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. 2005 Dec;142(3):419–425. doi: 10.1111/j.1365-2249.2005.02944.x

Immunological control of methicillin-resistant Staphylococcus aureus (MRSA) infection in an immunodeficient murine model of thermal injuries

T Katakura *, T Yoshida *, M Kobayashi *,†, D N Herndon †, F Suzuki *,†
PMCID: PMC1809536  PMID: 16297152

Abstract

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is a major cause of sepsis in patients who are immunosuppressed by their burns. In this study, an immunological regulation of MRSA infection was attempted in a mouse model of thermal injury. SCIDbg mice were resistant to MRSA infection, while SCIDbgMN mice (SCIDbg mice depleted of neutrophils and macrophages (Mφ)) were susceptible to the same infection. Also, thermally injured SCIDbg mice were shown to be susceptible to MRSA infection. On the other hand, the resistance of SCIDbgMN mice to the infection was completely recovered after an inoculation with Mφ from normal mice. However, anti-MRSA resistance was not shown in SCIDbgMN mice inoculated with Mφ from thermally injured mice. Mφ from MRSA-infected thermally injured mice were identified as alternatively activated Mφ, and Mφ from MRSA-infected unburned mice were characterized as classically activated Mφ. Mφ from thermally injured SCIDbg mice previously treated with 2-carboxyethylgermanium sesquioxide (Ge-132) protected SCIDbgMN mice against MRSA infection. Ge-132 has been described as an inhibitor of alternatively activated Mφ generation. These results suggest that MRSA infection in thermally injured patients is controlled immunologically through the induction of anti-MRSA effector cells and elimination of burn-associated alternatively activated Mφ, which are cells that inhibit the generation of classically activated Mφ.

Keywords: MRSA, macrophages, thermal injury

Introduction

Infections remain a common cause of morbidity and mortality in thermally injured patients with severe immunosuppression. These patients are very susceptible to infection from various pathogens [1–3]. Staphylococcus aureus is a major cause of infection in thermally injured patients. Methicillin-resistant S. aureus (MRSA), especially, has become a grave concern for these patients [4–6]. MRSA was represented in 40% of wound isolates and 14% to 17% of thermally injured patients would become infected, once they were colonized with MRSA [4]. MRSA is resistant not only to β-lactams, such as methicillin, but also to most other antimicrobial agents [7]. Vancomycin has been used, solely, as the drug to treat MRSA infection [7]. However, the increasing prevalence of multidrug resistant strains and the more recent appearance of strains resistant to vancomycin [8,9], raise the spectre of untreatable staphylococcal infections. Therefore, to regulate MRSA infection, a new strategy different from antibiotic therapy is required.

Classically activated macrophages (Mφ) are important for the host's innate immune responses against various infections [10,11]. These Mφ were generated from resident Mφ in response to the engagement of Toll-like receptors or binding of IFN receptors by IFN-α/β or IFN-γ [11]. Classically activated Mφ exhibit high oxygen consumption, killing activity against intracellular pathogens, cytotoxicity against tumour cells, and the expression of inducible nitric oxide synthase. They also secrete nitric oxide, pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) and Th1 response-associated cytokines (IFN-γ, IL-12, IL-18 and CCL3) [12,13]. In addition, classically activated Mφ induce Th1 cells by secreting IFN-γ, IL-12 and CCL3. In fact, these Mφ have been shown to eradicate infection from Mycobacterium avium complex [14], Salmonella typhimurium [15], Trypanosoma cruzi [16] and lymphocytic choriomeningitis virus [17] in animal studies. In contrast, alternatively activated Mφ play a role on the negative regulation of Th1 cells and classically activated Mφ [18,19]. These Mφ are generated from resident Mφ in response to Th2 cytokines (IL-4, IL-10 and/or IL-13) or glucocorticoids. Alternatively activated Mφ express foreign antigen receptors, such as a mannose receptor, β-glucan receptors and scavenger receptors. They also produce IL-1 receptor antagonist, IL-10, CCL17, CCL18, CCL22 and arginase [12,13]. Numerous papers have described immunosuppression following thermal injuries [20–26]. This includes: diminished T helper type 1 cell-associated cellular responses [21, 22, 24], increased lymphocytic apoptosis [25], impaired phagocytic and chemotactic activities of neutrophils (PMN) [23] and Mφ [26], and impaired functions of natural killer (NK) cells [20].

Recently, the critical role of Mφ against MRSA infection has been reported [27]. Injection of liposome-encapsulated dichloromethylene diphosphate to deplete Mφ decreased the survival rate of mice infected with MRSA. In addition, we have reported that classically activated Mφ were important effector cells to eliminate not only intracellular pathogens but also various bacterial infections [28]. All SCIDbgMN mice inoculated with alternatively activated Mφ from severely burned mice died after exposure to Enterococcus faecalis, MRSA or caecal ligation and puncture (CLP)-induced sepsis, while all SCIDbgMN mice inoculated with classically activated Mφ survived after the same infection. Therefore, in this study, we investigated the control of MRSA infection in thermally injured mice by modifying the host's anti-MRSA innate immune responses.

Materials and methods

Animals

Seven- to 8-week-old SCID-beige mice (SCIDbg mice) purchased from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study. SCIDbgMN mice were SCIDbg mice treated with carrageenan (0·4 mg/mouse, i.v., once daily for 5 days starting 5 days before X-irradiation), trypan blue (1 mg/mouse, i.p., 1 day before and 1 day after X-irradiation), anti-Ly6G mAb (100 µg/mouse, i.p., 1 day before cell inoculation) and whole body X-irradiation (4 Gy, 2 days before cell inoculation). SCIDbgMN mice are immunodeficient and lack functional T cells, B cells, NK cells, Mφ and PMN [29]. These mice were infected with MRSA. Functional PMN were not recovered in these mice 1–7 days after the X-irradiation, even when they were exposed to pathogens [29]. When bone marrow cells or peripheral blood cells taken from these mice were tested morphologically for residual PMN, after Wright-Giemsa and alkaline phosphatase stainings no PMN were detected until 7 days after the combination treatment. In addition, myelocytes (PMN precursor cells) were not demonstrated in the bone marrow of SCIDbgN mice until 7 days after the depletion of PMN, even when these mice were inoculated with PMN. Three to 7 days after the final treatment, no functional Mφ could be found in the reticuloendothelial system [30]. It has been previously reported that cells transferred to SCIDbg mice disseminated to most of their organs within 1 day of the inoculation [30]. SCIDbg mice with thermal injuries and SCIDbgMN mice with or without an inoculation of Mφ or PMN from various sources, were exposed to a lethal amount of MRSA. The Institutional Animal Care and Use Committee of The University of Texas Medical Branch approved all procedures utilizing animals (IACUC approval number: 02-04-024).

Reagents and MRSA

Recombinant mouse IL-10, IL-12 and CCL3 were purchased from PeproTech (Rocky Hill, NJ, USA) and mAbs for IL-10, IL-12 and Ly6G were obtained from BD PharMingen (San Diego, CA, USA). CCL17, anti-CCL3 mAb and anti-CCL17 mAb were purchased from R & D Systems (Minneapolis, MN, USA). Carrageenan and trypan blue purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as Mφ blockers. MRSA (biotype 21777) isolated from the clinical specimen of a thermally injured patient in Shriners Hospital for Children at Galveston was utilized in this study. This MRSA was defined as a vancomycin sensitive strain. The growth of MRSA was performed on mannitol salt agar supplemented with oxacillin for 48 h at 37 °C in aerobic conditions. Master cultures of the strains were stored at −70 °C. Primary and secondary cultures were stored at 4 °C for up to 4 weeks.

Cell preparations and cytokine assays

For Mφ isolation, peritoneal exudate cells suspended in 2 ml of RPMI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum (FBS) and antibiotics (maintenance medium) were cultured in fibronectin-coated Petri-dishes (60 × 15 mm) for 90 min at 37 °C [19]. At the end of cultivation, dishes were washed 3 times with maintenance medium warmed to 37 °C. After the dishes were kept for 60 min at 4 °C, adherent cells (Mφ populations) were recovered from the dishes using a rubber policeman. The purity of Mφ recovered in this preparation by a FACScan flow cytometer was 92% or more [19]. Mφ were cultured with RPMI-1640 medium supplemented with 10% FBS, 2 mM l-glutamine and antibiotics (complete medium). As previously described [31], PMN were isolated from whole peripheral blood using Ficoll-Hypaque and dextran sedimentations. The resulting PMN fraction was further treated with erythrocyte-lysing kits (R & D Systems) to eliminate small amounts of erythrocytes. The purity of the PMNs obtained was routinely more than 93%, when analysed by flow cytometry with FITC-conjugated anti-Gr-1 mAb and Wright-Giemsa/alkaline phosphatase stainings. For the production of cytokines, various Mφ populations (1 × 106 cells/ml) were cultured for 24 h without any stimulation. Culture fluids harvested were assayed for their cytokines and chemokines using enzyme-linked immunosorbent assay (ELISA). The detection limits of IL-10, CCL17, IL-12 and CCL3 in the culture fluids were 8, 16, 20 and 18 pg/ml, respectively. Mφ were considered as alternatively activated Mφ when they produced CCL17 and IL-10 [12,13]. Mφ were considered as classically activated Mφ when they produced CCL3 and IL-12 [12,13].

Ge-132 treatment

2-Carboxyethylgermanium sesquioxide (Ge-132) was generously supplied by the Asai Germanium Research Institute (Tokyo, Japan). Ge-132, O3n(GeCH2CH2COOH)2n, is a hydrolysate of trihalogermanium originally synthesized by Tsutsui et al. [32]. In our previous studies [33,34], Ge-132 has been shown to inhibit alternatively activated Mφ generation in mice immunized with allogeneic lymphocytes. In this study, the compound was used as an inhibitor of burn-associated alternatively activated Mφ generation. Ge-132 was dissolved in physiological saline and orally administered using a feeding needle. The dose and schedule of Ge-132 administered to mice were determined in our previous study [35].

Thermal injury

Thermally injured mice were prepared as follows: SCIDbg mice were anaesthetized with pentobarbital (40 mg/kg, i.p). Electric clippers were used to shave the hair on the back of each mouse from groin to axilla. Thermal injury was produced by pressing a custom-made insulated mold (with a 2·5 × 3·5-cm window) firmly against the shaved back of each mouse and subsequently exposing the area to a gas flame for 9 s. A Bunsen burner equipped with a flame-dispersing cap was used as a gas flame source. A third degree burn, on approximately 15% of total body surface area for a 26 g mouse, was consistently produced by this procedure [19,24]. Immediately after thermal injury, physiological saline (1 ml/mouse, i.p) was administered for fluid resuscitation. Animals were then housed until used in experiments. Control mice, not exposed to the gas flame, had their back hair shaved and received physiological saline (1 ml/mouse, i.p).

Infection experiments

Thermally injured SCIDbg mice treated with or without Ge-132 were exposed to MRSA at a dose of 2 × 103 CFU/mouse. The Ge-132 treatment (100 mg/kg, p.o) was performed 1 day before and 1 day after thermal injury. These mice were exposed to MRSA 2 days after being injured. To compare the anti-MRSA functions of Mφ or PMN, SCIDbgMN mice inoculated i.v. with 1 × 104 cells/mouse of Mφ or PMN from normal or thermally injured SCIDbg mice were infected i.v. with 2 × 103 CFU/mouse of MRSA. This amount of MRSA corresponded to 0·1 LD50 in normal SCIDbg mice, 10 LD50 in thermally injured SCIDbg mice and 10 LD50 in SCIDbgMN mice. The results were evaluated utilizing the following criteria; (i) the mean survival time in days (MSD) and (ii) the survival percentage at 7 days after infection.

Statistical analysis

The survival of mice exposed to the pathogen was analysed by the Kaplan-Meier test. Other data were expressed as mean ± SD, and the results were statistically analysed by anova using Statview 4·5 (Brain Power, Calabasas, CA, USA). A P-value ≤ 0·05 was considered to be significant.

Results

The susceptibility of thermally injured SCIDbg mice to MRSA infection

In preliminary studies, normal BALB/c mice were infected with various doses of MRSA, and their survival rates were observed for 72 h. When mice were infected i.v. with 1 × 108 CFU/mouse of MRSA, 50% of them died within 48 h of infection. However, 1 × 107 CFU/mouse of MRSA was not sufficient to kill these mice (100% survived). These results suggest that the use of normal mice to study host anti-MRSA innate immunities was not feasible because of the very large amounts of pathogen required to induce disease. Therefore, SCIDbg mice were utilized throughout this study. We have already demonstrated that the generation of alternatively activated Mφ in SCIDbg mice, in response to burns, was identical to that in normal mice undergoing a burn injury [19,36]. As the first series of experiments, the effect of thermal injury on the susceptibility of SCIDbg mice to MRSA infection was examined. Normal SCIDbg mice and SCIDbg mice 2 days after thermal injury were infected i.v. with 2 × 103 CFU/mouse of MRSA. As shown in Fig. 1, when 100% of normal SCIDbg mice infected with MRSA survived, 100% of thermally injured SCIDbg mice died within 3 days of MRSA infection (MSD, 1·9 ± 0·6 days). All of the unchallenged thermally injured SCIDbg mice remained alive more than 7 days. These results indicated that, in response to the exposure to thermal injury, SCIDbg mice became greatly susceptible to MRSA infection.

Fig. 1.

Fig. 1

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The susceptibility of thermally injured SCIDbg mice to MRSA infection. SCIDbg mice with thermal injury (•, n = 16) were infected with 2 × 103 CFU/mouse of MRSA. Normal SCIDbg mice infected with the same amount of MRSA (▪, n = 16) and thermally injured SCIDbg mice without infection (○, n = 16) served as controls. All mice were monitored every 12 h for 7 days after infection. Data are representative of 3 observations. *P < 0·001 compared with controls.

Mφ as a major effector cell on the host's anti-MRSA immune responses

In the above experiments, SCIDbg mice were shown to be resistant against MRSA infection. This indicates that Mφ, PMN or both are effector cells on the host's anti-MRSA innate immune responses. Next, SCIDbgMN mice were separately inoculated with Mφ or PMN and then exposed to MRSA infection. SCIDbgMN mice were SCIDbg mice depleted of Mφ and PMN. Mφ and PMN were prepared from normal SCIDbg mice. Two h after cell inoculation, these mice were infected i.v. with 2 × 103 CFU/mouse of MRSA. As shown in Fig. 2, all SCIDbgMN mice infected with MRSA survived after Mφ inoculation. However, only 25% of SCIDbgMN mice inoculated with PMN survived after the same infection. These results indicate that, in SCIDbg mice, Mφ are effector cells on the host's anti-MRSA innate immune responses. However, Mφ from thermally injured SCIDbg mice did not show any protective activities against MRSA infection in SCIDbgMN mice. Thus, all SCIDbgMN mice inoculated with Mφ from thermally injured mice died within 3 days after MRSA infection (Fig. 2). These results indicate that Mφ from thermally injured mice function differently on the anti-MRSA resistance, when compared to those from normal mice.

Fig. 2.

Fig. 2

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Survival of MRSA-infected SCIDbg mice after inoculation with PMN or Mφ from SCIDbg mice with or without thermal injury. SCIDbgMN mice not exposed to thermal injury were infected with MRSA (2 × 103 CFU/mouse, i.v). Two hours before infection, SCIDbgMN mice were inoculated i.v. with 1 × 104 cells/mouse of PMN (□, n = 12) from normal SCIDbg mice, Mφ (○, n = 10) from normal SCIDbg mice, PMN (▪, n = 12) from thermally injured SCIDbg mice or Mφ (•, n = 12) from thermally injured SCIDbg mice. As a control, SCIDbgMN mice treated with saline (▴, n = 10) were infected with the same amount of MRSA. Data are representative of 3 observations. *P < 0·05 or **P < 0·001 compared with SCIDbgMN mice treated with saline.

Characterizations of Mφ from SCIDbg mice with or without thermal injury

The ability to produce cytokines and chemokines was examined in cultures of Mφ from SCIDbg mice with or without thermal injury. In these experiments, two cytokines (IL-10 and IL-12) and two chemokines (CCL3 and CCL17) were measured in culture fluids from Mφ preparations. IL-12 and CCL3 have been shown as specific parameters of classically activated Mφ, while alternatively activated Mφ have been reported as a producer of IL-10 and CCL17 [12,13]. Mφ were prepared from SCIDbg mice with or without thermal injury 1 day after MRSA infection. Normal SCIDbg mice were shown to be resistant against MRSA infection, while thermally injured SCIDbg mice were susceptible to the infection (Fig. 1). Mφ from MRSA-resistant mice produced IL-12 and CCL3, but not IL-10 and CCL17. In contrast, Mφ from MRSA-susceptible mice produced IL-10 and CCL17, but not IL-12 and CCL3 (Table 1). These data clearly indicate that Mφ from MRSA-resistant mice are classically activated Mφ, and Mφ from MRSA-susceptible mice are identified as alternatively activated Mφ.

Table 1. Production of cytokines and chemokines in cultures of Mφ from SCIDbg mice with or without thermal injury.

Mφ source IL-10 IL-12 CCL3 CCL17
Normal SCIDbg mice 838 ± 15 983 ± 244 2021 ± 573 < 16
Thermally injured SCIDbg mice 604 ± 130* < 20** < 18** 527 ± 104*
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Mφ (1 × 106 cells/ml) from normal or thermally injured SCIDbg mice 1 day after MRSA infection were cultured with complete media. Culture fluids harvested 24 h after cultivation were assayed for cytokines and chemokines using ELISA. Data are expressed as mean (pg/ml) ± SD and representative of 3 experiments.

*

P < 0·05

**

P < 0·01 compared with normal SCIDbg mice.

Effect of alternatively activated Mφ depletion on the resistance of thermally injured SCIDbg mice against MRSA infection

Ge-132 has been shown to inhibit alternatively activated Mφ generation [34]. Therefore, this compound was used in thermally injured SCIDbg mice as an inhibitor of alternatively activated Mφ. As shown in Table 2, Mφ from thermally injured SCIDbg mice treated with Ge-132 did not produce IL-10 and CCL17, while these soluble factors were produced by Mφ from untreated thermally injured SCIDbg mice. These results indicate that alternatively activated Mφ are not generated in thermally injured SCIDbg mice treated with Ge-132. On the other hand, after treatment with Ge-132, Mφ from thermally injured SCIDbg mice 1 day after MRSA infection produced IL-12 and CCL3 (Table 2). These results indicate that, in response to MRSA infection, classically activated Mφ are generated in thermally injured SCIDbg mice treated with Ge-132. Furthermore, the resistance of thermally injured SCIDbg mice treated with Ge-132 to MRSA infection was examined. While 88% of thermally injured SCIDbg mice died from MRSA infection, all of the Ge-132-treated thermally injured SCIDbg mice or control SCIDbg mice survived (Fig. 3). These results suggest that the resistance of thermally injured SCIDbg mice against MRSA infection is completely improved when the generation of burn-associated alternatively activated Mφ are suppressed and classically activated Mφ are induced.

Table 2. Classically activated Mφ generation in thermally injured mice treated with Ge-132.

Mφ from thermally injured SCIDbg mice treated with IL-10 IL-12 CCL3 CCL17
Saline 604 ± 130 < 20 < 18 527 ± 104
Ge-132 < 8* 616 ± 74* 2136 ± 277** < 16*
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Thermally injured SCIDbg mice were treated with or without Ge-132 and infected with MRSA. Mφ from these mice 1 day after infection were cultured for 24 h. Culture fluids harvested 24 h after cultivation were assayed for cytokines and chemokines using ELISA. Data are expressed as mean (pg/ml) ± SD and representative of 3 experiments.

*

P < 0·01

**

P < 0·001 compared with thermally injured SCIDbg mice treated with saline.

Fig. 3.

Fig. 3

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Effect of alternatively activated Mφ depletion on MRSA infection in thermally injured SCIDbg mice. SCIDbg mice with (•, n = 16) or without (▪, n = 12) thermal injury and thermally injured SCIDbg mice treated with Ge-132 (100 mg/kg, p.o., 1 day before and 1 day after burn injury, ∇, n = 12) were exposed to MRSA (2 × 103 CFU/mouse, i.v). Ge-132 has been shown to be an inhibitor of alternatively activated Mφ generation. Data are representative of 3 observations. *P < 0·001 compared with normal SCIDbg mice.

Discussion

As compared with healthy individuals, thermally injured hosts have been described as hosts with a greatly increased susceptibility to various infections [28]. In this study, utilizing a mouse model of thermal injury, the prospect of immunologically controlling MRSA infection was investigated in SCIDbg mice with thermal injuries. Alternatively activated Mφ that appeared in response to thermal injury were shown to inhibit the generation of classically activated Mφ, an anti-MRSA effector cell in the host's innate immunity. Classically activated Mφ were not generated in mice whose alternatively activated Mφ predominated. However, classically activated Mφ were generated in thermally injured mice after the depletion of alternatively activated Mφ by treatment with Ge-132, and these mice became resistance to MRSA infection. From these results, the prospect of immunologically controlling MRSA infection was indicated in mice with thermal injuries.

PMN have been described as critical effector cells in the host's innate immune response against S. aureus [37]. However, in recent reports, PMN did not kill S. aureus effectively because the bacteria carry staphylococcal MprF gene, which resists the cationic antimicrobial peptides produced by PMN [38]. Furthermore, interaction of PMN and viable-opsonized S. aureus results in the activation of p38-mitogen-activated protein kinease in PMN. This causes apoptosis of PMN within an hour [39]. These facts strongly suggest that PMN might not be a major effector cell in anti-MRSA innate immune responses. In our study, PMN were not shown to significantly protect SCIDbgMN mice infected with MRSA. Instead of PMN, Mφ were shown to be more important as anti-MRSA effector cells. In our study, we used MRSA which was isolated from the clinical specimen of a thermally injured patient in our hospital. To clarify whether classically activated Mφ are generally effector cells for the host antibacterial innate immune responses against S. aureus infection, future experiments utilizing various clinical isolates of S. aureus are needed.

We have previously reported that classically activated Mφ are effector cells for the host's innate immunities against MRSA infection [28,36]. Severely (full-thickness) burned mice were susceptible to MRSA infection, while normal mice were resistant [28,36]. Severely burned mice were shown to be carriers of alternatively activated Mφ, while classically activated Mφ were obtained from mice with partial thickness burn injuries. Classically activated Mφ were induced by CpG DNA in normal mice. However, these Mφ were not elicited in severely burned mice even when they were treated with CpG DNA. IL-10 and CCL17 released from alternatively activated Mφ were shown to be an inhibitor of classically activated Mφ generation [19]. In the results shown in this paper, classically activated Mφ were generated in severely burned mice after an injection of Ge-132, an inhibitor of alternatively activated Mφ generation. After depletion of alternatively activated Mφ by Ge-132 treatment, severely burned mice became resistant against MRSA infection at the same level observed in normal SCIDbg mice.

Recently, we have demonstrated two different subsets of neutrophils (PMN-I and PMN-II) [36]. These neutrophils were distinguished from each other in the following ways: PMN-I (Gr-1+CD11b−CD49d+) produced IL-12/CCL3 and expressed Toll-like receptor 2 (TLR2)/TLR4/TLR5/TLR8; PMN-II (Gr-1+CD11b+CD49d−) produced IL-10/CCL2 and expressed TLR2/TLR4/TLR7/TLR9. PMN-I have an ability to direct resident Mφ to polarize classically activated Mφ, while resident Mφ are polarized to alternatively activated Mφ by PMN-II. Normal PMN (Gr-1+CD11b−CD49d−) did not produce any of these cytokines, expressed TLR2/TLR4/TLR9 and directed resident Mφ to polarize neither classically activated Mφ nor alternatively activated Mφ. PMN-I were discovered in mice with partial-thickness burn injuries, while PMN-II were demonstrated in mice with severe burn injuries. Normal PMN were obtained from normal mice. PMN-I or PMN-II, converted from normal PMN in response to host circumstance, may be a key to the appearance of classically activated Mφ or alternatively activated Mφ in thermally injured hosts.

Major thermal injury has been shown to induce a pathophysiological response that has a marked inflammatory component, with the release of a wide range of inflammatory mediators (such as TNF-α, IL-6, PGE2 and nitric oxide) from macrophages [26]. In our previous studies [19], alternatively activated Mφ derived from mice 48 h after thermal injury produced IL-6, but classically activated Mφ induced by CpG DNA did not. Both Mφ subsets produced PGE2 equally. Typical classically activated Mφ produced TNF-α and nitric oxide, while alternatively activated Mφ did not. Interestingly, TNF-α and IL-1β were produced by PMN-I (inducer cells for classically activated Mφ generation) and PMN-II (inducer cells for alternatively activated Mφ generation) equally [36]. PMN-II appeared in the peripheral blood of mice as early as 18 h after burn injury. The increase in the production of proinflammatory mediators following thermal injury, shown in earlier studies, may be the result of a mixed cell population in response to the injury. To explain the difference between earlier studies and our studies, studies on the appearance of each pro-inflammatory cytokine producing cells will be needed.

In our earlier studies, Mφ from thermally injured mice suppressed IFN-γ production in vivo and in vitro [40,41]. In this study, Mφ from thermally injured mice showed no anti-MRSA activities in SCIDbgMN mice, while SCIDbgMN mice exposed to MRSA survived after inoculation with Mφ from normal SCIDbg mice. Mφ from thermally injured mice were confirmed as alternatively activated Mφ, while Mφ from normal SCIDbg mice infected with MRSA were characterized as classically activated Mφ. Classically activated Mφ were not generated in thermally injured SCIDbg mice whose alternatively activated Mφ predominated. The regulation of alternatively activated Mφ generation may be a crucial step in the improvement of thermally injured patient resistance against MRSA infection.

Acknowledgments

This work was supported by Shriners North American Grant 8690 (to F. S.).

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