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Staphylococcal strain 8325-4, unlike other staphylococcal strains, fails to induce cytokine IL-1 and IL-6 gene expression in human endothelial cells. In the present investigation, this strain was shown to release a product that inhibited cytokine gene expression in endothelial cells infected with another staphylococcal strain. This inhibition was due to prevention of internalization, but not adherence, of bacteria by endothelial cells. Induction of endothelial cell cytokine gene expression by lipopolysaccharide was not affected by the staphylococcal supernatant. In contrast to endothelial cells, 8325-4 did not inhibit Wb-induced cytokine gene expression in monocytes. Further characterization of the inhibitory factor suggests that it is a lipoprotein and that both protein and lipid components play a role in its inhibitory function.

The ability of extracellular matrix heparan sulfate to alter the susceptibility of human endothelial cells to S. aureus was investigated. Endothelial cells grown on extracellular matrix synthesized by S. aureus-infected endothelial cells were more susceptible to subsequent staphylococcal infection than endothelial cells grown on the extracellular matrix synthesized by untreated endothelial cells. Endothelial cells were more susceptible to S. aureus infection when 1) grown on heparitinase-treated extracellular matrix that removed heparan sulfate chains, 2) grown on extracellular matrix produced by chlorate-treated endothelial cells that reduced sulfation in the matrix heparan sulfate proteoglycans, 3) grown on heparan sulfate purified from extracellular matrix elaborated by infected endothelial cells, and 4) endothelial cells were chlorate-treated and therefore expressed desulfated cellular heparan sulfate proteoglycans. Extracellular matrix produced by S. aureus-infected endothelial cells contained heparan sulfate proteoglycans with reduced sulfation. The altered extracellular matrix with reduced sulfated heparan sulfate proteoglycans signalled the uninfected endothelial cells to produce under sulfated cellular heparan sulfate proteoglycans that increased S. aureus adherence to the endothelial cells.

Interleukin-8 (IL-8) plays a crucial role in the migration of neutrophils to bacterium-infected sites. This study investigated IL-8 induction in Staphylococcus aureus-infected endothelial cells (EC). We observed that S. aureus-infected EC were induced to express both IL-8 mRNA and protein, which can promote transmigration of neutrophils across an EC monolayer.

The ability of the vascular endothelium to elaborate cytokines in response to gram-positive sepsis has received limited attention. This study examined cytokine expression by human umbilical vein endothelial cells (EC) following infection with a gram-positive bacterial pathogen, Staphylococcus aureus. S. aureus infection of EC resulted in the production of interleukin-6 (IL-6) and IL-1 beta. For IL-6, message was detected at 3 h after infection, protein was present at 24 h, and both message and protein persisted for 72 h. IL-1 beta message was detected at 12 h, IL-1 beta protein was detected at 24 h, and both persisted for 72 h. Message for colony-stimulating factor 1 remained unaltered. UV-killed S. aureus also elicited IL-1 beta and IL-6 message and protein expression at 24 and 48 h. Twenty-one clinical isolates of S. aureus were tested, and all induced IL-6 release by 48 h. However, the laboratory strain 8325-4 did not induce cytokine expression at any time point and was internalized by EC 1,000-fold less than other strains were. Internalization of latex beads by EC did not induce IL-6 gene expression. Furthermore, cytochalasin D treatment of the EC prevented IL-1 and IL-6 induction by S. aureus but not by tumor necrosis factor alpha or lipopolysaccharide. These results indicate that S. aureus is a potent inducer of IL-1 and IL-6 in EC and that internalization of S. aureus by EC is necessary for their cytokine expression. Thus, our data suggest that the vascular endothelium may play an important role in the pathogenesis of septicemia caused by gram-positive organisms.

The adherence of Staphylococcus aureus to human endothelial cells is saturable in both dose- and time-dependent assays. Staphylococcal surface components which bound to endothelial cells in vitro were identified by using biotin-labeled, solubilized staphylococcal proteins. Four trypsin-sensitive components with molecular sizes of 30, 55 to 57, 70, and 85 kDa were recognized. These proteins did not label with the glycan detection system. When staphylococci were harvested during the exponential phase of growth, staphylococcal adherence to endothelial cells was significantly increased and increased expression of the S. aureus binding proteins was observed. Preincubation of endothelial cells with protein A did not reduce S. aureus adherence in an in vitro infection assay. Four S. aureus surface components whose expression is growth phase dependent adhere to human endothelial cells in vitro.

A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.

Vasculitis, a recognized complication of staphylococcal-endovascular infections, may result in part, from the expression of FcR by Staphylococcus aureus-infected endothelial cells. FcR were measured using [51]Cr labeled SRBC preincubated with rabbit anti-SRBC IgG. FcR were not detected on uninfected endothelial cells, but were demonstrated on S. aureus infected cells using IgG, but not IgM labeled SRBC. FcR expression was dependent on the initial bacterial density (greater than or equal to 8 x 10(7) cfu/ml) and on phagocytosis of the staphylococci, but not on new protein synthesis. IgG labeled SRBC binding was blocked by aggregated IgG but not IgM. SRBC coated with the F(ab')2 portion of IgG did not bind, thus confirming that FcR were specifically involved in this interaction. FcR are expressed after S. aureus invasion of human endothelial cells and may contribute to the vasculitis which often accompanies S. aureus-endovascular infections.

We have investigated S. aureus adherence to human endothelial cells utilizing an in vitro model. Staphylococcus binding to confluent endothelial cell monolayers was saturable in both dose and time response studies suggesting that the binding interaction was specific. We have developed a technique, based on the pH dependent affinity of iminobiotin for streptavidin, for the isolation of an endothelial cell membrane component that binds S. aureus, in vitro. A 50-kD membrane component was isolated and purified using this approach. This component was trypsin sensitive, periodate insensitive, and did not label with [3H]glucosamine. [35S]Methionine and [125I]iodine labeling confirmed that the protein was synthesized by and expressed on the endothelial cell surface. Functional binding studies demonstrated that staphylococci, but not endothelial cells, bound to the protein when immobilized on microtiter wells. Preincubation of staphylococci with the purified protein significantly (P less than 0.001) reduced staphylococcal binding to cultured endothelial cells. The capacity of S. aureus to colonize and invade endovascular surfaces may in part be a consequence of staphylococcal interaction with this endothelial cell membrane protein.

Staphylococcus aureus is an invasive pathogen capable of causing life-threatening disease. A major component of this pathogen's virulence is its ability to invade normal endovascular tissue. We examined the interaction of S. aureus with cultured human endothelial cells and with human and rabbit endovascular tissue. Our ultrastructural study demonstrated a sequence of steps which occurred with staphylococcal invasion of human endothelial cells; adhesion, endocytosis, and intracellular replication. Ultimately, this resulted in cell disruption and death. Cytochemical staining of lysosomes demonstrated lysosomal fusion with both viable and killed intracellular bacteria without evidence of staphylococcal degradation. Quantitative studies using an in vitro infection assay demonstrated comparable rates of adhesion by viable and ultraviolet-killed bacteria, phagocytosis at a slower rate, and intracellular replication. The present study demonstrates an active role for the endothelial cell in the development and spread of endovascular staphylococcal infections. It also supports the use of this in vitro tissue culture system as a model for the study of bacterial invasion of the endothelium.

Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo.