Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune to Rickettsia conorii exerted specific major histocompatibility complex (MHC) class I-matched CTL activity against R. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells from Rickettsia australis-immune mice exerted specific CTL activity against an R. australis-infected macrophage-like cell line. Gamma interferon (IFN-gamma) gene knockout mice were more than 100-fold more susceptible to R. australis infection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome of R. australis infection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-gamma. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-gamma gene knockout mice into R. australis-infected IFN-gamma gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-gamma. CTLs appear to be crucial to recovery from rickettsial infection.

Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection.

The mechanism of killing of obligately intracellular Rickettsia conorii within human target cells, mainly endothelium and, to a lesser extent, macrophages and hepatocytes, has not been determined. It has been a controversial issue as to whether or not human cells produce nitric oxide. AKN-1 cells (human hepatocytes) stimulated by gamma interferon, tumor necrosis factor alpha, interleukin 1β, and RANTES (regulated by activation, normal T-cell-expressed and -secreted chemokine) killed intracellular rickettsiae by a nitric oxide-dependent mechanism. Human umbilical vein endothelial cells (HUVECs), when stimulated with the same concentrations of cytokines and RANTES, differed in their capacity to kill rickettsiae by a nitric oxide-dependent mechanism and in the quantity of nitric oxide synthesized. Hydrogen peroxide-dependent intracellular killing of R. conorii was demonstrated in HUVECs, THP-1 cells (human macrophages), and human peripheral blood monocytes activated with the cytokines. Rickettsial killing in the human macrophage cell line was also mediated by a limitation of the availability of tryptophan in association with the expression of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase. The rates of survival of all of the cell types investigated under the conditions of activation and infection in these experiments indicated that death of the host cells was not the explanation for the control of rickettsial infection. This finding represents the first demonstration that activated human hepatocytes and, in some cases, endothelium can kill intracellular pathogens via nitric oxide and that RANTES plays a role in immunity to rickettsiae. Human cells are capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by one or a combination of three mechanisms involving nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation.

Mechanisms of Intracellular Killing of Rickettsia conorii in Infected Human Endothelial Cells, Hepatocytes, and Macrophages

A mouse model of spotted fever group rickettsiosis, in which disease results from disseminated rickettsial infection of endothelial cells and vascular damage, was developed by intravenous inoculation of 6- to 8-week-old, male, Balb/c mice with Rickettsia australis. Animals developed progressively severe vasculitis, interstitial pneumonia, and multifocal hepatic necrosis. These lesions correlated with early disseminated infection of endothelial cells followed by growth and invasion of rickettsiae into perivascular cells. The dose of 2 x 10(6) organisms was uniformly lethal. Serum interleukin- (IL) 1, IL-6, and interferon (IFN) increased by day 3 and tumor necrosis factor (TNF) on day 5. TNF, IL-6, and IFN declined on day 7. Spleen cells responded to Rickettsia australis antigen by producing IFN, TNF, IL-1, and IL-6 on day 5, followed by lower quantities of these cytokines on day 7. Despite the production of antibodies, IFN, TNF, IL-1, and IL-6, a lethal outcome occurred frequently. A decreased ability to secrete IL-2 suggests an element of infection-associated immunosuppression.

Rickettsia australis infection: a murine model of a highly invasive vasculopathic rickettsiosis.

Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Fragments representing the genes of the two major outer membrane proteins of spotted fever group rickettsiae (rOmpA and rOmpB) were tested as DNA vaccines. Immunizations with each of three fragments (rompA4999-6710, rompB1550-2738, and rompB2459-4123) conferred a degree of protection on vaccinated mice against virulent rickettsial challenge. Protection was achieved when DNA immunizations were followed by booster immunizations with the homologous recombinant protein. Proliferation and gamma-interferon secretion were detected after in vitro stimulation of lymphocytes from immunized animals with whole Rickettsia conorii antigen. The data validate particular segments of rOmpA and rOmpB as potent immunogens and hence as sources of immunostimulatory elements with specificity for T lymphocytes, which are the key effectors of protective immunity against rickettsial infections.

Identification of protective components of two major outer membrane proteins of spotted fever group Rickettsiae.

In order to determine the definitive importance of T- and B-lymphocytes in immunity to Rickettsia conorii, mice genetically deficient in T-cells, B-cells, or both T- and B-cells were infected experimentally. T-lymphocytes rather than humoral antibodies were crucial to rickettsial clearance and a reduced mortality rate. Mice incapable of an antibody response to polysaccharide capsular antigens effectively controlled rickettsial infection with no mortality. In contrast, nude mice produced antibody to thymus-independent antigens early in the course of infection, yet experienced severe rickettsial infection resulting in deaths. The observed hepatic lesions are similar to those of boutonneuse fever. This model offers the opportunity to investigate rickettsial immune mechanisms and hepatic injury.

N. R. Montenegro

Papers

Infection of genetically immunodeficient mice with Rickettia conorii