3 5 5 - - 3 6 3


© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press


C R A I G D. A L L R E D , A N N O. S H I G E O K A a n d H A R R Y R. H I L L *

Division o f Clinical Immunology and Allergy, Department o f Pediatrics and the Department o f Pathology, University o f Utah, Salt Lake City, UT 84132, U.S.A. (Received 7 August 1978, accepted 8 October 1978)

T h e i n c i d e n c e of n e o n a t a l g r o u p B s t r e p t o c o c c a l sepsis a n d m e n i n g i t i s has increased m t h e last decade. A n t i b i o t i c t h e r a p y has p r o v e d i n a d e q u a t e in c o n t r o l l i n g this p r o b l e m , a n d in d e v e l o p i n g i m m u n o l o g i c a l m e t h o d s o f disease p r e v e n t i o n a n d c o n t r o l it is i m p o r t a n t to d e f i n e t h e critical bacterial a n d h o s t factors involved in t h e i m m u n e response. We have used a r a d i o l a b e l e d bacterial u p t a k e m e t h o d to assess t h e p r e s e n c e o f f u n c t i o n a l o p s o n i n s for a s t r a i n o f t y p e III g r o u p B s t r e p t o c o c c i . Bacteria, labeled w i t h t r i t i a t e d leucine, were o p s o n i z e d in fresh, h e a t e d or M g - E G T A t r e a t e d s e r u m . T h e o p s o n i z e d s t r e p t o cocci were a d d e d to m o n o l a y e r s o f a d u l t h u m a n p h a g o c y t e s (> 90% PMN) a d h e r e n t to glass coverslips. A t 10-rain intervals for 60 rain t h e coverslips were w a s h e d free o f uningested b a c t e r i a a n d placed in a s c i n t i l l a t i o n c o u n t e r to d e t e r m i n e t h e degree o f phagocytosis. M a x i m u m o p s o n i c activity was o b t a i n e d w i t h specific a n t i b o d y c o n t a i n i n g serum. No u p t a k e o f u n o p s o n i z e d b a c t e r i a was observed. M a r k e d l y r e d u c e d activity with h e a t e d a n d M g - E G T A t r e a t e d s e r u m i n d i c a t e d a m a j o r role for t h e classical c o m p l e m e n t p a t h w a y in h o s t d e f e n s e against g r o u p B s t r e p t o c o c c i . S e r u m lacking specific a n t i b o d y d e m o n s t r a t e d a l m o s t n o u p t a k e i n d i c a t i n g a m i n o r role for t h e a l t e r n a t i v e p a t h w a y in o p s o n i z i n g t h e s e organisms. R e d u c e d levels o f p h a g o c y t o s i s were also m e a s u r e d in every case w h e r e sera f r o m p a t i e n t s w i t h k n o w n t y p e III i n f e c t i o n s were used for o p s o n i z a t i o n . These results agree w i t h p r e v i o u s d a t a f r o m o u r l a b o r a t o r y using a c h e m i l u m i n e s c e n c e assay o f o p s o n i e activity.


Reported attack rates indicate that between 12,000 and 15,000 infants will develop group B streptococcal disease during the next year. Up to 50% of these infants will die (Feigin, 1976). Although group B streptococci are quite sensitive to several available antibiotics, their use often fails to prevent fatal progression of an infection. This problem, among others, has shifted emphasis towards preventive intervention in combating the disease. Immunological aspects relevant to prevention and control have become particularly important. Research is currently aimed at identifying the critical corn* Dr. Hill is a n I n v e s t i g a t o r o f t h e H o w a r d H u g h e s Medical I n s t i t u t e . S u p p o r t e d , in part, b y U.S. Public H e a l t h Service grants A I 1 3 1 5 0 a n d A M 2 1 1 4 0 a n d a g r a n t f r o m t h e Thrasher Foundation.

356 ponents of the human immune response to group B streptococci. These studies are prerequisite to the development of vaccines or serum components that will be effective against these organisms. For the past 2 years we have been investigating humoral and cellular aspects of the immunological response to group B streptococci. The present study had 2 objectives. The first was to evaluate functional opsonins in h u m a n serum with an assay directly measuring phagocytic uptake of radiolabeled bacteria. The second was to compare results of this assay with findings obtained in our previous studies of chemiluminescence techniques to evaluate group B streptococcal opsonins (Hemming et al., 1976; Shigeoka et al., 1978a). Our results indicate that (1) specific serum opsonins are required for effective phagocytosis of these organisms, (2) an intact classical complem e n t p a t h w a y is necessary for optimal opsonization, (3) there is a good correlation between the radiolabeled bacterial uptake and chemiluminescence assays in evaluating opsonins to group B streptococci, and (4) patients who develop group B streptococcal disease usually lack opsonins to their infecting strains by both techniques. MATERIALS AND METHODS

Leukocyte preparation Heparinized blood (10 U/ml) from healthy h u m a n donors was allowed to settle for 30--60 min at 37°C. The leukocyte rich plasma was removed, the cells sedimented and washed twice in sterile phosphate-buffered saline (PBS = 4500 ml distilled water, 5.2 g Na2HPO4, 0.9 g KC1, 0.9 g KH2PO4, 36 g NaC1) by centrifugation at 100 X g for 10 min and resuspended in PBS. The final concentration was 1.0 X 107 PMN/ml for cells used in the chemiluminescence experiments and 5.0 X 106 PMN/ml for cells used in the radiolabeled uptake tests.

Bacterial preparation A strain of group B streptococci type III isolated from an infected patient was made radioactive by incubating 0.1 ml of fresh bacterial suspension in Todd-Hewitt medium containing [3H]leucine (5 pCi/ml) at 37°C for 2 4 h . The concentration of bacteria was determined by streak plating serial dilutions of the suspension. The bacteria were killed by heating at 60°C for 1 h, washed 3 times in PBS, resuspended in PBS at 1.7 X 10S/ml and stored in 5 ml aliquots at --20 ° C. Organisms prepared as described were used in all radiolabeled uptake experiments. Bacteria used in the chemiluminescence procedure were identically prepared with the exception t h a t the radioactive amino acid was omitted from the 24 h incubation.

Opson izing procedure Immediately prior to use, bacteria were thawed at room temperature and mixed with test serum at the desired concentration (% volume) in a sterile

357 capped tube. The mixture was rotated at 37°C the bacteria were pelleted and washed once in nisms used in chemiluminescence experiments volume in PBS and those used in uptake studies

for 30 min. After incubation PBS by centrifugation. Orgawere resuspended to original in medium 199.

Serum treatment Serum frozen at --70°C was slowly thawed at room temperature immediately prior to use. Inactivation of both c o m p l e m e n t pathways was accomplished by heating serum at 56°C for 30 min. The classical c o m p l e m e n t pathway was inhibited by treating serum with 10 mM MgC12 and 10 mM ethylene glycol tetraacetic acid (EGTA, Sigma, St. Louis, MO) (Des Prez et al., 1975; Hill et al., 1977). Reconstitution of heated serum was attempted by adding whole human c o m p l e m e n t (Cordis Laboratories, Miami, FL) in a ratio of 25 #l complement to 0.1 ml serum. This concentration of complement has previously been found to maximally enhance the opsonic activity of heated rabbit or human serum (Hemming et al., 1976). Serum was also absorbed with the test organism for 30 min at 4°C to remove specific opsonins to the strain b u t leave the alternative c o m p l e m e n t p a t h w a y intact. In cases where serum had been thawed more than once, whole human complement (25 pl/ 0.1 ml serum) was added to control and test serum in that experiment. Clinical specimens The serum samples used in these experiments were obtained directly from donors and patients and frozen at --70°C before use. Thirteen sera which had been previously tested by chemiluminescence for opsonins to the type III strain mentioned above were selected for study with the radiolabeled uptake system. Four of the sera were from patients with proven t y p e III infections and 9 were from apparently healthy donors. Radiolabeled bacterial uptake procedure Monolayers o f human phagocytes (>90% PMNs) were prepared by incubating the leukocyte suspension on 18 mm X 18 mm glass coverslips at room temperature for 30 rain. Non-adhering cells were washed away with PBS and 0.3 ml (5 X 10 s organisms) of the opsonized, labeled bacterial suspension was added to each coverslip. At 10-min intervals coverslips were vigorously washed with PBS to remove uningested bacteria, directly placed in Poly Q scintillation vials (Beckman, Fullerton, CA) containing 5.0 ml aquasol and counted 5 min in a Beckman LS-100 scintillation counter (Fullerton, CA). Microscopic examination of coverslips stained at each time interval showed the monolayers to be evenly distributed. The number of adherent cells was estimated by counting cells in 5 random grids at 400 X magnification and multiplying the average by the n u m b e r o f grids (20,227) per coverslip. The range of adherent cells was 5 X 105--1 X 106 per coverslip, giving a bacteria to phagocyte ratio o f approximately 100 : 1--50 : 1. The 2-sample mean of accumulated cpm/106 phagocytes was calculated at each interval and plotted

358 against time. Peak activity measured by this technique can vary significantly between experiments and depends u p o n m any factors including source of d o n o r leukocytes, age of leukocytes before use and ambient temperature. To overcome this difficulty, all related test conditions were run in a single e x p e r i m e n t using the same serum sample as a source of opsonins. To enable meaningful comparison between activity from clinical specimens, where experiments were p e r f o r m e d separately, an a n t i b o d y containing control serum was always included and data calculated as per cent of control. The reference serum was shown to contain specific opsonins to the test bacteria by chemiluminescence and indirect i m m u n o f l u o r e s c e n c e against antibody.

Chemiluminescence procedure The chemiluminescence pr oc e dur e has been previously described (Hemming et al., 1976; Hill et al., 1977; Shigeoka et al., 1978a). Briefly, the reaction mixtures contained 2.5 ml PBS, 0.5 ml of opsonized bacterial suspension and 0.5 ml l e u k o c y t e preparation (5 × 100 PMNs) for a total volume of 3.5 ml. The bacterial suspension was c o n c e n t r a t e d in PBS by centrifugation to contain a p p r o x i m a t e l y 5 × l 0 s organisms. This provided a bacteria to p h ag o cy te ratio near 100 : 1. The mixtures were made in scintillation vials which were wrapped in aluminum foil and stored in the dark at least 24 h prior to use. Vials were immediately capped u p o n i n t r o d u c t i o n of the reaction m i x t u r e and placed in a Beckman LS-100 scintillation counter, out of phase, with one p h o t o m u l t i p l i e r t ube disconnected. Duplicate vials for each test condition were c o u n t e d at 10-min intervals for 60--90 rain and results were r eco r d ed as m e a n counts per minute. The cont rol serum used in the uptake studies was also used in the corresponding chemiluminescence experiments. When clinical specimens were being compared, data was calculated as per cent o f this control. RESULTS A representative example of the kinetics of opsonized, radiolabeled group B streptococcal upt ake is shown in Fig. 1 and com pared to the chemiluminescence profile induced by the same organism. Experiments were p e r f o r m e d where bacteria were opsonized with different concentrations of a n t i b o d y containing serum. This serum was shown to contain a n t i b o d y to the t y p e III test strain by indirect immunofluorescence. As can be seen in Fig. 2, a progressive decrease in phagocytic uptake occurred when the c o n c e n t r a t i o n o f opsonizing serum was decreased from 25% to 0.5%. No upt ake was measured using unopsoni zed bacteria. Qualitatively identical results were obtained with the chemiluminescence assay. Fig. 3 shows the influence of c o m p l e m e n t in opsonizing group B streptococci as measured by the radiolabeled upt ake technique. Bacteria opsonized in fresh a n t i b o d y containing serum were readily ingested by the phagocyte


50O02 a.. ~_ 4000-


Opsonized TyperlTstrep +PMNs

%1o ~ ~ 3o00-


¢~ 8-

Opsonized Type~I strep* PMNs



~ 6-

olon I Opsonized Type 1Tr strep olone















Fig. 1. The results of m a n y e x p e r i m e n t s s h o w e d that peak p h a g o c y t i c u p t a k e and chemil u m i n e s c e n c e appeared b e t w e e n 50 and 70 min incubation. There was no passive a c c u m u lation o f radioactivity measured w h e n o p s o n i z e d or u n o p s o n i z e d bacteria were incubated on coverslips lacking p h a g o c y t e m o n o l a y e r s .

monolayers and demonstrated m a x i m u m accumulation of radioactivity. In contrast, bacteria opsonized with heated serum, which inactivates components of both complement pathways, showed only 50% of optimal uptake. Similarly reduced activity was observed when serum was treated with MgEGTA, a procedure which inactivates the classical pathway but leaves the alternative intact (Des Prez et al., 1975; Hill et al., 1977). When whole human complement was added to heated serum, phagocytic uptake was UPTAKE OF OPSONIZEDRADIOLABELED GROUP8 STREPBY HUMANPMNs PERCENTSERUM PRESENTDURING OPSONIZATION








4 2 1/2

"J ~4- ~ ~




















Fig. 2. Progress:re decrease in p h a g o c y t i c u p t a k e and c h e m i l u m i n e s c e n c e w i t h falling conc e n t r a ~ , u , ol ~psonizing serum.


TYPETITstrep+ freshserum

~3ooc ! ype ]]J.strep+

heated serum+ complement 2o(>

TypeITI strep + MgEGTAserum


I00( ~

TypeTITstrep+ heated Serum psonizedType TIT strepalone PMNs olone i I Mediumalone



Fig. 3. E f f e c t o f c o m p l e m e n t o n m a x i m u m p h a g o c y t i c u p t a k e .

restored to levels obtained with fresh serum. Serum absorbed at 4°C with the same strain before opsonization, which removes specific opsonins but leaves the alternative complement pathway intact, showed very low levels of uptake comparable to t h a t obtained with unopsonized bacteria (not shown). These findings are similar to results we have reported with the chemiluminescence assay (Shigeoka et al., 1978a, b). We have previously used the chemiluminescence technique to evaluate the presence of opsonins in serum from normal donors and patients with group B streptococcal infections. Thirteen sera which had been tested for opsonins to the type III wild strain by chemiluminescence were studied with the radiolabeled bacterial uptake serum. Nine of these sera were from apparently healthy donors and 4 were from infants with known type III septicemia. Fig. 4 shows the association between the results of the uptake and chemiluminescence assays with these sera. The correlation between the methods was highly significant with an R value of 0.79. Reduced levels of chemiluminescence and phagocytosis were f o u n d in all patients with infection.




o 0


~ 80



2oi 40 ' ' 8'o ' ,6o ' ,2'o CHEMILUMINESCENCE% OF CONTROLSERUM

Fig. 4. T h i r t e e n sera were t e s t e d f o r t h e p r e s e n c e o f f u n c t i o n a l o p s o n i n s t o a t y p e III wild strain of group B streptococci by the chemiluminescence and phagocytic techniques. The a s s o c i a t i o n b e t w e e n results o f t h e assays was highly s i g n i f i c a n t w i t h a n R value o f 0.79. R e d u c e d levels o f c h e m i l u m i n e s c e n c e a n d p h a g o c y t o s i s were o b s e r v e d in every p a t i e n t w i t h k n o w n i n f e c t i o n t o a t y p e III organism.


The goal of immunological control or prevention of group B streptococcal disease requires that we clearly understand the immune mechanisms involved in defense against these organisms. In previous studies we have used a chemiluminescence technique to evaluate serum opsonins to group B streptococci (Hemming et al., 1976; Shigeoka et al., 1978). Basically, this technique measures the light emitted by stimulated leukocytes. The exact mechanism which causes these cells to luminesce is unknown, but it u n d o u b t e d l y involves generation of several oxidized molecular species capable of emitting light (Cheson et al., 1976; Babior, 1978). Although leukocytes produce chemiluminescence upon exposure to ingestible bacteria or other particles, it is n o t clear that actual uptake is required for light emission. In previous studies, we and others have shown that phagocytosis and chemiluminescence can be disassociated (Goldstein et al., 1977; Allred and Hill, 1978). It is essential that chemiluminescence be correlated with more direct measures of phagocytosis if it is going to be used to assess opsonic activity. In the present studies, a radiolabeled bacterial uptake assay was developed and shown to correlate with data obtained with the chemiluminescence assay. This technique, a modification based upon a procedure described by Mande'2 ~ 5 ~ 5 ) for S. aureus, overcomes the weaknesses of m a n y other

362 assays used to evaluate phagocytosis and opsonic activity. Excellent separation of uningested from ingested bacteria is possible with this m e t h o d because the phagocytic cells are tenaciously adherent to glass coverslips. Uningested particles can be vigorously washed away while the phagocytes remain attached. Microscopic examination of washed coverslips showed that most bacteria are intracellular, very few being bound to the membrane outside the cell and almost none remaining attached directly to the glass. Conditions of inflammation present in the body during infection are more realistically approximated in this than in other assays of phagocytosis. When leukocytes are adherent to a substrate, as in the body tissues, they move towards and ingest target particles. Target bacteria in this assay are radiolabeled and the uptake of radioactivity recorded reflects the efficiency of ingestion and/or opsonization. Kinetic studies of opsonization of radiolabeled group B streptococci by h u m a n PMNs demonstrated activity which linearly increased to a m a x i m u m between 50 and 70 min and remained relatively stable afterwards for up to 100 min. These results agree closely with those of Mandell (1975) with S. a u r eu s . The ratio of bacteria to PMNs used in our assay was more than any single cell could hold (approximately 1 0 0 : 1 ) and unwashed coverslips examined at peak activity showed that most cells were full of ingested organisms. We believe that uptake remained unchanged after an hour because cells could not incorporate more. A nutrient medium was provided during incubation so energy limitation would not influence activity and lack of trypan blue uptake (

Evaluation of group B streptococcal opsonins by radiolabeled bacterial uptake.

Journal o f Immunological Methods, 26 ( 1 9 7 9 ) 3 5 5 - - 3 6 3 355 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press E V A...
520KB Sizes 0 Downloads 0 Views