Abstract
Rickettsia rickettsii is a small, Gram-negative, obligate intracellular bacillus and the causative agent of a fulminating human disease, Rocky Mountain spotted fever (RMSF) (Silverman, 1984). RMSF is a tick-borne disease wherein rickettsiae grow in the gut lining of ticks, often without harming the host. Human infection results either from a tick bite (commonest) or rarely by contamination of cut skin or a wound with faeces of the ticks (Bleck, 1999). In humans, R. rickettsii grows principally in endothelial cells of small blood vessels, producing vasculitis, thrombosis of vessels, fever, typical skin rash (petechial), organ dysfunctions and cell death (Walker & Raoult, 2000). Although the primary target of R. rickettsii is the vascular endothelial cell, often there is multi-organ and system involvement in the later stages of RMSF pathogenesis.
The central nervous system (CNS) appears to be one of the major systems involved during the later stages of RMSF pathogenesis (Walker et al., 2001). During infection, the incidence of major dysfunctions pertaining to the CNS has been summarized as headache (7991 %), signs of meningoencephalitis and meningismus (18 %), stupor (2126 %), ataxia (518 %), seizures (8 %), hearing loss (7 %), coma (910 %) and death (48 %) (Walker & Raoult, 2000). In RMSF, restlessness and irritability often lead to mental confusion and delirium, followed by coma in later stages of the disease. Cortical blindness, central deafness, spastic paralysis and sixth nerve palsy have also been reported (Walker et al., 2001). Among all rickettsial diseases, RMSF has emerged as an important cause of status epilepticus in children in the USA (Bleck, 1999). A confirmed case of acute disseminated encephalomyelitis after RMSF has also been reported (Wei & Baumann, 1999). For RMSF, among the non-human hosts and reservoirs required to complete the natural cycle are rodents, dogs and rabbits (Bleck, 1999).
The cerebellum is involved in important functions, mainly dealing with balance, voluntary muscle coordination, and gait, so damage to the cerebellum may lead to loss of these functions. Ataxia, hypotonia, paresis and paralysis are frequently found to be associated with cerebellar dysfunction. As similar types of symptoms are observed in the later stages of RMSF (Bleck, 1999), one may suspect the involvement of the cerebellum. In order to investigate whether cerebellar neurons are infected or damaged during R. rickettsii infection, we used primary cultures of rat cerebellar granular neurons (CGNs). Our results demonstrate that R. rickettsii efficiently infects neuronal cells and that the infection causes apoptotic death of neurons.
Cultures of human umbilical vein endothelial cells (ECs) were established as described previously (Joshi et al., 2003; Silverman, 1984). Near-confluent cultures of ECs at second passage were used for various experiments. Primary cultures of cerebellar granule cells were prepared from 8-day-old SpragueDawley rat cerebellum essentially as described previously (Kovács et al., 2002) and plated at 5x105 cells ml1 into 24-well plates (1 ml per well). Cytosine-ß-arabinofuranoside (Ara-C; 10 µM) was added 24 h after plating to limit the number of non-neuronal cells.
A plaque-purified seed stock (1x1075x107 p.f.u. ml1) of R. rickettsii (Sheila Smith strain) was prepared from infected Vero cells as described previously (Joshi et al., 2003; Silverman, 1984). The endothelial cells or CGNs were infected with 5x104 p.f.u. R. rickettsii diluted in 80 µl culture medium per cm2 of cell culture area. After 2 h incubation, the medium containing infective organisms was aspirated, and the cell monolayer was washed twice to remove Vero cell debris and then retained quickly in fresh culture medium for the remainder of infection. CGN cultures were quickly retained in conditioned (pre-saved) medium. Infection of ECs plated on Thermanox coverslips (Nalge-Nunc International) was monitored in parallel by staining with polyclonal anti-rickettsia serum [anti-RR; Centers for Disease Control and Prevention (CDC)] by indirect immunofluorescence, as described elsewhere (Clifton et al., 1998; Silverman, 1984). Some of the cultures were exposed to 30 µg tetracycline /ml1 for 30 min prior to, and then maintained in throughout, the infection to inactivate R. rickettsii (Silverman, 1984). In some experiments, incubation of uninfected cells with staurosporine (STS; 2 µM) was included as a positive control for induction of apoptotic cell death (Joshi et al., 2003).
For immunocytochemical analyses, ECs or CGNs plated on glass coverslips (as described above) were left uninfected or infected with R. rickettsii in the presence or absence of tetracycline, or treated with apoptosis-inducing agent(s). The cells were then processed for fixation, permeabilization, blocking and washing, essentially as described previously (Joshi et al., 2003), for specific identification of a neuronal marker (ß-III tubulin) and/or R. rickettsii-infected cells, with the following primary antibodies: mouse anti-ß-III tubulin (anti-TUJ1) (1 : 300; Covance) or anti-R. rickettsii rabbit polyclonal serum (1 : 1000; CDC). In the case of double labelling, first the cell-bearing coverslips were treated with anti-TUJ1, followed by treatment with the corresponding secondary antibodies (1 : 400, 37 °C, 1 h). The coverslips were then stained with anti-R. rickettsii rabbit polyclonal serum (1 : 1000, 37 °C, 1 h) followed by the corresponding secondary antibodies (1 : 200, 37 °C, 30 min). Secondary antibodies were either rhodamine-conjugated or fluorescein-conjugated goat anti-rabbit or anti-mouse IgG (Calbiochem) or Texas-red-conjugated anti-mouse IgG (Southern Biotechnology Associates). The coverslips were then processed for examination by fluorescent, bright-field or phase-contrast microscopy.
The extent of apoptotic cell death of immunocytochemically identified neurons or ECs was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labelling (TUNEL) assay. The use of TUNEL has been reported to detect nuclear DNA fragmentation during the course of apoptotic cell death. TUNEL was performed using the ApopTag-Fluorescein in situ apoptosis detection kit (Intergen) as per the manufacturer's directions and as described previously (Joshi et al., 2003). A minimum of 500 cells, located in multiple, randomly selected fields, were counted for each condition in three different sets of experiments, and data were calculated as the arithmetic mean values of percentages of TUNEL-positive cells.
Fluorescent, phase-contrast or bright-field microscopy was performed, and digitalized images were captured using an Olympus CR40 fluorescent microscope and a Qimaging cooled digital charge-coupled device (CCD) video camera, with appropriate settings. Digitalized images were saved as TIFF files and edited using Adobe Photoshop (version 6.0). The datasets in this study were calculated as a percentage of the corresponding control levels. Statistical significance was determined by one-way ANOVA followed by Bonferroni's test for multiple comparisons.
To test whether R. rickettsii infects CGNs from a non-human rodent host, rat primary CGN cultures on day 8 in vitro were exposed to R. rickettsii. A double immunofluorescent-staining technique was employed to identify infected neurons, using anti-RR serum and a neuron-specific (anti-neuronal ß-III isoform of tubulin; anti-TUJ1) antibody. Fig. 1 shows that CGNs were infected (65 % of the neurons) during a 6 h exposure period to R. rickettsii, and the infection induced widespread neuronal degeneration, as evident from the bright-field images. Overlay of the anti-RR and neuron-specific anti-TUJ1 staining of the same field (Fig. 1f) demonstrates that R. rickettsii infects neurons. The small arrows in the bright-field image of the same field (Fig. 1c) point to infected neurons, immunopositive for both R. rickettsii and neuron-specific ß-III tubulin. Some of the neuronal cells are heavily infected and degenerated (large arrowheads), but weakly immunopositive for the neuron-specific ß-III tubulin, most likely because the advanced degeneration and/or bacterial overload has destroyed or masked the epitope recognized by the monoclonal anti-TUJ1 antibody. To verify that the observed neuronal degeneration was R. rickettsii-induced, CGN cultures were exposed to R. rickettsii in the presence of tetracycline, an antibacterial agent, which efficiently kills R. rickettsii and has been in use for this purpose. As expected, the neurons exposed to R. rickettsii in the presence of tetracycline remained intact (data not shown).