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Departments of Laboratory Medicine and Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
Extracellular adherence protein (Eap) has been suggested as an important virulence factor of Staphylococcus aureus because it enhances bacterial adherence and internalization into eukaryotic cells, interference with T cells, and neutrophil adherence to endothelial cells. We demonstrate that Eap has dual effects on peripheral blood mononuclear cells, depending on its concentration. At low concentrations (up to 9 g/mL), Eap induces a proliferative response; at higher concentrations, it causes a significant inhibition of T cell proliferation induced by S. aureus supernatants toxic shock syndrome toxin1 or phytohemagglutinin. A marked increase in apoptotic (i.e., Annexin V and propidium iodide positive) T and B cells could be demonstrated after exposure to the inhibitory concentration of Eap. Human antiEap antibodies prepared from polyspecific immunoglobulin G (IgG) blocked the immunomodulatory effects of Eap. Our results demonstrate novel immunomodulatory activities of Eap and identify potential mechanisms of action of intravenous IgG therapy in the treatment of S. aureus infections.
Staphylococcus aureus has the capacity to infect and disseminate into the bloodstream and distant sites in the host when skin or mucosal barriers are breached or when host immunity is affected. The range of diseases caused by S. aureus is broad and includes endocarditis, osteomyelitis, and septic shock [1]. S. aureus produces a number of cell surfacelocalized proteins that are responsible for binding to fibronectin, collagen, fibrinogen, and vitronectin, among others. These extracellular matrixbinding proteins have been proposed to contribute to successful colonization and persistence at various sites in the host. In addition, 4 proteins with the ability to bind to fibrinogen are produced by and released from S. aureus coagulase [2]: extracellular fibrinogenbinding protein (Efb) [35], extracellular matrix proteinbinding protein [6], and extracellular adherence protein (Eap, also referred to as Map and P70) [711]. We have shown that Eap causes an aggregation of the bacteria and enhances adherence and internalization of the bacteria into eukaryotic cells [8, 9]. Internalization of the bacteria into nonprofessional phagocytic cells has been associated with (1) persistent and relapsing infections, because of their intracellular location, which shields the bacteria from host defense and antibiotic treatment; (2) apoptosis, leading to engulfment by macrophages; and (3) necrosis, leading to dissemination of the bacteria in the host [12].
In more recent investigations, Eap has been revealed to be an immune-modulating protein [1315]. Chavakis et al. [13] showed an interaction between Eap and intercellular adhesion molecule (ICAM)1 in vitro, whereas no interaction could be seen with molecules such as the -integrin Mac-1 (CD11b/CD18) or leukocyte function antigen1 on leukocytes. The binding of Eap to ICAM-1 inhibits the adherence of leukocytes to the endothelial tissue and thereby inhibits the extravasation of leukocytes from the bloodstream into the site of infection [13]. Similarly, Eap inhibited the binding of neutrophils to stimulated endothelial cells, which resulted in an inhibition of the transendothelial migration of neutrophils. Antibodies against ICAM-1 had the same blocking effect as Eap, which confirmed that Eap's blocking effect is ICAM-1 dependent [14]. Lee et al. [15] showed that the presence of Map (i.e., Eap) interfered with T cell proliferation, leading to chronic diseases such as arthritis, osteomyelitis, and abscess formation. Eap is not the only molecule from S. aureus that compromises the immune system. Chemotaxis inhibitory protein of S. aureus is an exoprotein that has been shown to inhibit the response of neutrophils and monocytes toward chemoattractants, such as C5a and formylated peptides [16, 17]. In addition, it has been shown that Efb binds to the C3d fragment of C3 and inhibits both the classical and alternative pathways of complement activation [18]. The consequence of the presence of Eap during S. aureus infection, whether it is on the endothelial tissue or on the process of antigen presentation to T cells, is a hampered protective cellular immunity, which is the main defense used by the body to clear bacterial infections. In the present study, we have taken these aspects into account and looked more closely into the direct influence of Eap on peripheral blood mononuclear cells (PBMCs). We investigated the effects of Eap at various concentrations and identified novel immunomodulatory activities of Eap that are concentration dependent.
MATERIALS AND METHODS
Preparation of bacterial culture supernatant.
S. aureus strain Newman was grown in Luria broth overnight at 37°C. The supernatant was separated by centrifugation at 5000 g, and proteins in the culture supernatants were precipitated by ethanol (1 : 3 supernatant : ethanol). The precipitate was dissolved in 1 mL of distilled water and dialyzed extensively against multiple changes of distilled water. The dialysates (supernatants) were filter sterilized and stored at -20°C until their use.
Purification of Eap.
Native Eap was purified as described elsewhere [7]. Briefly, a supernatant from S. aureus strain Newman was subjected to affinity chromatography on Fg-Sepharose (Pharmacia). Proteins were eluted with 0.7% acetic acid and further purified on a Mono S column (Pharmacia), by use of a gradient of 01 mol/L NaCl in 40 mmol/L phosphate buffer (pH 6.5). Three peaks of proteins were eluted. The first eluted at a salt concentration of 0.150.25 mol/L NaCl (coagulase), the second at 0.350.45 mol/L NaCl (Efb), and the third at 0.50.7 mol/L NaCl (Eap). The eluate (third peak) was dialyzed against PBS. Eap purified in this way was subjected to silver-stained PAGE, to assess its purity. No band other than Eap was detected. The Eap-negative strain Newman AH12 (eap::EryR) [9] was also used for purification of what we here call "pseudo Eap." Fast-protein liquid chromatography (FPLC) fractions corresponding to the third peak from strain Newman mAH12 (eap::EryR) were collected in the same way and were used as controls. This preparation contained trace amounts of protein, which, after extensive concentration (Minicon; Millipore), could be used at a concentration of 4 g/mL, which is far above what could be present in the Eap preparation. Eap was also purified from S. carnosus T300, which carries a plasmid that encodes Eap (provided by M. Hussain, Institute of Medical Microbiology, University of Münster, Münster, Germany).
Antibodies against Eap.
A sheep was immunized intramuscularly with 150 g of Eap in Freund's complete adjuvant. Booster doses were administered 2 and 4 weeks later by use of Freund's incomplete adjuvant. Blood samples were obtained 2 weeks after the last booster. A protein G Sepharose 4 Fast Flow (Pharmacia) was used to obtain IgG by use of the procedure recommended by the manufacturer.
Human antiEap antibodies were purified from polyspecific IgG (IVIG) (Gamimune N 10%; Bayer), which is a pool of human polyspecific IgG intended for therapeutic use. Eap-Sepharose was prepared by coupling 5 g of Eap to 0.5 g of CNBr-activated Sepharose 4B (Pharmacia), according to the procedure recommended by the manufacturer. Two hundred microliters of IVIG stock diluted in 2 mL of PBS was loaded on the Eap-Sepharose. Bound IgG was eluted by adding 0.7% acetic acid and 0.5 mol/L NaCl in water. IgG eluted from the Eap-Sepharose was dialyzed against PBS. The presence of specific antibodies against Eap was determined by use of ELISA.
Reagent.
Purified toxic shock syndrome toxin (TSST)1 was provided by R. Mllby (Karolinska Institutet, Stockholm, Sweden).
Preparation of PBMCs and proliferation assay.
PBMCs were isolated from heparinized blood from healthy donors by Ficoll-Hypaque gradient centrifugation. The PBMCs were cultured in RPMI 1640 medium supplemented with 25 mmol/L HEPES, 4 mmol/L L-glutamine, 100 U/mL penicillin/streptomycin, and 5% heat-inactivated fetal calf serum. PBMCs (2 × 106 cells/mL) were cultured for 72 h, after which they were pulsed for 6 h with 1 Ci/well of [3H]-thymidine (specific activity, 5.0 Ci/mmol; Amersham Pharmacia Biotech). Phytohemmagglutinin-L (PHA; Sigma) was used as a general positive control for stimulation in all experiments, at a concentration of 2 g/mL. All samples were assayed in triplicate, and the data are presented as counts per minute. The experiments were repeated 3 times by use of cells from different individuals. For stimulation of proliferation, PBMCs were cultured with the predetermined optimal concentration of sterile culture supernatant (1 : 50 dilution) or Eap at concentrations of 081 g/mL (final concentration) from S. aureus or recombinant from S. carnosus. Material corresponding to the third FPLC fraction from the Eap-negative strain Newman mAH12 (eap::EryR; pseudo Eap) was also used as a negative control at concentrations of 04 g/mL (final concentration). Sheep antibodies against Eap (0, 62, or 1000 g/mL final concentration) or affinity-purified human IgG against Eap (020 g/mL final concentration) were used in combination with Eap (9 or 81 g/mL). PBMCs were also incubated with different concentrations of TSST-1 from S. aureus (1.25, 2.5, and 5 ng/mL) in the presence of Eap. Different concentrations of pseudo Eap were also added to PBMCs, together with TSST-1 (2.5 ng/mL). PHA (500 ng/mL) was also used to stimulate PBMCs, which were then incubated in combination with different concentrations of Eap (081 g/mL). After 72 h, cells were pulsed for 6 h with 1 Ci/well of [3H]-thymidine (specific activity, 5.0 Ci/mmol).
Flow-cytometric (FACS) analysis of PBMCs.
PBMCs were purified as described for the proliferation assay and were then challenged with different concentrations of Eap (081 g/mL final concentration) from S. aureus or S. carnosus. After 72 h of incubation, cells were analyzed by use of a FACScalibur flow cytometer (Becton Dickinson). The acquisition and processing of data from 15,000 cells was analyzed by use of Quest software (Becton Dickinson). Dot plots of forward scatter (cell size) versus side scatter (internal complexity) were used to assess the viability of the cells. To determine the effect of Eap on individual cells, 3-color flow cytometry was used. Fluorescein isothiocyanate (FITC)conjugated antihuman CD19 (B cells) or CD3 (T cells) (Becton Dickinson Immunocytometry Systems) were added to the cells and incubated on ice for 30 min. For the analysis of apoptosis, cells were further washed once and stained with 5 L of Annexin Vbiotin (Boehringer Mannheim) for 15 min at room temperature, followed by incubation with 1 L of streptavidinallophycocyanin (Becton Dickinson) for 15 min. Finally, 1 L of propidium iodide (PI; Sigma) was added to the cells, and analysis was performed as described above.
Statistical methods.
Unpaired Student's t test and repeated-measures 1-way analysis of variance were used to determine the significance of the results.
RESULTS
Dose-dependent differential effect of Eap on PBMCs.
To assess the effect of Eap on human immune cells, PBMCs were cultured for 72 h in the presence of different concentrations of Eap (081 g/mL). Eap was found to have a stimulatory effect at concentrations of 09 g/mL (P < .05), whereas, at higher concentrations, an inhibitory effect was seen (P < .01) when PBMCs were incubated for 72 h (figure 1). These dose-dependent stimulatory and inhibitory effects were seen regardless of cell proliferation time48, 72, or 96 h.
Effect of antibodies against Eap.
Eap exerts either a stimulatory or inhibitory effect on PBMCs, depending on the concentration used. We set about to determine whether antibodies against Eap could block these effects. PBMCs were incubated with either the stimulatory (9 g/mL) or the inhibitory (81 g/mL) concentration of Eap, together with increasing concentrations of sheep antibodies against Eap. Preimmune sheep IgG was used as negative control, and no blocking effect on Eap was detected; neither was any effect of sheep IgG itself on the PBMCs seen (data not shown). The results showed that the stimulatory effect elicited by Eap at 9 g/mL was significantly blocked (P < .05) by antibodies at 62 g/mL (figure 3A). The inhibitory effect of the higher Eap concentration (81 g/mL) turned into a stimulatory effect by antibodies, presumably because of the reduction in the Eap concentration, rather than complete neutralization (figure 3A). A preparation of human IgG was found to contain antibodies against Eap (data not shown), although at a low level. Such antibodies were purified by affinity chromatography and were found to inhibit the stimulatory effect of Eap on PBMCs at concentrations of 5 (P < .05) and 20 (P < .01) g/mL (figure 3B).
To investigate the effect of Eap on specific cell types, cells were differentially stained for markers of B or T cells. A clear shift from viable cells to apoptotic/necrotic cells was seen in both cell types (figure 5). An increase in Annexin VPIpositive B cells, from 28% to 76%, was demonstrated when Eap concentrations were increased from 0 to 81 g/mL (figure 5A). Similarly, an increase in the frequency of apoptotic T cells, from 6% to 42%, was found at the same Eap concentrations (figure 5B).
DISCUSSION
The present results demonstrate that Eap from S. aureus has a concentration-dependent effect on PBMCs from healthy donors. We were able to demonstrate in a proliferation assay that (1) at low concentrations, Eap elicits a stimulatory effect on PBMCs and, at high concentrations, it has an inhibitory effect through the induction of apoptosis of T and B cells; (2) the inhibitory effect of Eap on PBMCs was efficient enough to inhibit the stimulatory effects of TSST-1 or S. aureus supernatant, as well as PHA; and (3) the effects elicited by Eap could be blocked by antibodies against Eap.
Eap has previously been reported to have several functions that are believed to be important in the pathogenesis of S. aureus infections, including the adherence and internalization of host cells [8, 9], as well as immunomodulatory effects [1315]. The present results identify additional immunomodulatory activities of Eap on human PBMCs and, importantly, show that these activities depend on the concentration of Eap. The immunostimulatory effect of Eap on human PBMCs, here assessed by a proliferation assay, is in agreement with the results of Jahreis et al. [10], who reported the induction of interleukin-2 by p70 (i.e., Eap, in a range of concentrations similar to what we used) resulting in lymphocyte proliferation. Other studies have shown that concentration-related effects are not unusual among S. aureus toxins. The -toxin of S. aureus has been shown to induce apoptosis or necrosis in endothelial cells, depending on the extracellular concentrations of the toxin. Lower concentrations induce apoptosis, whereas higher concentrations induce necrosis, of endothelial cells [19, 20]. Similarly, TSST-1 has also been shown to have different effects on host cells, depending on the concentration. At low concentrations, TSST-1 stimulates immunoglobulin synthesis by PBMCs from healthy subjects, and, at high concentrations, TSST-1 induces B cell apoptosis [21]. Interestingly, several of the concentration-dependent effects are linked to apoptotic and necrotic effects, which are likely to be important events in pathogenesis, considering that S. aureus has evolved several ways to induce apoptosis and necrosis. These methods include the induction of pathologic levels of inflammatory cytokines and intracellular bacterial localization that results in apoptosis [20, 2224], as well as the current finding of the Eap-induced apoptosis of B and T cells.
It will be of major importance to define the receptors involved in the stimulatory and inhibitory effects of Eap. Previous studies have identified a direct effect on anti-inflammatory machinery through the binding of Eap to ICAM-1 and the consequent interference in the T cellmediated responses [1315]. Furthermore, Verdrengh et al. [25] showed, in an in vivo model, that ICAM-1-/- mice developed less-frequent and less-severe arthritis than their wild-type littermates when they were infected with S. aureus. At the same time, Lee et al. [15] showed that Map- (i.e., Eap) bacterial strains less frequently cause arthritis and osteomyelitis. These data indicate that ICAM-1 is a receptor that is involved in some of the responses induced by Eap, but further studies are required to define the receptors involved in the immunostimulatory and inhibitory responses.
Evidence that Eap is expressed in vivo and can challenge the host immune cells was obtained from analyses of the presence of antiEap antibodies in serum, which revealed that patients with ongoing staphylococcal infections had significantly increased acute-phase antibody levels against Eap, compared with those in healthy control subjects (A.H. and J.-I.F., unpublished data). The levels of antibodies against Eap increased even more during the convalescent phase, 1430 days after the onset of disease. In the present study, we were able to show that human IgG against Eap could block the stimulatory effect on PBMCs. We have previously shown that antibodies against Eap (in sheep) were able to block the adherence and internalization of S. aureus into eukaryotic cells [8, 9]. The human antiEap antibodies were purified from IVIG, which has been suggested to be an efficacious adjunctive therapy in staphylococcal and streptococcal toxic shock syndrome [2629]. The main mechanisms of action of IVIG in this setting have been suggested to involve antisuperantigen antibodies and anti-inflammatory effects of IVIG, but we have demonstrated that blockade of the immunomodulatory effects of Eap may be yet another mechanism that contributes to the efficacy of IVIG in the treatment of staphylococcal toxic shock syndrome.
Taken together, the present data suggest that S. aureus virulence factors may have differential effects, depending on bacterial density and the stage of infection. A hypothetical model would be that, at low concentrations, during the early stage of infection (which is characterized by a lower bacterial density), Eap is present at levels that correspond to the stimulatory concentration. This would, together with the action of other bacterial factors recognized by the immune system, result in an expansion and proliferation of human immune cells and, consequently, bacterial clearance. In contrast, during a later stage of infection, when the bacterial population is dense, Eap, along with other secreted staphylococcal products, may accumulate to reach levels in the extracellular environment that are capable of triggering apoptosis and necrosis. This would generate an immunosuppressive state in the host, which would be advantageous to the bacteria and allow the establishment of a long-term persistence and, therefore, relapsing or chronic infections.
Acknowledgment
We thank Nahla Ihendyane for technical advice about the peripheral blood mononuclear cell proliferation assay.
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