School, University of Glasgow, Royal Infirmary, Glasgow,. Scotland. centrifugation. The bacteria were washed three times with sterile distilled water before being.
INFECTION AND IMMUNITY, June 1981, p. 1298-1300 0019-9567/81/061298-03$02.00/0
Vol. 32, No. 3
Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages C. G. GEMMELL,t* P. K. PETERSON, W. REGELMAN, D. SCHMELING, J. R. HOIDAL, AND P. G. QUIE Departments of Pediatrics and Medicine, University ofMinnesota Medical School, Minneapolis, Minnesota
In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.
Strong evidence exists for the role of the M protein of group A streptococci in determining their resistance to phagocytosis by polymorphonuclear leukocytes (PMN). This protein is present on the surface of the Streptococcus cell in the "fuzz" or fimbria-like structures (6, 11), and recent studies suggest that resistance to phagocytosis is due to inhibition of bacterial opsonization by M protein (8). The presence of M protein impairs complement activation and concomitant binding of 03b to the bacterial cell wall during opsonization in normal human serum (2; C. G. Gemmell, P. K. Peterson, D. Schmeling, Y. Kim, J. Mathews, L. Wannamaker, and P. G. Quie, J. Clin. Invest., in press). The inability of Streptococcus pyogenes to elaborate M protein in the presence of clindamycin renders the bacterial cells susceptible to phagocytosis by human leukocytes. In the present study, we extended our investigation of the effect of M protein on phagocytosis by evaluating two populations of mononuclear phagocytes, circulating blood monocytes (MN), and resident alveolar macrophages (AM) in relation to PMN. (This paper was presented in part at the Annual Meeting of the American Society for Microbiology, Miami, Fla., 11-16 May 1980 [C. G. Gemmell, P. K. Peterson, D. Schmeling, J. Hoidal, and P. G. Quie, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, D15, p. 40].) S. pyogenes (M type 6), kindly provided by L. Wannamaker, University of Minnesota, was enriched for M protein by rotation in human blood by the method of Lancefield (7). This strain carried significant amounts of M protein as measured serologically and ultrastructurally by transmission electron microscopy (Gemmell et al., in press). It was grown in and inoculated into Todd-Hewitt broth overnight before culture in the same medium containing [3H]thymidine (12) prewarmed to 37°C for 4 h before harvesting by
centrifugation. The bacteria were washed three times with sterile distilled water before being standardized to a concentration of 5 x 108 colony-forming units per ml. The bacteria were opsonized with either 10% normal human serum lacking specific streptococcal antibodies, 10% heated (56°C for 30 min) human serum, or Hank's balanced salt solution enriched with 0.1% gelatin (gel-HBSS) for 15 min at 370C with shaking. The opsonized bacteria were washed with gel-HBSS before use in the phagocytosis assay. Bacteria which were not preincubated in either serum of gel-HBSS were also used. By using previously described methods, relatively pure (>95%) preparations of PMN (9) and MN (1) separated from healthy human blood donations and bronchopulmonary lavage was used to obtain AM (4) with a purity of >88%; all three cell types were obtained from the same donor. After being washed three times, all cell types were suspended to a final concentration of 5 x 106 phagocytes per ml. Phagocytosis was determined by using a previously described method (9). Bacterial killing was measured by constituting mixtures as for the phagocytosis assay and determining the colony-forming units of streptococci at specified times of incubation after lysis of the phagocytes with distilled water and making appropriate dilutions before plating onto 10% horse blood agar plates. The rate of uptake of opsonized or untreated bacteria was monitored for 30 min, and the kinetics are illustrated in Fig. 1, which represents results with PMN, MN, and AM obtained from each of three different donors. From these studies it was apparent that AM could take up opsonized streptococci more readily than PMN or MN, and although a heat-labile serum factor markedly enhanced the rate of uptake by AM, bacteria opsonized in heated serum and nonopsonized bacteria were also t Present address: Department of Bacteriology, Medical School, University of Glasgow, Royal Infirmary, Glasgow, phagocytosed. Phagocytosis was also assessed visually by Scotland. 1298
VOL. 32, 1981
NOTES
1299
a
S
0.5
0
Sc 06
15 30 0 30 0 15 40 Time (minutes) Time (minutes) Time (minutes) FIG. 1. Phagocytosis of S. pyogenes by PMN, MN, and AM. The rate of uptake of [3H]thymidine-labeled S. pyogenes after opsonization with 10% normal serum, 10% heated serum, and gel-HBSS was monitored for ) Normal serum-opsonized bacteria; (- -) 30 min of interaction with PMN (a), MN (b), and AM (c). ( heated serum-opsonized bacteria; (-) gel-HBSS-opsonized bacteria; (* ..) bacteria treated with neither
O
serum nor gel-HBSS.
examination of Wright-stained smears of the various reaction mixtures by using methods described earlier (10). The results obtained correlated closely with the measurements of uptake of labeled bacteria. To help differentiate between attached and ingested bacteria, a modification of the method of Verhoef et al. (12) was employed by using a specific phage-associated lysin (6), obtained from a group C streptococcal culture, to destroy extracellular bacteria. Briefly, three identical vials were made up as for the phagocytosis assay, containing 0.1 ml of opsonized or nonopsonized bacteria and 0.1 ml of AM, and incubated at 370C for 60 min. After this incubation, the vials were divided as follows: one vial was processed to determine the total bacterial population, the second vial received 3 ml of phosphate-buffered saline (pH 7.4), and the third received 3 ml of phosphate-buffered saline containing a 1:10 dilution of phage lysin. The other two vials were then incubated an additional 30 min at 370C, after which they were washed three times in cold phosphate-buffered saline by differential centrifugation and finally suspended in 3 ml of scintillation fluid. Radioactivity in the vials receiving phosphate-buffered saline alone directly after incubation represented AM-associated bacteria (both attached and ingested bacteria), whereas radioactivity in the vial receiving phosphate-buffered saline with phage-associated lysin represented only ingested bacteria. The results of this experiment are shown in Table I. It is clear from this experiment that the measured uptake of radiolabeled streptococci by AM
TABLE 1. Effect ofphage-associated lysin on uptake of S. pyogenes S43 by AMF % Uptake by AM without
Opsonic sourceb 10% PHS
phage lysin treatment at
% Uptake by AM with
phage lysin
60 min
treatment at 60 min
90
94
50 57 10%AHS 33 29 gel-HBSS a Unpurified phage-associated lysin was kindly prepared by John Hill, Department of Pediatrics, University of Minnesota, by the method of Fischetti et al. (5) and used at a dilution of 1:10 to treat the Streptococcus-AM mixtures at 37°C for 30 min before measurement of the uptake of radiolabeled bacteria by the method of Verhoef et al. (12). b PHS, Pooled human serum; AHS, heat-inacti-
vated serum.
largely reflects the ability of AM to ingest this organism. Treatment of AM-streptococci interaction mixtures with phage-associated lysin failed to significantly diminish the levels of AMassociated streptococci, whereas free bacteria were readily destroyed by the lysin (>80% lysis of bacteria which were incubated without AM). Taken together, these findings substantiate the concept that AM, unlike PMN and MN, are capable of phagocytosing M protein-positive streptococci. In addition, we looked at the ability of PMN, MN, and AM to kill S. pyogenes. In this situation, a significant fall in colony-forming units was observed only with organisms opsonized
1300 NOTES with unheated human serum and presented to AM (Fig. 2). However, the relatively slow rate of killing compared with the rate of phagocytosis by AM may reflect the diminished capacity of AM to generate bactericidal oxygen radicals (3). As with phagocytosis per se, PMN and MN failed to ingest and kill M protein-positive S. pyogenes even after serum opsonization (data not shown). These studies have shown that human AM are able to phagocytose and kill M protein-positive streptococci more effectively than either PMN or MN. M protein is known to inhibit bacterial opsonization via the alternative compllement pathway (2, 8) and would explain the imipairment of phagocytosis by PMN and MN. So)me C3b does bind onto this strain of S. p Tenes (7), but not in sufficient amounts to re,verse its resistance to phagocytosis by PMN anId MN. On the other hand, sufficient opsonization must have taken place for uptake by AM to oCCur. occur.
It is conceivable that these differences might explain why this organism rarely causes infectic)n in the lower respiratory tract, whereas it equently d oes so in the pharynx. In fr4 equent1y does i the In contrast, exuperiments with Streptococcus pneumoniae so
100
2~t_
.
~~~--_4*
INFECT. IMMUN.
(Gemmell et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, D15, p. 40) revealed that AM had a markedly decreased capacity to phagocytose and failed to kill this organism. It js possible that the polysaccharide capsule of S. pneumoniae is a more effective inhibitor of opsonization and phagocytosis by AM, compared with M protein, and this may partly explain why S. pneumoniae is a relatively important pathogen in the lower respiratory tract. This work was supported by Public Health Service grant 2R01 AI.08821-02 from the National Institutes of Health and by the Weilcome Foundation, United Kingdom.
R 2.
Immun. 26:1172-1176. 3. Cohen, A. B., and M. J. Clne. 1971. The human alveolar cultivation in vitro, and studies isolation, macrophage: of morphologic and functional characteristics. J. Clin. Invest. 50:1390-1398. 4. Finley, T. N., E. W. Swenson, W. S. Curran, G. L Huber, and A. J. Ladmnan. 1967. Bronchopulmonary lavage in normal subjects and patients with obstructive lung disease. Ann. Int. Med. 66:651-658. 5. Fischetti, V. A., J. B. Zabriskie, and E. C. Goshlich. 6 6.
n
0-
8.
0 .t
50
-
9n
0
9.
10.
0
10
20
30
40
50
Time (minutes)
60
of human monocytes on microexudate-coated surfaces. J. Immunol. 120:1372-1374. Bisno, A. L 1979. Alternate complement pathway activation by group A streptococci: role of M protein. Infect.
7.
it)
CiTED
1. Ackerman, S. K., and S. D. Douglas. 1978. Purification
1971. Purification and physical properties of group C streptococcal phage-associated lysin. J. Exp. Med. 133: 1105-1117. Fox, E. N. 1976. M-proteins of group A streptococci. Bacteriol. Rev. 38:5748. Lancefield, R. C. 1957. Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J. Exp. Med. 106:525-544. Peterson, P. K., D. Schmeling, P. P. Cleary, B. J. Wilkinon, Y. Kim, and P. G. Quie. 1979. Inhibition of alternative complement pathway opsonization by group A streptococcal M-protein. J. Infect. Dis. 139: 575-585. Peterson, P. K., J. Verhoef, D. &hmeling, and P. G. Quie. 1977. Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 136:502-509. Peterson, P. K., B. J. Wilkinson, Y. Kim, D. Schmeling, and P. G. Quie. 1978. Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes. Infect. Immun. 19:943-949.
FIG. 2. Killing of S. pyogenes by AM. The rate of 11. Swanson, J., K. C. Hsu, and E. C. Gotschlich. 1968. Electron microscopic studies on streptococci. I. M ankilIling of S. pyogenes after opsonization with 10% no ormal serum, 10% heated serum, and gel-HBSS wa1s tigen. J. J., Exp.P.Med. 130:1063-1091. 12. K. Peterson, Verhoef, and P. G. Quie. 1977. .ntored for minofinteractionwih for 60 60 mm of interaction With AM. ( ) mc)nitored Kinetics of staphylococcal opsonization, attachment, NcDrmal serum-opsonized bacteria; (- -) heated ingestion, and killing by human polymorphonuclear leukocytes: a quantitative assay using ['HIthymidine se?rum-opsonized bacteria; (-) gel-HBSS-opsoni;zed bacteria.
labelled bacteria. J. Immunol. Methods 14:303-311.
