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Originally published online as doi:10.1189/jlb.0705396 on December 30, 2005

Published online before print December 30, 2005
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(Journal of Leukocyte Biology. 2006;79:463-472.)
© 2006 by Society for Leukocyte Biology

An in vitro Shwartzman reaction-like response is augmented age-dependently in human peripheral blood mononuclear cells

Akira Motegi*, Manabu Kinoshita{dagger}, Kengo Sato*, Nariyoshi Shinomiya{ddagger}, Satoshi Ono§, Shigeaki Nonoyama*, Hoshio Hiraide{dagger} and Shuhji Seki{ddagger},1

* Departments of Pediatrics,
{ddagger} Microbiology, and
§ Surgery 1, National Defense Medical College, and
{dagger} Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa, Saitama, Japan

1 Correspondence: Department of Microbiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, 359-8513, Japan. E-mail: btraums{at}ndmc.ac.jp

ABSTRACT

A lethal human septic shock model, mouse generalized Shwartzman reaction (GSR), was elicited by two consecutive lippolysaccharide (LPS) injections (24 h apart) in which interferon-{gamma} (IFN-{gamma}) induced by interleukin (IL)-12 played a critical role in the priming phase, and tumor necrosis factor (TNF) was an important effector molecule in the second phase. We recently reported IL-12/LPS-induced mouse GSR age-dependently enhanced. We herein demonstrate that human peripheral blood mononuclear cells (PBMC) from healthy adults/elderly, cultured with IL-12 for 24 h and with LPS for an additional 24 h, produced a much larger amount of TNF (which increased age-dependently) than did PBMC without IL-12 priming. Whereas macrophages mainly produced TNF following LPS stimulation, macrophages and lymphocytes were necessary for a sufficient TNF production. IL-12-induced IFN-{gamma} up-regulated Toll-like receptor 4 (TLR-4) on macrophages of adults. Although the PBMC from children produced a substantial amount of IFN-{gamma} after IL-12 priming, the GSR response, with augmented TNF production and an up-regulated TLR-4 expression of macrophages, was not elicited by LPS stimulation. CD56+natural killer cells, CD56+T cells, and CD57+T cells (NK-T cells), which age-dependently increased in PBMC, produced much larger amounts of IFN-{gamma} after IL-12 priming than that of conventional CD56–CD57–T cells and also induced cocultured macrophages to produce TNF by subsequent LPS stimulation. The elder septic patients were consistently more susceptible to lethal shock with enhanced serum TNF levels than the adult patients. The NK cells, NK-T cells, and macrophages, which change proportionally or functionally with aging, might be involved in the enhanced GSR response/septic shock observed in elderly patients.

Key Words: aging • CD56+ T cell • CD57+ T cell • interleukin-12 • LPS

INTRODUCTION

Over the past several years, the mortality of patients from septic shock has not changed significantly [1 2 3 ]. Furthermore, the incidence of gram-negative bacterial infections has conversely increased in recent years, and these infections often result in septic shock caused by lipopolysaccharide (LPS; endotoxin) [1 2 3 ]. To develop an effective therapy for septic shock, many studies have been performed vigorously using experimental animals. The generalized Shwartzman reaction (GSR) in animals is one of the most well-known and useful septic shock models induced by LPS [4 5 6 7 8 9 ]. Historically, Shwartzman first described a local tissue reaction in rabbits elicited by a subcutaneous injection of LPS, followed by an intravenous (i.v.) injection 24 h later, resulting in the necrosis of the tissue, which is now known as the localized Shwartzman reaction (LSR) [10 ]. The GSR, which causes disseminated vascular coagulation (DIC) and death, was also initially elicited in rabbits by two i.v. injections of LPS 24 h apart, and individual LPS injection is not lethal per se [11 ]. Many clinical case reports have suggested the occurrence of the GSR/LSR following gram-negative septicemia in humans [12 13 14 15 16 17 18 19 20 21 22 ]. In addition, a univisceral LSR is suggested to be involved in acute/early graft rejection [21 , 22 ], and this remains a serious problem in transplantation [23 24 25 ]. A small amount of LPS (2–4 ng/kg) administered i.v. to human volunteers reportedly provokes mild but variable responses characteristic of gram-negative sepsis in humans, although the LPS dose is at least 100 times less than that given to experimental animals [26 27 28 29 ]. The response of humans to LPS thus appears to be much more sensitive than that of animals, and the GSR/LSR may possibly cause a more dramatic effect in humans. Nevertheless, little information and evidence regarding the mechanisms of human GSR/LSR have been reported so far, as an appropriate experimental model has not yet been established for humans.

However, the detailed mechanisms of the Shwartzman reaction, including the cytokine milieu and related lymphocyte subsets, have been explored recently in mice [4 5 6 7 8 ]. An interleukin (IL)-12 injection or interferon-{gamma} (IFN-{gamma}) induced by IL-12 was thus found to play an important role in the first LPS priming in the GSR in mice, and tumor necrosis factor (TNF) is a final effector molecule in the lethality of mice, which have already been sensitized by IFN-{gamma}. We have demonstrated that natural killer (NK)T cells and their IFN-{gamma} production are involved in the mouse GSR induced by IL-12 and subsequent LPS challenge [8 ]. Furthermore, the CD8+ CD122+ T cells with a potent capacity to produce IFN-{gamma}, which increases with age, also play a crucial role in the age-dependent enhancement of mouse GSR [30 ]. In humans, in addition to CD56+ NK cells, T cells with NK cell markers, namely, CD56+T cells and CD57+T cells (NK-T cells), have a potent capacity to produce IFN-{gamma}, and they also increase age-dependently. We proposed these NK-T cells to be functional counterparts of mouse NK-T cells and CD8+ CD122+ T cells, respectively [31 ]. These T cells in mice and humans have intermediate intensities of T cell receptor (TCR), which are lower than those of conventional T cells [32 , 33 ]. These findings led us to hypothesize that these cells in human peripheral blood mononuclear cells (PBMC) may also be closely involved in the age-related susceptibility to septic shock/GSR [31 , 33 ].

In the present study, we reproduce a GSR-like response (GSR response) in vitro using human PBMC and thereby demonstrate that a mechanism essentially identical to that of the mouse model may underlie the GSR response, thus providing additional validity that GSR/LSR can indeed take place in patients with certain diseases and conditions. The results may also provide the plausible mechanism by which elderly patients tend to be more susceptible to septic shock than younger patients.

MATERIALS AND METHODS

Peripheral blood samples from healthy volunteers
Peripheral blood samples were obtained from healthy adult volunteers ranging from 20 to 40 years of age (mean, 35 years). To examine the effects of aging on the GSR response, blood samples were also obtained from elderly volunteers, aged 70 years or older, and child volunteers, aged 7 years or younger, without any significant clinical features and who visited the out-patient clinic of the National Defense Medical College Hospital (Japan) and the collaborating clinics for routine examinations. We obtained informed consent from all volunteers or their parental authorities and also received approval from the ethical committee of our medical school.

Peripheral blood samples from elderly or adult septic patients
Blood samples were also obtained from 38 septic patients who had been transferred to our surgical intensive care unit (ICU) in the National Defense Medical College Hospital as a result of intra-abdominal infections. All patients were diagnosed to have gram-negative infections by a culture examination. They were divided into two groups according to their ages: adults (<70) and elderly (≥70). No differences in the patient backgrounds such as gender or causative disease were observed between the adult and elderly patient groups (see Table 2 ). For the diagnosis of sepsis and septic shock, the criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference were used [34 ]. Blood samples were obtained from the patients at the time of admission to our surgical ICU, i.e., within a few hours after sepsis diagnoses in all cases. The sera or plasma samples were stored at –80°C to measure the serum TNF or plasma endotoxin levels. We also obtained informed consent from all patients or their parental authorities and received approval from the ethical committee of our medical school.


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  		Table 2. Comparison of the Serum TNF Levels and the Incidence of Septic Shock between Adult and Elderly Patients with the Intra-Abdominal Infections

 
Reagents
LPS (Escherichia coli 0111:B4) was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human IL-12 and IFN-{gamma} were purchased from PeptroTech EC (London, UK). Anti-IFN-{gamma} antibody was purchased from R&D Systems (Minneapolis, MN).

Isolation of PBMC and the separation of adherent macrophages and nonadherent lymphocytes
PBMC were obtained from the peripheral blood using a lymphocyte separation medium (ICN Biomedicals Inc., Aurora, OH). To separate the macrophages and lymphocytes, the PBMC were suspended at 1 x 106/ml in RPMI 1640 containing 20% human serum and then were cultured on collagen-coated plastic plates for 2 h at 37°C in 5% CO2. Thereafter, lymphocytes were obtained by gentle pipetting to collect nonadherent cells, and subsequently, adherent macrophages were obtained with a cell scraper.

Induction of GSR response by IL-12 or IFN-{gamma} priming and subsequent LPS stimulation
After isolation, 2 x 105 cells in 200 µl RPMI 1640 containing 20% human serum were cultured with priming reagents, 20 ng/ml IL-12 (vol 5 µl) or control phosphate-buffered saline (PBS; 5 µl), in a 96-well flat-bottomed plate for 24 h at 37°C in 5% CO2. In our previous study [31 , 35 ], we confirmed that this concentration of IL-12 is appropriate to stimulate/prime the human lymphocytes. The cells were also cultured with 5 ng/ml IFN-{gamma} for priming. Subsequently, the cells were cultured with LPS (10 ng/ml) for 24 h, and then the supernatants were harvested and maintained at –80°C for enzyme-linked immunosorbent assay (ELISA).

Neutralization of IFN-{gamma} in the priming phase
To neutralize the IFN-{gamma} induced by IL-12 priming, PBMC were preincubated with 10 mg/ml anti-IFN-{gamma} antibody immediately before the priming stimulation by IL-12. PBMC were also preincubated with 10 mg/ml mouse immunoglobulin G2a (IgG2a; R&D Systems) as an isotype control.

Analysis of Toll-like receptor-4 (TLR-4) expression on the adherent macrophages
Adherent macrophages were separated from PBMC by the method mentioned above and were stained with fluorescein isothiocyanate (FITC) anti-TLR-4 monoclonal antibody (mAb; Serotec Ltd., Oxford, UK). FITC-conjugated mouse IgG2a (Caltag Laboratories, Burlingame, CA) was used as an isotype control. The TLR-4 expression was analyzed using a flow cytometric analyzer (EPICS XL, Beckman Coulter, Miami, FL).

Assays for IFN-{gamma} and TNF levels
The IFN-{gamma} and TNF levels of the culture supernatants and the serum TNF levels in the septic patients were measured by cytokine-specific ELISA kits (OptEIATM, PharMingen, San Diego, CA).

Determination of endotoxin levels in the septic patients
The blood samples of the septic patients were separately obtained by venipuncture into sterilized, pyrogen-free glass tubes containing disodium EDTA for the determination of endotoxin. The plasma endotoxin level was determined by a chromogenic limulus amebocyte assay (Endospec-SP test, Seikagaku Kogyo, Tokyo, Japan) [36 ]. Briefly, plasma sample (0.1 mL) was diluted 1:2 with 0.32 mol/L perchloric acid at 37°C for 20 min to remove interfering factors and then centrifuged at 4000 g for 10 min. The supernatant (25 µL) was pipetted into each well on a microtiter plate and neutralized with 25 µL 0.18 mol/L NaOH. The sample was then added to 50 µL limulus coagulation factors specific to endotoxin and dissolved in 0.2 mol/L Tris-HCL buffer, pH 8.0. The mixture was incubated at 37°C for 30 min. Absorbance was measured using an automatic reader at 545 nm after diazotization to avoid interference from bilirubin and other yellow pigments, and sensitivity was enhanced by addition of 50 µL of each of the following reagents: 0.04% (w/v) sodium nitrite in 0.48 nmol/L HCl, 0.3% (w/v) ammonium sulfate, and 0.07% (w/v) N-1-naphthylethylene-diamine dihydrochloride. The standard curve was plotted using E. coli 0111:B4 endotoxin.

Flow cytometric analysis and cell sorting
PBMC were obtained from children, adults, and elderly individuals and then were stained with FITC anti-CD57 mAb (Beckman Coulter), phycoerythrin (PE) anti-{alpha}ß TCR mAb (Beckman Coulter), and PC5 anti-CD56 mAb (Beckman Coulter). The surface phenotypes of the PBMC were analyzed using a flow cytometric analyzer (EPICS XL, Beckman Coulter).

To deplete the CD56+ cells or CD57+ cells, whole PBMC were incubated with magnetic bead-conjugated anti-CD56 mAb (Dynal A.S., Oslo, Norway) or magnetic bead-conjugated anti-PE mAb (Dynal A.S.) with PE anti-CD57 mAb (Immunotech, Marseille, France), respectively, and then the CD56+ cells or CD57+ cells were sorted out by magnetic cell sorter (MACS; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). To obtain CD56+ NK cells, CD56+ T cells, or CD57+ T cells, whole PBMC were stained with FITC anti-CD57 mAb, PE anti-{alpha}ß TCR mAb, and PC5 anti-CD56 mAb, and then CD56+ NK cells (CD56+, {alpha}ßTCR– cells), CD56+ T cells (CD56+, {alpha}ßTCR+ cells), and CD57+ T cells (CD56–CD57+, {alpha}ßTCR+ cells) were purified by EPICS Altra (Beckman Coulter; purity of each population was more than 95%).

Statistical analysis
Differences amongst the groups were analyzed by the Mann-Whitney U-test or an ANOVA analysis with Fisher’s protected least significant difference using the StatView 5 software package (SAS Institute Inc., Cary, NC). Differences were considered to be significant at a value of P < 0.05.

RESULTS

IL-12 priming and subsequent LPS stimulation induced human PBMC to enhance TNF production, thus suggesting the presence of a GSR response
IL-12 priming and a subsequent LPS challenge elicited the GSR in mice. The production of IFN-{gamma} after IL-12 to priming and TNF after LPS challenge is crucial for GSR [6 , 8 , 30 ]. We thereby cultured PBMC from healthy adult volunteers with IL-12 (20 ng/ml) to prime the incubation and then examined their IFN-{gamma} production, which from PBMC, increased remarkably until 24 h and then reached a plateau at 36 h after the priming incubation with IL-12 (Fig. 1A ). Therefore, PBMC were cultured with IL-12 for 24 h and with LPS (10 ng/ml) for a subsequent 24 h and examined their TNF production, which is an effector molecule in the second phase of the GSR [6 , 8 , 30 ]. The control PBMC were also primed with PBS and subsequently cultured with LPS (10 ng/ml). Although both primed PBMC showed peaks at 24 h after LPS stimulation, the IL-12-primed PBMC remarkably enhanced TNF production more than those of the PBS-primed PBMC (Fig. 1B) , thus suggesting the occurrence of a GSR response as assessed by an enhanced TNF production. To identify the most effective dose of LPS to enhance TNF production, PBMC were stimulated with 1, 10, and 100 ng/ml LPS for 24 h after priming by IL-12. Stimulation with 10 ng/ml LPS induced a significantly higher TNF production from the IL-12-primed PBMC than the stimulation with 1 ng/ml LPS and showed similar TNF production from the primed PBMC stimulated with 100 ng/ml LPS (Fig. 1C) . We thereby cultured PBMC with IL-12 (20 ng/ml) for 24 h and with LPS (10 ng/ml) for a subsequent 24 h to induce the GSR response in the following experiments.


Figure 1
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  		Figure 1. A time-course analysis of the cytokine production from PBMC by IL-12 priming and subsequent LPS stimulation. (A) IFN-{gamma} production from PBMC after IL-12 priming. PBMC from healthy adult volunteers were cultured with IL-12 (20 ng/ml) to measure the IFN-{gamma} levels in the supernatant at the indicated time. (B) TNF production from the IL-12-primed PBMC after subsequent LPS stimulation. After priming incubation by IL-12 (20 ng/ml) or control PBS for 24 h, the PBMC were cultured with LPS (10 ng/ml) for 24 h to measure the TNF levels in the supernatant at the indicated time. (C) Relationship between the concentration of LPS and the enhancement of TNF production from IL-12-primed PBMC. After priming incubation by IL-12 or PBS for 24 h, the PBMC were cultured with 1, 10, or 100 ng/ml LPS for 24 h to measure the TNF levels in the supernatant. The data are the means ± SE from five independent experiments. *, P < 0.01.

 
Adherent macrophages and nonadherent lymphocytes played a crucial role in the GSR response, and adherent macrophages produced TNF after LPS stimulation
To examine the participation of macrophages and lymphocytes in the human GSR response, plastic adherent macrophages, nonadherent lymphocytes, and whole PBMC were obtained from healthy adults and were then cultured with IL-12 for 24 h and with LPS for a subsequent 24 h. Neither the macrophages nor the lymphocytes produced as much TNF by themselves as did whole PBMC (Fig. 2A ), thus suggesting that the macrophages and lymphocytes play an indispensable role in the induction of the GSR response. We mixed the plastic adherent macrophages and the nonadherent lymphocytes again and confirmed that the IL-12/LPS-induced TNF production of those re-mixed cells did not differ from that of whole PBMC (data not shown). To examine which cells produce TNF after LPS stimulation in this reaction, adherent macrophages and nonadherent lymphocytes were separated from whole PBMC at 24 h after the priming by IL-12 or PBS, and those separated cells were subsequently cultured with LPS for 24 h, respectively. The adherent macrophages from IL-12-primed PBMC produced a remarkably larger amount of TNF than the other cell groups (Fig. 2B) , thus suggesting that macrophages mainly produce TNF following LPS stimulation in the second phase. To examine the role of IFN-{gamma} on the enhanced TNF production, we next cultured whole PBMC, adherent macrophages, and nonadherent lymphocytes with IFN-{gamma} (5 ng/ml) and subsequently stimulated with LPS. IFN-{gamma} priming and LPS stimulation induced adherent macrophages as well as whole PBMC to produce large amounts of TNF, although they did not cause the nonadherent lymphocytes to enhance TNF production (Fig. 2C) , suggesting that macrophages primed by IFN-{gamma} but not IL-12 enhance LPS-induced TNF production, and IFN-{gamma} produced by IL-12-primed lymphocytes is important for macrophages to enhance TNF production.


Figure 2
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  		Figure 2. The role of adherent macrophages and nonadherent lymphocytes in the GSR response. (A) TNF production from whole PBMC, adherent cells, and nonadherent cells by the IL-12 priming and subsequent LPS stimulation. The cells were cultured with IL-12 (20 ng/ml) for 24 h and with LPS (10 ng/ml) for an additional 24 h to measure TNF production. (B) TNF production from adherent macrophages and nonadherent lymphocytes after LPS stimulation. After priming incubation by IL-12 (20 ng/ml) or PBS for 24 h, whole PBMC were separated to plastic-adherent macrophages and nonadherent lymphocytes. Subsequently, each cell group was cultured with LPS (10 ng/ml) for 24 h to measure TNF production. (C) TNF production from whole PBMC, adherent cells, and nonadherent cells by the IFN-{gamma} priming and subsequent LPS stimulation. The cells were cultured with IFN-{gamma} (5 ng/ml) for 24 h and with LPS (10 ng/ml) for an additional 24 h to measure TNF production. The data are the means ± SE from five independent experiments.*, P < 0.01, versus other groups.

 
IL-12-induced IFN-{gamma} production and following an up-regulation of TLR-4 expression, played a crucial role in the GSR response
To further confirm the importance of IFN-{gamma} in the priming phase of the GSR response, PBMC were preincubated with anti-IFN-{gamma} antibody or mouse IgG2a (isotype control) followed by IL-12 priming. Thereafter, the neutralization of IFN-{gamma} in the priming phase was found to inhibit the enhanced TNF production almost completely from PBMC after LPS stimulation (Fig. 3A ). As TLR-4 is a cell surface receptor for LPS to transmit its signaling [37 38 39 40 ], the TLR-4 expression on adherent macrophages was examined after IL-12 priming, which remarkably up-regulated the TLR-4 expression on the macrophages (Fig. 3B 3a) , and the neutralization of IFN-{gamma} in the IL-12 priming phase suppressed its expression (Fig. 3B 3b) . IFN-{gamma} priming also up-regulated the TLR-4 expression on the macrophages (Fig. 3B 3c) , thus suggesting that IL-12 priming induces IFN-{gamma}, and in addition, IFN-{gamma} up-regulates the TLR-4 expression in the TNF-producing macrophages.


Figure 3
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  		Figure 3. The roles of IFN-{gamma} induced by IL-12 priming in the GSR response. (A) The effect of neutralization of IFN-{gamma} on TNF production in the GSR response. After preincubation with anti-IFN-{gamma} antibody (Ab) or mouse IgG2a, PBMC were cultured with IL-12 or PBS for 24 h. Subsequently, each cell group was cultured with LPS for 24 h to measure the TNF production. The data are the means ± SE from five independent experiments. *, P < 0.05, versus other groups. (B) TLR-4 expression on adherent macrophages after IL-12 or IFN-{gamma} priming and the effect of neutralization of IFN-{gamma} on the IL-12-induced TLR-4 expression. PBMC were cultured with IL-12 (a), IL-12 + anti-IFN-{gamma} antibody (b), IFN-{gamma} (c), or PBS (d) for 24 h, and then TLR-4 expression on adherent macrophages was examined. The fluorescence intensity of TLR-4 expression was shown as a solid line, and the intensity of isotype control (mouse IgG2a) was shown as the shaded area. The representative data of five independent experiments are shown.

 
The GSR response was augmented in the PBMC from elderly individuals but not in the PBMC from children
The GSR is age-dependently augmented in mice [30 ]. We thereby examined the effect of aging on the human GSR response. PBMC were obtained from children, adults, and elderly volunteers and then were cultured with IL-12 priming and subsequent LPS stimulation. It is surprising that IL-12 priming did not enhance the TNF production by the LPS stimulation in the PBMC from children, but it was significantly enhanced in the PBMC from adult and elderly volunteers (Fig. 4 ). Furthermore, this enhanced TNF production was augmented significantly in the PBMC from elderly volunteers compared with that from adult volunteers (Fig. 4) , thus suggesting the presence of an age-dependent augmentation of the human GSR response. However, this age-dependent augmentation of the TNF production from PBMC was not observed by a single LPS stimulation without IL-12 priming (Fig. 4) .


Figure 4
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  		Figure 4. TNF production from PBMC in the GSR response of each age group. PBMC were obtained from children (n=7), adults (n=10), and elderly individuals (n=6). They were cultured with PBS or IL-12 (20 ng/ml) for 24 h and with LPS (10 ng/ml) for a subsequent 24 h, and then their TNF productions were measured. The data are the means ± SE; *, P < 0.05;**, P < 0.01.

 
Age-dependently increasing CD56+NK cells, CD56+T cells, and CD57+T cells in PBMC played a crucial role in the GSR response
In our previous study [31 ], we demonstrated that CD57+T cells (CD57+, {alpha}ßTCR+ cells), CD56+T cells (CD56+, {alpha}ßTCR+ cells), and CD56+NK cells (CD56+, {alpha}ßTCR– cells) increase with age in humans. Furthermore, these age-dependently, increasing cells also have a high potential to produce IFN-{gamma}. In the present study, we confirmed that these cells were significantly less in the PBMC from children than in the PBMC from adults and elderly individuals (Table 1 ). Nonadherent lymphocytes and their IFN-{gamma} played a crucial role in inducing the GSR response. Conversely, the GSR response was enhanced age-dependently. We thereby examined the participation of these age-dependently developed cells in the GSR response. CD56+ cells or CD57+ cells were sorted out from the PBMC of healthy adults using MACS. Control, whole PBMC were only run through the column of MACS. The cells were then cultured with IL-12 priming and subsequent LPS stimulation. The depletion of not only CD56+ cells but also CD57+ cells significantly decreased the TNF production in comparison with that of whole PBMC (Fig. 5A ). As NK, NK-T, and T cells are the main producer of IFN-{gamma}, which play a crucial role in the priming phase of the GSR response, CD56+NK cells, CD56+T cells, CD57+T cells, and CD56–CD57–T cells were sorted from the PBMC of adults using the cell sorter. Whole lymphocytes were also run through a cell sorter column as a control. Thereafter, these sorted cells and whole lymphocytes were cultured with IL-12 for 24 h to examine their IFN-{gamma} productions by IL-12 priming. CD56+NK cells, CD56+T cells, and CD57+T cells produced as much an amount of IFN-{gamma} after IL-12 priming as the whole lymphocytes produced, whereas they also significantly produced a larger amount of IFN-{gamma} than those of CD56–CD57–T cells (Fig. 5B) . Furthermore, the CD56+NK cells, CD56+T cells, CD57+T cells, CD56–CD57–T cells, and whole lymphocytes were cocultured with adherent macrophages under the stimulus of IL-12 for 24 h and subsequently under the stimulus of LPS for an additional 24 h to examine TNF production. In line with the IFN-{gamma} production in the priming phase, the combination culture of macrophages with CD56+NK cells, CD56+T cells, and CD57+T cells produced as much TNF as the combination culture with whole lymphocytes produced, whereas they significantly produced a larger amount of TNF than that produced by combination culture with CD56–CD57–T cells (Fig. 5C) .


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  		Table 1. The Effects of Aging on the Proportion (%) of CD56+NK, CD56+T, and CD57+T Cells in the Human PBMC

 

Figure 5
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  		Figure 5. The roles of CD56+NK cells, CD56+T cells, and CD57+T cells in the GSR response. (A) The effect of a depletion of CD56+ cells or CD57+ cells on the TNF production in the GSR response. CD56+ cells or CD57+ cells were sorted out from the healthy adult PBMC using MACS. These sorted cells and whole PBMC were then cultured with IL-12 (20 ng/ml) for 24 h and with LPS (10 ng/ml) for a subsequent 24 h to measure TNF production. The data are the means ± SE from five independent experiments. *, P < 0.05, versus whole PBMC. (B) IFN-{gamma} production from CD56–CD57–T cells, CD56+NK cells, CD56+T cells, and CD57+T cells by IL-12 priming. Each lymphocyte subset was purified by a cell sorter and then was cultured with IL-12 (20 ng/ml) for 24 h. The data are the means ± SE from five independent experiments. *, P < 0.05, versus CD56–CD57–T cells. (C) TNF production in coculture with adherent macrophages and the following lymphocytes: CD56–CD57–T cells, CD56+NK cells, CD56+T cells, CD57+T cells, or whole lymphocytes. Each lymphocyte subset purified by the cell sorter was cocultured with adherent macrophages, and those cocultured cells were stimulated by LPS (10 ng/ml) for 24 h after IL-12 priming. The data are the means ± SE from five independent experiments. *, P < 0.05, versus CD56–CD57–T cells.

 
Macrophages from children did not up-regulate the TLR-4 expression after IL-12 priming or enhance the TNF production after subsequent LPS stimulation
As the PBMC from children did not induce the GSR response, we examined IL-12-induced IFN-{gamma} in the PBMC of children. Although it was not significant, the PBMC from children tended to show a lower IFN-{gamma} production after IL-12 priming than that of the PBMC from adults and elderly individuals (Fig. 6A ). The TLR-4 expression of macrophages after IL-12 priming was also examined in the children. After IL-12 or PBS priming, the macrophages were separated from the primed PBMC of children. The macrophages from children did not up-regulate the TLR-4 expression by IL-12 priming as much as those by the control PBS priming (Fig. 6B) . In addition, we examined the TNF production after LPS stimulation in the macrophages that were separated from the IL-12- or PBS-primed PBMC of children. In line with the TLR-4 expression, macrophages from IL-12-primed PBMC of children did not enhance the TNF production following LPS stimulation as much as that from the PBS-primed PBMC (Fig. 6C) .


Figure 6
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  		Figure 6. GSR response was not induced in the PBMC from children. (A) IFN-{gamma} production of the PBMC after IL-12 priming in each age group. PBMC obtained from children (n=7), adults (n=10), and elderly individuals (n=6) were cultured with IL-12 (20 ng/ml) for 24 h. The data are the means ± SE. (B) TLR-4 expression on the adherent macrophages after IL-12 priming in the PBMC of children. PBMC from children were stimulated with IL-12 (20 ng/ml) or PBS for 24 h, and then the TLR-4 expression on the adherent macrophages was examined. The fluorescence intensity of TLR-4 expression was shown as a solid line, and the intensity of isotype control (mouse IgG2a) was shown as a shaded area. The representative data of five independent experiments are shown. (C) TNF production by the LPS stimulation in the adherent macrophages separated from IL-12-primed PBMC of children. After culturing with IL-12 (20 ng/ml) or PBS for 24 h, whole PBMC from children were separated into adherent macrophages and nonadherent lymphocytes. Subsequently, the adherent macrophages were cultured with LPS (10 ng/ml) for 24 h. The data are the means ± SE from five independent experiments.

 
Elderly patients with intra-abdominal infections were susceptible to septic shock accompanied with enhanced serum TNF levels
We examined the serum TNF levels and the incidence of septic shock in elderly and adult patients with the intra-abdominal infections. As expected, the elderly patients were remarkably more susceptible to septic shock and also showed a higher shock-induced lethality in comparison with the adult patients (Table 2 ). It is interesting that the elderly patients showed significantly higher serum TNF levels than the adult patients, although both patient groups showed similar plasma endotoxin levels (Table 2) , thus suggesting an enhancement of the endotoxin-induced TNF production in the elderly patients.

DISCUSSION

We found that the GSR response, which is characterized by an enhanced TNF production, elicited by an IL-12 priming and subsequent LPS stimulation, can be induced in human PBMC in vitro. Although the macrophages mainly produced TNF following LPS stimulation, macrophages and nonadherent lymphocytes were necessary to obtain a sufficient TNF production. IL-12-induced IFN-{gamma} in the priming phase and IFN-{gamma}-induced up-regulation of TLR-4 expression on the macrophages may thus play a crucial role in the GSR response. Furthermore, the GSR response was age-dependently augmented in human PBMC, and it was not elicited in the PBMC from children. NK cells and NK-T cells, which increased in the PBMC age-dependently, produced large amounts of IFN-{gamma} by the IL-12 priming and induced cocultured macrophages to enhance TNF production by the following LPS stimulation, thus suggesting that these cells play a crucial role in the induction of human GSR/LSR. Despite no difference in the plasma endotoxin levels, the elderly septic patients with intra-abdominal infections were remarkably susceptible to lethal shock and also showed significantly higher serum TNF levels than those of the adult patients, thus suggesting the occurrence of human GSR.

As the bilateral renal cortical necrosis in the presence of DIC is a typical, pathological change of the GSR in animals [11 ], early reports suggested that the GSR also occurs in humans as a result of the presence of these pathologic findings, even when neither any evident priming stimulation nor any subsequent endotoxic insult was found in the patients [12 , 15 16 17 , 41 ]. Conversely, several case reports demonstrated that GSR was suspected in the patients with endotoxin shock as a result of gram-negative bacteria following an abortion, which demonstrated DIC and renal cortical necrosis, thus suggesting that the abortion and/or its clinical procedure can be a priming stimulation preceding endotoxemia and thus cause GSR [41 ]. Furthermore, a previous report in which renal cortical necrosis was induced in pregnant rabbits by a single injection of LPS alone also suggests that a less severe immunological insult can thus be a priming factor for GSR. Other reports have also demonstrated that the LSR was involved in severe enterocolitis, e.g., necrotizing enteritis or ulcerative colitis [18 19 20 ]. The gastrointestinal tract is continuously exposed to bacterial LPS, which is absorbed by the intestinal mucosa and detoxicated in the liver [42 43 44 ]. Bacterial LPS derived from E. coli and other gram-negative bacteria may possibly prime the intestinal mucosa and the intraepithelial lymphocytes of the intestine.

In line with the findings of mouse GSR, human NK and NK-T cells play an important role in the priming phase for the induction of GSR response, as these cells produced large amounts of IFN-{gamma} as a result of IL-12 priming, and IFN-{gamma} elicited an enhanced TNF production from macrophages after LPS stimulation via the up-regulation of their TLR-4 expression. In our recent study, lethal GSR does not occur in young mice, but it does occur in middle-aged and elderly mice [30 ]. CD8+CD122+T cells increase in number in line with the mouse age, and they are also potent IFN-{gamma} producers [33 ]; thereby, these cells cause an age-dependent enhancement of the GSR in mice [30 ]. In the present study, PBMC from children did not induce the GSR response either. Human NK cells and NK-T cells increase in number with aging, and these cells also have high potential to produce IFN-{gamma} [31 ]. In the present study, the macrophages from children did not up-regulate the TLR-4 expression by the IL-12 priming, and thereby, these macrophages might not enhance TNF production by any subsequent LPS stimulation. The IFN-{gamma} production after IL-12 priming tended to be low in the PBMC from children in comparison with that in the PBMC from adults or elders. Therefore, macrophages as well as NK cells, and NK-T cells play a crucial role in the age-dependent enhancement of the human GSR response.

Burke et al. [21 , 22 ] reported that an elevation of the serum IFN-{gamma} and the subsequent elevation of serum TNF are characteristic findings in acute rejection in liver or kidney transplantation, thus suggesting that a univisceral LSR is involved in acute rejection. It is empirically known that child donors and recipients show a better graft acceptance in terms of acute rejections, in which LSR might be involved, and there have been no case reports so far suggesting the occurrence of GSR in children. It is interesting that age-dependently increased human NK and NK-T cells are relatively abundant in the liver and bone marrow in comparison with the peripheral blood, lymph nodes, or spleen in mice and humans [45 46 47 48 ]. In fact, mouse NK cells and NK-T cells are reportedly involved in the acute rejection in bone marrow transplantation [49 , 50 ]. Furthermore, the amounts of IFN-{gamma} produced in a one-way mixed lymphocyte reaction of human PBMC, which is one of the useful predictors of graft-versus-host disease (GVHD) [51 , 52 ], correlated well with age as well as the proportion of CD57+T cells in responder PBMC [52 ]. These findings thus suggest that NK cells and NK-T cells are indeed involved in the rejection of transplanted organs and acute GVHD.

It is noteworthy that the elderly septic patients with an intra-abdominal infection showed significantly higher serum TNF levels than the adult patients, despite no difference in the plasma endotoxin levels. Furthermore, these elderly septic patients were susceptible to lethal shock, thus suggesting the occurrence of GSR in the elderly. Conversely, the TNF production of the PBMC from the elderly volunteers was not enhanced by a single LPS stimulation without IL-12 priming. In the cases of intra-abdominal infections, the patients/lymphocytes may therefore already be primed by the primary diseases before the insult of a massive dose of endotoxin (LPS) in the peritoneal cavity.

However, the possibility has been raised that the occurrence of lethal GSR in mammals may reflect an aspect of the mature immune function in adults. In fact, TLR-4-deficient mice are highly resistant to endotoxin shock but are conversely extremely susceptible to bacterial infections themselves [53 ]. We also recently found that most young mice (4 weeks and 6 weeks old) survive IL-12/LPS-induced GSR, whereas a substantial proportion of mice from 8 weeks to 20 weeks of ages, which are considered to be mature and physiologically most immunocompetent, died from IL-12/LPS-induced GSR [30 ]. The macrophages, NK cells, NK-T cells, and cytokines, produced by them, play an important role in the protection against infections. Therefore, the absence of a GSR response in child PBMC may also partly reflect a lack of maturity in their immune function. In addition to the levels of TNF production from macrophages, the response/sensitivity to the TNF of the tissues in elderly mammals should be evaluated and investigated regarding the enhancement of the GSR and lethality of them. Finally, it should be noted that human and mouse NK cells and NKT cells are involved in the IFN-{gamma} production after the superantigen stimulation of Staphylococcus aureus [54 , 55 ], and superantigen-activated human NK cells and NK-T cells but not conventional T cells had a cytotoxic effect on human umbilical vein endothelial cells [55 ]. These findings suggest that NK cells and NKT cells play a role in the GSR induced by gram-positive and gram-negative bacteria infections, and these cells, as well as macrophages, should thus be considered as potential targets in developing future therapeutic strategies.

ACKNOWLEDGEMENTS

This work was supported in part by a grant-in-aid for Special Research Program (Host Stress Responses to Internal and External Factors) from the National Defense Medical College to N. S. and S. S.

Received July 20, 2005; revised October 14, 2005; accepted November 15, 2005.

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