INFECTION AND IMMUNITY, Feb. 1978, p. 607-612 0019-9567/78/0019-0607$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 19, No. 2

Printed in U.S.A.

Kinetics of Phagocytosis of Chlamydia psittaci by Mouse Fibroblasts (L Cells): Separation of the Attachment and Ingestion Stages GERALD I. BYRNEt Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 8 August 1978

The kinetics of phagocytosis of Chlamydia psittaci (6BC) by monolayers of fibroblasts (L cells) was studied with an assay that distinguished between the attachment and ingestion phases of phagocytosis. At multiplicities of 10 and 100 50% infectious doses (ID5o) per L cell, virtually all of the inoculated C. psittaci had been attached and ingested after 60 min at 370C. At multiplicities of 500 to 5,000 IDso per L cell, the initial rates of attachment and ingestion of C. psittaci to L cells increased with the multiplicity of infection, but phagocytosis stopped even though many chlamydial cells remained free in suspension and readily available for attachment to the host-cell monolayers. Phagocytosis probably ceased because the L cells were injured when they took up large numbers of chlamydial cells. This injury prevented direct determination of the number of potential binding sites for C. psittaci on each L cell. However, this number is large enough to make the rates of chlamydial attachment and ingestion predominantly dependent on the multiplicity of infection. mouse

In the accompanying paper (3), it was hypothesized that members of the genus Chlamydia enter nonprofessional phagocytes with unusual efficiency because they carry on their surfaces ligands with high affinity for normal, ubiquitously distributed structures on the surface of host cells. This mode of chlamydial entry was called parasite-specified phagocytosis to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes. Since phagocytosis by professional phagocytes has been shown to occur in distinct stages of attachment and ingestion (10, 11), a method for separating the attachment and ingestion phases in the phagocytosis of Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was devised and used to study the kinetics of the process. MATERIALS AND METHODS Growth of L cells and C. psittaci Methods for propagating L-cell monolayers and growing, harvesting, titrating, partially purifying, and preparing radioisotopically labeled suspensions of Chlamydia psittaci (6BC) have already been described (2, 3, 6). Measurement of the phagocytosis of "C-labeled C. psittaci by L-cell monolayers: separation of attachment and ingestion stages. L cells were suspended in medium 199 containing 0.1% sodium bicarbonate, 200 ,ug of streptomycin sulfate per t Present address: The New York Hospital-Comell Medical Center, Department of Medicine, New York, NY 10021.

mnl, and 10% heat-inactivated fetal calf serum (FCS) at a density of 106 cells per ml. Four-milliliter portions were added to plastic flasks (25-cm2 surface area) that had been previously equilibrated in an atmosphere of 5% CO2 and 95% air, and the flasks were incubated at 37°C for 4 h. After incubation, the monolayers were observed with a phase-contrast microscope to be sure that the cells had attached and spread confluently on the substrate as expected. Labeled chlamydiae were diluted in sucrose-phosphate buffer (1) containing 2% FCS and 2 ,ug of cycloheximide per ml so that appropriate infectious doses would be contained in volumes of 0.6 to 0.8 ml. Cycloheximide inhibited regeneration of the L-cell sites that bind chlamydiae (2) and was without effect on their uptake during the relatively brief periods of incubation employed (3). The medium was decanted from the monolayers and replaced with the chlamydial inoculum. For zerotime points, the inoculum was decanted immediately. The other flasks were incubated at 37°C for the required times with constant shaking at 125 strokes/min. After incubation, the inoculum was decanted and the monolayers were washed three times with 4 ml of icecold growth medium and three times with 4 ml of icecold phosphate-buffered saline (4). The washings were combined with the decanted inoculum, and 0.5 ml of 0.08% trypsin (Difco, 1:200) was added to each flask. The excess trypsin was decanted and added to the combined washings. After each monolayer had been at 25°C for 10 min, the cell layer was detached from the substrate by agitation with 2.5 ml of medium, and the L-cell suspension was transferred to a conical centrifuge tube. The flasks were washed once with 2.5 ml of medium, and the washings were combined with the cells already in the centrifuge tubes. The L607

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cell suspensions were then centrifuged for 5 min at 300 x g and separated into sedimented and supernatant fractions. Since experiments with flasks containing no host cells showed that the inoculum did not remain attached to the plastic substrate after the appropriate number of washes, the entire chlamydial inoculum must have been, at any time after infection, either free in suspension, attached to the host cell surface, or inside the host cell. The chlamydiae found in the combined fractions composed of the decanted inoculum, the first six washes, and the excess decanted trypsin solution were defined as free chlamydiae. The concentration of free chlamydiae was assessed by determining the total acid-insoluble counts in the combined fraction just described and by titrating the infectivity of a portion of this sample in fresh L cells (2, 6). Since trypsin-sensitive binding sites on the host cell surface are required for phagocytosis of chlamydiae by L cells (2, 3), those chlamydiae that remained associated with the L cells during the initial washings but were rendered nonsedimentable at 300 x g after trypsinization of the monolayers were defined as externally attached parasites and were measured by the same methods used to determine the concentration of free chlamydiae. Ingested chlamydiae were represented by the "C counts that sedimented with the trypsinized L cells during centrifugation at 300 x g and were, therefore, not susceptible to removal from the host cell surface by tryptic digestion. That the chlamydiae classified as ingested in this assay were truly taken into L cells was shown by measuring ingestion of "C-labeled C. psittaci both by the method just described and by determining the percentage of L cells infected (inclusion bearing) at low multiplicity. The good agreement between the two methods shows that the labeled chlamydiae that became associated with L cells and were not dissociated by trypsin actually entered host cells, multiplied, and formed visible inclusions. At higher multiplicities, the two methods were compared by measuring in the same sample the increase in "C label associated with L cells- and the decrease in the C. psittaci infectivity remaining free and unattached to L cells (Fig. 1). The specific activity (number of "C counts per 50% infectious dose [ID50]) of each labeled chlamydial preparation was measured each time it was used by titrating the infectivity and determining the number of counts in the ingested fraction after 2 h at 37°C for an inoculum (

Kinetics of phagocytosis of Chlamydia psittaci by mouse fibroblasts (L cells): separation of the attachment and ingestion stages.

INFECTION AND IMMUNITY, Feb. 1978, p. 607-612 0019-9567/78/0019-0607$02.00/0 Copyright © 1978 American Society for Microbiology Vol. 19, No. 2 Print...
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