The endothelial cell is the putative primary target cell in humans infected with Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever. Although ...
INFECTION AND IMMUNITY, June 1984, p. 545-553
Vol. 44, No. 3
0019-9567/84/060545-09$02.00/0 Copyright © 1984, American Society for Microbiology
Rickettsia rickettsii-Induced Cellular Injury of Human Vascular Endothelium In Vitro DAVID J. SILVERMAN Department of Microbiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 Received 3 January 1984/Accepted 17 February 1984
The endothelial cell is the putative primary target cell in humans infected with Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever. Although the clinical manifestations of infection by this organism are well documented, the mechanism of injury to the endothelial cell is not understood. The ability to culture human endothelial cells in vitro provides a unique system with which to study this host-parasite interaction directly. Human vascular endothelial cells derived from the umbilical vein, when infected by R. rickettsii, became severely damaged within a few days postinfection. The primary lesion observed at the ultrastructural level appeared to occur at intracellular membranes, specifically, the rough-surfaced endoplasmic reticulum. Widespread dilatation of these membranes eventually led to the creation of large intracellular cisternae and the apparent circumscription of rickettsiae and cellular organelles by the roughsurfaced endoplasmic reticulum. Small membrane-bound fragments of host cytosol created by dilating membranes also were present within the cisternae. Within 5 to 6 days postinfection, cells lost their osmoregulatory control and lysed. Some possible mechanisms of cell injury directed at the level of intracellular membranes are discussed. The infection, proliferation, and cellular destruction caused by Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, have been documented in studies utilizing numerous host cell types (4, 13, 16, 18-20, 25, 27). As a result of these in vitro studies, R. rickettsii was shown to exhibit specific properties which served to distinguish this organism, and possibly other members of the spotted fever group, from other species of the genus Rickettsia. Intranuclear infection and replication and the ability of the organism to move bidirectionally through the plasma membrane, resulting in the rapid spread of the rickettsiae in cultured cells, are well-established properties of R. rickettsii which have been discovered through the use of light microscopy. Rickettsiae-induced cellular destruction at the electronmicroscopic level has also been studied with R. rickettsii and R. prowazekii. The cell damage elicited in chicken embryo fibroblasts in response to infection by these two organisms is strikingly different (18-20). R. prowazekii, because of its apparent inability to escape from the host cell during initial phases of infection, remains confined to its intracellular environment, replicates to large numbers in the host cell cytoplasm, and, as a rule, is released only when the cell lyses. R. rickettsii, on the other hand, with its capacity for bidirectional movemnent through the plasma membrane, spreads rapidly to adjacent cells, does not accumulate to large numbers within the infected host cell, and induces early cytopathic changes which eventually lead to cell lysis and death (18, 25). Recent studies in my laboratory have focused on the characterization of the infection of human vascular endothelial cells by R. rickettsii, with the purpose of using this system as a possible in vitro model for studying the interaction of R. rickettsii with the putative target cell in human infections (16a). We have recently shown that the basic growth properties of R. rickettsii in human endothelial cells are similar to those observed in the chicken embryo cell model system previously described by Wisseman et al. (25). There are a number of advantages in using human endothelial cells as a model for infection by R. rickettsii: (i)
endothelial cells are easily obtained from the human umbilical vein; (ii) they represent cells which are of the same species and ontogenic type as those which are thought to be principally parasitized in vivo; (iii) they retain many of their in vivo properties in an in vitro culture system, e.g., synthesis of prostaglandins (22), expression of angiotensin converting enzyme activity (8), synthesis of factor VIII-associated protein (9), and retention of the Weibel-Palade body, an endothelial cell-specific cytoplasmic organelle (5); and (iv) they provide the opportunity to examine the interaction between the infected target cell and other blood-derived cells such as granulocytes, macrophages, and platelets which likely modulate the course of infection in humans. I report here electron-microscopic studies of the cell injury induced by R. rickettsii in human endothelial cells with the purpose of extending knowledge of this hostparasite interaction. I propose some possible mechanisms involved in cell injury and advocate the potential usefulness of this in vitro system for studying the pathogenesis of Rocky Mountain spotted fever at the cellular level. MATERIALS AND METHODS Rickettsiae. The rickettsial seed used in these experiments was a plaque-purified isolate of the Sheila Smith strain of R. rickettsii. The organism was propagated in the yolk sacs of 5day-old embryonated chicken eggs and harvested and purified on Renografin gradients as previously described (7). Quantitation of rickettsiae in the seed stock was carried out by the method of Silverman et al. (17), and infectivity was determined by plaque titration in primary cultures of chicken embryo cells and umbilical vein endothelial cells by a modification of the method of Wike and Burgdorfer (23). The purified seed was found to have a rickettsial body count of 2.2 x 109/ml and a titer of 1.1 x 109PFU/ml for a rickettsial body/PFU ratio of 2. Isolation and culture of human umbilical vein endothelial cells. Endothelial cells were isolated from human umbilical veins by the methods of Jaffe et al. (9) and Gimbrone (5). The 545
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FIG. 1. R. rickettsii-infected human umbilical vein endothelial cell in an early stage of infection, showing organisms (R) in the cytosol. Rickettsiae are easily distinguished from mitochondria because of the "halo" which surrounds them and which appears to prevent contact between the organisms and the host cytosol. Arrowheads depict RER. Bar, 2 ,um.
umbilical vein was cannulated from both ends with intramedic polyethylene tubing (Clay Adams, Parsippany, N.J.) and gently flushed with isotonic saline to remove remaining blood cells and plasma. The vein was filled with a 0.1% collagenase solution, type CLS (Worthington Biochemicals Corp., Freehold, N.J.), prepared in phosphate-buffered saline. The cannulas were clamped shut, and the umbilical cord was suspended and incubated at 37°C in a beaker of sterile phosphate-buffered saline for 20 min. Cells were collected in sterile plastic centrifuge tubes, washed by centrifugation in Hanks balanced salt solution (HBSS) and seeded into 25-cm2 plastic tissue culture flasks (Corning Glass Works, Corning, N.Y.). Endothelial cells were cultured in medium 199 with Earle salts containing 20% pooled human serum which had been heat inactivated at 56°C for 30 min, and tested for antirickettsial antibodies by a modification of the method of Bozeman and Elisberg (2). No antibacterial or antifungal agents were used in either the isolation or the culture of the cells. The cells were grown at 37°C in an atmosphere of 5% CO2 in air and passaged using a 0.05% trypsin-0.02% EDTA solution. For these experiments, endothelial cells were not used beyond the second passage.
Identification of cells isolated from the umbilical vein as endothelial cells was confirmed by demonstrating the presence of factor VIII-associated protein on the cell surface by an indirect immunofluorescence assay and the presence of Weibel-Palade bodies by electron microscopy of thin sec*tions through cultured cells (5, 9). For detection of factor VIII-associated protein, cells were cultured on Thermanox (Lux Scientific Corp., Newbury Park, Calif.) plastic cover slips and fixed in acetone for 10 min. Rabbit antiserum to human factor VIII-associated protein (Calbiochem-Behring, La Jolla, Calif) was added to the cover slips and incubated at room temperature for 30 min. The cover slips were counterstained with rhodamine-labeled bovine serum albumin, treated with fluorescein-conjugated goat antibody to rabbit immunoglobulins (Calbiochem-Behring Corp.), mounted on slides, and examined by fluorescence microscopy on an American Optical model 120 Microstar microscope equipped with an epifluorescence attachment. A line of human embryonic skin and muscle cells, F-1000 (Flow Laboratories, Inc., McLean, Va.), was used as a control. Infection of endothelial cells by R. rickettsii. To determine the uptake of R. rickettsii and to monitor growth of the
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