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Department of Microbial Pathogenesis and Vaccine Research, GBFGerman Research Center for Biotechnology, Braunschweig, Germany
Natural killer (NK) cells are critical components of the innate immune system and have been implicated in the pathogenesis of septic shock. In the present study, the relative contribution of NK cells to the development of Streptococcus pyogenesinduced septic shock was investigated in a mouse model of group A streptococcal infection that resembles the development of this condition in humans. C3H/HeN mice were depleted of NK cells by in vivo administration of antiasialo ganglio-N-tetraosylceramide antibodies and then were examined for their response to infection with S. pyogenes. NK celldepleted mice exhibited increased survival times and slower development of disease during group A streptococcal infection than did nondepleted control mice. The augmented resistance to S. pyogenes observed in NK celldepleted mice was associated with serum levels of proinflammatory cytokines such as interferon-, interleukin (IL)12, and IL-6 during the early phase of infection that were much lower than those detected in nondepleted control mice. NK celldeficient mutant mice were also more resistant to S. pyogenes than were the corresponding control mice. We conclude that NK cells, by amplifying the inflammatory response, significantly contribute to the progression of S. pyogenesinduced septic shock.
Group A streptococci (Streptococcus pyogenes) are important human pathogens that are responsible for a wide spectrum of human diseases, ranging from mild clinical illnessessuch as pharyngitis, impetigo, scarlet fever, and cellulitisto severe life-threatening diseasessuch as necrotizing fasciitis and streptococcal toxic shock syndrome (STSS) [1]. Since the late 1980s, a marked increase in the incidence of severe streptococcal infections, including STSS, has been reported worldwide [26]. Despite attempts at early diagnosis, the use of antimicrobial treatment, and intensive-care support, the mortality rate associated with STSS remains high [2, 7]. The resurgence of severe streptococcal infections and the associated high mortality rate have renewed interest in understanding the development of STSS and in elucidating the host immune mechanisms involved in these disease processes.
It has become clear that the innate immune system plays an important role in protective immunity against S. pyogenes [8, 9]. Along with macrophages and polymorphonuclear neutrophils, NK cells are critical components of the innate immune response. NK cells are attracted to infected tissue by cytokines and chemokines [10] and can mediate the lysis of autologous infected cells without previous sensitization [1113]. By their production of interferon (IFN), NK cells can also contribute to the immune defense against pathogens [1417].
Through their secretion of high amounts of IFN-, NK cells can be activated during septic shock events and can contribute to the pathogenesis of this reaction [18]. NK cells have also been implicated in mice as important contributors of IFN- during priming of a generalized Shwartzman reaction, which is a lethal cytokine-induced shock response elicited by sequential priming and challenge with bacteria or bacterial components [19, 20]. Thus, the purpose of the present study was to evaluate the contribution of NK cells to the pathogenesis of S. pyogenesinduced septic shock in a previously described mouse model of group A streptococcal infection [8, 21].
MATERIALS AND METHODS
Bacterial strain.
S. pyogenes strain A20 (M type 23), a human isolate obtained from the German Culture Collection (DSM 2071), was used in the present study. Stock cultures were maintained at -70°C and were cultured at 37°C in Todd Hewitt broth (Oxoid) supplemented with 1% yeast extract (THY). Bacteria were collected at the midpoint of the log phase, were washed twice with sterile PBS, and were diluted to the required concentration, and the number of viable bacteria was determined by diluting and plating the bacteria on blood agar plates (GIBCO) containing 5% horse blood and then counting the colony-forming units.
Mice.
C3H/HeN, NIH, and Hsd:NIH-bg-nu-xid female mice, 810 weeks old, were purchased from Harlan-Winkelmann. They were housed in microisolator cages and were given food and water as needed. All experiments were approved by the appropriate ethical board.
Infection model.
A murine model of group A streptococcal infection, described elsewhere, was used [8, 21]. Mice were inoculated with 1 × 105 cfu of S. pyogenes in 0.2 mL of PBS through a lateral tail vein. At selected times after infection, groups of mice (5 mice/group) were killed by CO2 asphyxiation, and bacteria were enumerated in specific organs by plating 10-fold serial dilutions of tissue homogenates on blood agar plates. Colonies were counted after incubation at 37°C for 24 h. Viable bacterial counts in the blood of infected mice were also determined by collecting blood samples from the tail veins at different times after infection and plating serial dilutions on blood agar plates. In some experiments, the survival rate was monitored over time by studying 18 mice/group (NK celldepleted C3H/HeN mice) or 15 mice/group (NK celldeficient Hsd:NIH-bg-nu-xid mice). Each set of experiments was repeated at least 3 times.
NK cell depletion.
Asialo ganglio-N-tetraosylceramide (asialo GM1) is a cell surface component that is expressed on the surface of NK cells at high levels [22]. Rat antiasialo GM1 antibodies (Wako Chemicals) were used to deplete NK cells [23]. C3H/HeN mice were injected intravenously with either 50 g of antiasialo GM1 antibodies or IgG control antibodies (Sigma), 3 and 7 days before infection. The efficiency of depletion was determined by flow-cytometric analysis of spleen cells labeled with fluorescein isothiocyanateconjugated antimouse NKG2D (DX5) antibodies (Bioscience).
Cytokine ELISA.
The serum levels of IFN-, interleukin (IL)6, and IL-12 in NK celldepleted C3H/HeN mice and nondepleted control C3H/HeN mice (3 mice/group/time point) were determined by a specific ELISA using matched antibody pairs (BD Pharmingen) and recombinant cytokines (BD Pharmingen) as standards. Briefly, 96-well microtiter plates were coated with the corresponding purified rat antimouse monoclonal antibodies against IFN-, IL-6, or IL-12 at a concentration of 2 g/mL in sodium bicarbonate buffer and were incubated at 4°C overnight. The wells were washed and then were blocked with 2% bovine serum albumin in PBS before the serum sample and the appropriate standard were added to each well. Biotinylated rat antimouse monoclonal antibodies against IFN-, IL-6, or IL-12 at a concentration of 2 g/mL were used during the second step. Detection was performed using streptavidin peroxidase, and the plates were developed using ABTS. Measurements of the different cytokines were performed in 3 independent experiments.
Clinical chemistry.
The extent of liver damage induced by S. pyogenes infection in NK celldepleted C3H/HeN mice and nondepleted control C3H/HeN mice was also determined by measuring the serum levels of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). Serum levels of GOT and GPT were measured by biochemical assay, in accordance with the manufacturer's instructions (Sigma).
Generation of effector cells.
C3H/HeN mice were infected intravenously with 1 × 105 cfu of S. pyogenes. One day later, spleen cells were isolated from the mice and were treated with a NH4Cl red blood celllysing solution. NK cells were purified by positive selection using miniMACS magnetic anti-NK (DX-5) microbeads, in accordance with the manufacturer's instructions (Miltenyi Biotec), and were used for the cytotoxicity assay.
Cytotoxicity assay.
NK cellsensitive YAC-1 lymphoma cells were used as the target in the cytotoxicity assay. Cells were harvested from an exponential growing culture, were labeled by incubation with 100 Ci of Na251CrCrO4 (Amersham) at 37°C for 1 h, were washed 3 times, and were plated in a U-bottom 96-well cell-culture plate at a density of 2 × 104 cells/well. Different numbers of effector cells were then added in triplicate at effector-to-target (E : T) cell ratios of 60 : 1, 40 : 1, and 20 : 1. After an incubation at 37°C and 5% CO2 for 4 h, 30 L of the supernatant was mixed with 150 L of scintillation liquid in a 96-well gamma-counter plate. The plates were sealed, were shaken for 15 min, and were measured with an automatic gamma counter. The percentage of lysis activity was calculated using the following formula:
where spon. 51Cr release is spontaneous release of 51Cr and max. 51Cr release is maximum release of 51Cr.
Histological examination.
Livers were removed from C3H/HeN mice at 48 h after infection, were fixed with 10% buffered neutral formaline solution, were embedded in paraffin, and were cut into 5-m-thick sections. Sections were stained with azure blue, and the tissue was examined microscopically.
Statistical analysis.
Comparisons between groups were performed by use of the analysis of variance (ANOVA) test. Comparison of survival time curves was performed by use of the Wilcoxon rank sum test. P < .05 was considered to be statistically significant.
RESULTS
Because the beneficial effect of the depletion of NK cells in this model of infection resulted in delayed death, we then investigated whether the depletion of NK cells altered bacterial growth. To test this possibility, we examined the bacterial growth in the livers and spleens of both groups of C3H/HeN mice after infection with 1 × 105 cfu of S. pyogenes. Changes in bacterial growth in the livers and spleens were not significantly different between NK celldepleted C3H/HeN mice and nondepleted control C3H/HeN mice at any time point (data not shown), as demonstrated after calculation of 95% confidence intervals for the differences between mean bacterial counts in the organs of NK celldepleted C3H/HeN mice and nondepleted control C3H/HeN mice (-0.17 to 0.40 for liver at 24 h after infection; -0.65 to 0.56 for spleen at 24 h after infection; -0.02 to 1.00 for liver at 48 h after infection; -0.29 to 0.80 for spleen at 48 h after infection). These data suggest that the absence of NK cells increased the survival time of C3H/HeN mice after infection with S. pyogenes but had no effect on the ability of these mice to inhibit bacterial growth.
The histological findings correlated with the serum levels of GOT and GPT observed in NK celldepleted C3H/HeN mice and nondepleted control C3H/HeN mice. Thus, the serum levels of GOT (figure 4C) and GPT (figure 4D) in NK celldepleted C3H/HeN mice were significantly lower than those in nondepleted control C3H/HeN mice (P < .05, by ANOVA). These results suggest that, during infection with S. pyogenes, NK cells may contribute to liver damage in C3H/HeN mice.
DISCUSSION
In the present study, we have provided evidence that NK cells play an important role in S. pyogenesinduced septic shock in mice. We have shown elsewhere [21] that challenge of C3H/HeN mice with S. pyogenes resulted in an exaggerated systemic inflammatory syndrome that resembled STSS in humans. In the present study, we have shown that depletion of NK cells significantly increased the survival time of susceptible C3H/HeN mice after group A streptococcal infection, which suggests that these cells may potentially contribute to the pathogenesis of S. pyogenesinduced septic shock. Similarly increased survival rates after depletion of NK cells have been also observed in other in vivo models of systemic inflammation [19].
NK cells are important components of the innate immune response and generally contribute to the innate immune defense against pathogens through the release of IFN- before an adaptive immune response can develop [1117]. However, when this defense system is inadequately activated, the uncontrolled production of proinflammatory cytokines causes several pathophysiological reactions, which ultimately lead to septic shock and death [1820].
Our results show that serum levels of IFN- were significantly lower in NK celldepleted C3H/HeN mice than in nondepleted control C3H/HeN mice during the first 48 h after infection. These data indicate that NK cells may be a major source of this cytokine during S. pyogenesinduced septic shock in C3H/HeN mice and are consistent with the findings of recent reports that have shown a central role for NK cells in the production of IFN- during endotoxemia [24].
To date, efforts to modulate the inflammatory response by inhibiting cytokines during sepsis caused by infections with gram-positive bacteria have been unsuccessful [25]. This leads to the conclusion that the mechanisms of STSS may be multifactorial and perhaps are more difficult to target than those involved in other infections. Indeed, our results suggest that STSS might not be caused by a single cell population and that different cellular subsets seem to participate in STSS in a hierarchical manner. In this regard, we suspect that the development of sepsis in this model of infection suggests a positive feedback regulation between cytokines. First, macrophages or dendritic cells are stimulated by bacteria or bacterial components to produce NK cellactivating cytokine (IL-12), then NK cells are activated to produce IFN-, and the presence of IFN- promotes further activation of macrophages. It is well known that IL-12 produced by macrophages or dendritic cells early during the course of infection can strongly activate NK cells and seems particularly potent at inducing production of IFN- [2628]. Furthermore, Miettinen et al. [29] have shown that S. pyogenes can induce secretion of IL-12 by monocytes. The early activation of NK cells may accelerate the influx of inflammatory cells in infected tissues and, thus, organ injury. Our results are consistent with those of previous reports, in which administration of high doses of IL-12 to mice before infection with S. pyogenes induced elevated systemic levels of IFN-, which resulted in increased susceptibility to S. pyogenes and decreased survival rates [30].
Administration of IL-12 has also been reported to enhance natural immune protection against S. pyogenes in the mouse model and, thus, is proposed as a potential strategy for the treatment of infections caused by gram-positive bacteria [31]. However, the results of the present study indicate that it may be prudent to exercise caution when considering the use of IL-12 for immunotherapy of group A streptococcal infections, because this cytokine might affect the course of infection in different ways in different hosts.
IL-6 is an important mediator of the immune response during the acute phase of infection, and the presence of high serum levels of IL-6 at the time of death has been found to correlate inversely with survival time in patients with STSS [32, 33]. Levels of IL-6 were significantly higher in nondepleted control C3H/HeN mice than in NK celldepleted C3H/HeN mice during the first 48 h after infection. Because IL-6 is a cytokine produced by activated macrophages [34], these data further corroborate the involvement of NK cells in promoting macrophage activation. The identification of host cell populations that are critically involved in the development of STSS could be highly valuable for future clinical applications.
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