Vol. 19, No. 2
INFECTION AND IMMUNITy, Feb. 1978, p. 598-606 0019-9567/78/0019-0598$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Parasite-Specified Phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa Cells GERALD I. BYRNEt* AND JAMES W. MOULDER Department of Microbiology, University of Chicago, Chicago, Illinois 60637
Received for publication 8 August 1977
Phagocytosis of the 6BC strain of Chlamydia psittaci and the lymphogranuloma venereum 440L strain of Chlamydia trachomatis by L cells and HeLa 229 cells occurred at rates and to extents that were 10 to 100 times greater than those observed for the phagocytosis of Escherichia coli and polystyrene latex siiheres. Both species of Chlamydia were efficiently taken up by host cells of a type they had not previously encountered. Phagocytosis of chlamydiae was brought about by the interaction of parasite surface ligands with elements of the host cell surface. The chlamydial ligands were readily denatured by heat, were masked by antibody, and were resistant to proteases and detergents. The host cell components were reversibly removed by proteases. Chlamydial phagocytosis was inhibited when host cells were incubated for many hours with cycloheximide. It was suggested that the presence on the chlamydial cell surface of ligands with high affinity for normal, ubiquitously occurring structures on the surface of host cells is an evolutionary adaptation to intracellular existence. The term parasitespecified phagocytosis was used to describe the efficient phagocytosis of chlamydiae by nonprofessional phagocytes and to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes.
Although all intracellular parasites have by definition the ability to get inside of potential host cells, most obligate intracellular parasites possess the rare additional ability to enter readily cells that are not actively phagocytic (4, 6, 7, 18, 31). These cells are often called nonprofessional phagocytes to set them apart from the professional phagocytes, which are macrophages, monocytes, and polymorphonuclear leukocytes. Despite the unusual ease with which many obligate intracellular parasites are taken up by nonprofessional phagocytes, phagocytosis has been the usual mechanism invoked to explain their ingestion (7, 18, 31). The electron microscope has shown that the obligate intracellular procaryotes Chlamydia psittaci (17, 23) and Chlamydia trachomatis (1, 2) enter nonprofessional phagocytes by a mechanism resembling classical phagocytosis. Friis (9) believed that C. psittaci entered L cells by passive phagocytosis, with the chlamydial cells playing no active role in the process. Doughri et al. (8) concluded that a systemically inoculated bovine strain of C. psittaci specifically attached to the brush borders of intestinal epithelial cells in newborn calves. Entry of trat Present address: New York Hospital-Cornell University Medical Center, Department of Medicine, New York, NY 10021.
choma strains of C. trachomatis was enhanced by pretreatment of HeLa 229 cells with the polycation diethylaminoethyl (DEAE)-dextran and inhibited by neuraminidase, whereas lymphogranuloma venereum strains of C. trachomatis were not similarly affected (22). DEAE-dextran also stimulated the uptake of some strains of C. psittaci by L cells (P. Spears and J. Storz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, D3, p. 70). Kuo and Grayston (21) reaffirmed the differential effect of DEAE-dextran on trachoma and lymphogranuloma venereum strains and also noted that centrifugation of the inoculum onto host cell monolayers stimulated attachment and ingestion of trachoma organisms. Byrne (4) found that tryptic digestion of L cells reversibly destroyed their ability to ingest C. psittaci. Implicit in most of these studies was the idea that the host cell and not the chlamydial cell ultimately determines how many and what kind of chlamydiae get ingested; that is, host-specified mechanisms of chlamydial phagocytosis were envisaged. However, the entry of C. psittaci into L cells (4) and of C. trachomatis into HeLa 229 cells (21) was inhibited by mild heating of the inoculum, which suggests that chlamydial phagocytosis is mediated by heat-labile components of the parasite surface. This investigation ex-
VOL. 19, 1978
tended earlier studies on the requirements for ingestion of C. psittaci by L cells (4) to the phagocytosis of C. psittaci and a lymphogranuloma venereum strain of C. trachomatis by both L and HeLa 229 cells. The results suggest that Chlamydia species enter nonprofessional phagocytes by a mechanism distinct from the hostspecified immunological and non-immunological phagocytosis practiced by professional phagocytes. The term parasite-specified phagocytosis is proposed to describe this mode of entry into host cells. MATERIALS AND METHODS Growth of host cells. The 5b clone (30) of L929 mouse fibroblasts (L cells) was grown as described elsewhere (15, 30). Suspension cultures were maintained in medium 199 containing 0.1% sodium bicarbonate, 200 jig of streptomycin sulfate per ml, and 5% heat-inactivated fetal calf serum (FCS). Monolayers were grown in medium 199 containing 10% FCS. HeLa 229 cells (human cervical carcinoma) were cultivated in modified Eagle medium containing nonessential amino acids, 0.1% sodium bicarbonate, 200,ug of streptomycin sulfate per ml, and 15% FCS. No contamination with mycoplasma was detected throughout these studies in tests made by an independent laboratory (Flow Laboratories, Rockville, Md.). Cell numbers were measured in a Coulter cell counter model ZB, and cell viability was estimated by the trypan blue exclusion test (19). Growth, purification, and titration of chlamydiae. The 6BC strain of C. psittaci was propagated in L-cell suspensions. L cells were infected and C. psittaci was partially purified and titrated according to Hatch (15). Partially purified C. psittaci was suspended in sucrose-phosphate buffer (SPB) (3) containing 2% FCS. Chlamydial infectivity was measured as the 50% infectious dose (ID50) for 5 x 106 L cells as previously described (4). The theoretical ratio of one ID5o unit (for a single L cell) for every 0.7 plaqueforming unit was approached closely. The preparation of L-'4C-amino acid-labeled C.psittaci has already been described (4). The 440L strain of C. trachomatis, isolated from a case of lymphogranuloma venereum (27), was grown in monolayers of HeLa cells in 75-cm2 flasks. An inoculum of 10 ID50 per HeLa cell was adsorbed to the monolayer for 2 h at 37°C. Then 15 ml of modified Eagle medium containing 2 ,ug of cycloheximide per ml and 0.25 ,uCi of an L-'4C-labeled amino acid mixture (New England NuclearCorp.; averagespecificactivity,259mCi/mmol) was added, and the infected HeLa cells were incubated for 44 h at 37°C in an atmosphere of 5% CO2 and 95% air. C. trachomatis was harvested by decanting the medium and holding it at 4°C. The cells were removed from the substrate by shaking with glass beads in a small volume of medium. The surface of the flask and the beads were washed twice with cold SPB, and the washes were combined with the decanted growth medium. All subsequent operations were carried out at 4°C. The combined cell suspension was centrifuged at 500 x g for 10 min, and the supernatant fluid was decanted and saved. The sedimented
PHAGOCYTOSIS OF CHLAMYDIA
cells were resuspended in 20 ml of SPB, glass beads were added, and the mixture was shaken for 30 min at 200 strokes/min. Beads and cell debris were removed by centrifugation, and the supernatant fluid was combined with the first 500 x g supernatant fraction. The pooled, C. trachomatis-containing fluids were centrifuged at 12,000 x g for 20 min, and the sedimented chlamydiae were resuspended in SPB. C. trachomatis was partially purified according to the procedure used for C. psittaci (15), and the infectivity of the partially purified suspensions was determined by ID50 titration in L cells as described for C. psittaci (4, 15). C. trachomatis suspensions gave identical ID5o titers in L and HeLa cells. Preparation of 14C-labeled Escherichia coli. E. coli (Hfr Hayes 3300, the gift of Alvin Markovitz) was grown in broth, and 1 ,uCi of L-14C-labeled amino acid mixture per ml of broth was added when the bacteria entered the exponential growth phase. When the stationary phase was reached, the bacteria were sedimented by centrifugation, resuspended in broth containing 10% glycerol, and held at -90°C until the day of use. The specific activity of the E. coli suspension was determined by measuring the total number of acid-insoluble counts and the number of viable bacteria. Measurement of the phagocytosis of chlamydiae by L cells and HeLa cells. Phagocytosis of 14C-labeled C. psittaci and C. trachomatis by host cell suspensions was determined by following the uptake of acid-insoluble, chlamydia-specific radioactivity in the presence of cycloheximide and by the subsequent microscopic observations of chlamydial inclusions in Giemsa-stained preparations (4). Phagocytosis of C. psittaci and C. trachomatis host cell monolayers was measured by the procedure described in the accompanying paper (5). The assay methods met the criteria set by Stossel (28) for valid measurement of phagocytosis. Measurement of phagocytosis of polystyrene latex spheres and of E. coli by L cells. Phagocytosis of 0.5- to 1-lpm-diameter polystyrene latex spheres (Dow Diagnostics) was followed by observation with phase-contrast or fluorescence microscopy (4). Phagocytosis of viable, 14C-labeled E. coli by L-cell suspensions was measured in the same way as was the uptake of "4C-labeled C. psittaci (4). Alteration of host cells. HeLa cell suspensions were treated at 37°C with 10 ,ig of crystaUline trypsin per ml (Worthington Biochemicals; 209 U/mg of protein; 1 U is equal to the hydrolysis of 1 ,um of ptoluenesulfonyl-L-arginine methyl ester in 1 min at 25°C and pH 8.1) in the same way that L cells had previously been treated (4). The rate of phagocytosis of 14C-labeled C. trachomatis was measured after trypsin treatment and compared with the rate obtained with untreated HeLa cells. Cycloheximide (2 ,Lg/ml) was added to minimize host cell recovery (4). To determine the effects of long-term incubation of L cells with cycloheximide on their ability to phagocytose C. psittaci, L-cell suspensions at a density of 106 cells/ml were incubated at 37°C in the presence of 2 yg of cycloheximide per ml. At intervals after addition of the antibiotic, portions of the suspension were removed, and chlamydial ingestion was measured as
BYRNE AND MOULDER
previously described and compared with that observed in L cells similarly incubated in the absence of cyclo-
heximide. Alteration of chlamydiae. C. psittaci and C. trachomatis were subjected to ultraviolet light (1,800 ergs/mm2) or heat (3 min at 60°C) (4). This treatment restulted in a 3-log drop in the infectivity of both chlamydial strains. C. psittaci suspensions were treated with homologous hyperimmune rabbit antiserum and the corresponding preimmunization serum as described by Moulder et al. (24). The two chlamydial strains were also treated with 0.125% solutions of the nonionic detergent Nonidet P-40 (NP-40; Shell Oil Co., Ltd.) for 15 min at 4°C. C. psittaci was incubated for 10 min at 37°C with 50 mM methyl methane sulfonate and then freed of the alkylating agent by washing in the centrifuge in SPB. C. psittaci was incubated in 0.05 M sodium carbonate buffer (pH 10.1) containing 1 mM ethylenediaminetetraacetic acid (EDTA) for 60 min at 25°C (25, 26). C. psittaci was also incubated with various concentrations of trypsin and Pronase (Sigma Chemical Co., 3 to 4 U/mg; 1 U will hydrolyze casein to produce 181 ,g of tyrosine per min at 25°C) for 60 min at 25°C. The effect of each of these treatments was determined by comparing the rate of phagocytosis by L-cell suspensions and monolayers of treated chlamydiae and untreated control populations. The effect of each treatment on chlamydial infectivity was ascertained by titration in L cells.
RESULTS Relative efficiency of phagocytosis of E. coli, polystyrene latex spheres, and C. psittaci by L cells. L cells phagocytose chlamydiae just as rapidly and to the same extent as do unelicited mouse peritoneal macrophages (M. Gardner, personal communication). However, L cells are not generally regarded as active phagocytes. They have been used infrequently in research on endocytosis, and little is known about their capabilities. Therefore, a direct comparison was made of the relative efficiency of phagocytosis by L cells of E. coli, polystyrene latex spheres, and C. psittaci. E. coli cells labeled with "4C-amino acids were mixed with L cells in suspension so that attachment and ingestion of E. coli would be manifested by the presence of counts in the L-cell fraction after a series of low-speed centrifugations. Supernatant fractions were also analyzed for the presence of labeled E. coli. Regardless of the length of incubation at 37°C or the number of E. coli cells added for each host cell, greater than 99% of the counts were recovered in supernatant fractions during three cycles of low-speed centrifugation, and less than 1% of the E. coli counts sedimented with the L cells after the third centrifugation. The same number of counts was associated with the L cells at the beginning of the incubation as after 2 h. If E.
coli were being phagocytosed by L cells, then the amount of bacteria-specific label should have increased with time, but, even when this discrepancy was ignored, the total L-cell-associated counts after "infection" with a multiplicity of 1,000 E. coli per L cell represented less than six bacterial cells per host cell, and at a multiplicity of infection of 100, less than one bacterium was "ingested" per L cell. As observed by phase-contrast microscopy, L cells also phagocytosed zymosan, Staphylococcus aureus, and Bacillus cereus with comparable low efficiency. Polystyrene latex spheres 0.5 to 1.0 jsm in diameter were incubated with L cells for 2 h at 37°C, and the cells and associated latex spheres were sedimented by centrifugation, allowed to attach and spread on a substrate, and washed extensively. The latex spheres still associated with the L cells were then counted by fluorescence or phase-contrast microscopy. Although L cells took up the spheres somewhat better than E. coli, the efficiency of ingestion was at least 10-fold less than observed for C. psittaci (Table 1). The figures in Table 1 are probably too optimistic, and the true efficiency of ingestion of the latex spheres could have been much lower. The method used to measure phagocytosis of the latex spheres was not specific enough to discriminate between spheres actually ingested or merely attached. At multiplicities of less than 100 ID5), per host cell, 31 to 43% of the total chlamydia-specific, acid-precipitable counts were associated with L cells after 2 h at 37°C (Table 1). This estimate of ingestion efficiency is misleading because the total parasite-specific 14C counts in any C. psittaci preparation represented not only infectious elementary bodies but also noninfectious reticulate bodies and nuclease- and trypsin-resistant products derived from the constantly occurring breakdown of chlamydial cells. When the phagocytosis of C. psittaci by L cells was estimated by titrating the infectivity remaining unassociated with L cells after 2 h at 37°C, all or almost all of the infectious C. psittaci were Lcell associated when the multiplicity of infection was less than 100 ID.5 per host cell (Table 1). Phagocytosis of Chlamydia species by homologous and heterologous host cells. To see whether chlamydiae were efficiently phagocytosed only by host cells identical with those in which the parasites had been propagated and not in heterologous hosts, the efficiency of phagocytosis of '4C-labeled C. psittaci and C. trachomatis was compared in L and HeLa cells. C. psittaci had never been passed in HeLa cells; C. trachomatis had never been passed in L cells. When monolayers of L cells and of HeLa cells were infected under identical
VO.L. 19, 1978
PHAGOCYTOSIS OF CHLAMYDIA
TABLE 1. Relative efficiency of phagocytosis of polystyrene latex spheres, E. coli, and C. psittaci by L cellsa Object phagocytosed
Type of L-cell culture
Multiplicity of infection (no. of objects added per L
Polystyrene latex spheres
l00b 500 1,000
100 500 1,000 10o 50 100
Added objects that became L-cell associated Added(1) Added
100 500 1,000
5c 15 11
4,000d 40,000 202,000 404,000
22' 181 790 1,600
25,600 51,200 103,000
6,760 12,700 20,300
Efficiency of phagocytosis
[(2)/(1)] 0.05 0.03 0.01