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The Journal of Immunology, 2008, 180, 4133 -4138
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Burns
*Mouth Disorders

Role of Polymorphonuclear Neutrophils on Infectious Complications Stemming from Enterococcus faecalis Oral Infection in Thermally Injured Mice1

Yasuhiro Tsuda*, Kenji Shigematsu*, Makiko Kobayashi*,{dagger}, David N. Herndon{dagger} and Fujio Suzuki2,*,{dagger}

* Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555; and {dagger} Shriners Hospitals for Children, Galveston, TX 77550


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Thermally injured mice are susceptible to Enterococcus faecalis translocation. In this study, the role of polymorphonuclear neutrophils (PMN) on the development of sepsis stemming from E. faecalis translocation was studied in SCID-beige (SCIDbg) mice depleted of PMN (SCIDbgN mice) or macrophages (M{phi}) and PMN (SCIDbgMN mice). Sepsis was not developed in SCIDbgN mice orally infected with E. faecalis, while the orally infected pathogen spread systemically in the same mice inoculated with PMN from thermally injured mice (TI-PMN). SCIDbgMN mice were shown to be greatly susceptible to sepsis caused by E. faecalis translocation, while orally infected E. faecalis did not spread into sepsis in the same mice that were previously inoculated with M{phi} from unburned SCIDbg mice (resident M{phi}). In contrast, orally infected E. faecalis spread systemically in SCIDbgMN mice inoculated with resident M{phi} and TI-PMN, while all SCIDbgMN mice inoculated in combination with resident M{phi} and PMN from unburned SCIDbg mice survived after the infection. After cultivation with TI-PMN in a dual-chamber transwell, resident M{phi} converted to alternatively activated M{phi}, which are inhibitory on the generation of classically activated M{phi} (typical effector cells in host antibacterial innate immunities). TI-PMN were characterized as immunosuppressive PMN (PMN-II) with abilities to produce cc-chemokine ligand-2 and IL-10. These results indicate that PMN-II appearing in response to burn injury impair host antibacterial resistance against sepsis stemming from E. faecalis translocation through the conversion of resident M{phi} to alternatively activated M{phi}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection is a leading cause of morbidity and mortality in thermally injured patients (1, 2). These patients are particularly susceptible to infection with bacterial pathogens, and local infections easily escalate into infectious complications (3, 4, 5). The translocation of bacteria beyond the intestinal lumen is one of the mechanisms that promotes infectious complications (6, 7). In fact, bacterial translocation has been demonstrated in 16 to 40% of patients with multiple organ dysfunction syndrome and intestinal ischemia (8). The manifestation of multiple organ dysfunction syndrome and intestinal ischemia is frequently demonstrated in thermally injured patients (9, 10). Bacterial translocation is postulated to occur in several clinical conditions, such as bacterial overgrowth in small bowel, damage to the gut barrier, and states of systemic immunosuppression (6, 7). Enterococci were viewed as a relatively avirulent endogenous flora with little potential for human infection when compared with other Gram-positive bacteria. However, Enterococcus faecalis has emerged as a clinically important pathogen that causes septic complications in immunocompromised hosts (11, 12). Therefore, treatment targeting the host’s antibacterial immune responses seems to be critical to successfully regulate infectious complications stemming from E. faecalis translocation.

The innate immune system is the first line of host defense against bacterial translocation (13, 14). The important roles of polymorphonuclear neutrophils (PMN)3 and macrophages (M{phi}) in antibacterial innate immunity have been proven in many papers (15, 16, 17, 18). Classically activated M{phi} (M1M{phi}), characterized as major killer cells for pathogens (19), are the main effector cells in innate immunities. M1M{phi} are generated from resident M{phi} following stimulation with invasive pathogens via pattern recognition receptors (14, 18, 19). However, M1M{phi} have never been generated in burn mice whose alternatively activated M{phi} (M2M{phi}) predominated, even when they are exposed to the pathogens or stimulated with M1M{phi} inducers (16, 20). M2M{phi} lack the ability to kill bacteria, and soluble factors released from M2M{phi} inhibit the conversion of resident M{phi} to M1M{phi} following stimulation with bacteria (21). In the recent studies, SCID-beige (SCIDbg) mice depleted of PMN (SCIDbgN) were shown to be resistant to sepsis stemming from E. faecalis translocation, while all SCIDbgN mice depleted of M{phi} (SCIDbgMN) died after the same oral infection with E. faecalis. However, SCIDbgN mice became highly susceptible to infectious complications stemming from E. faecalis translocation when they were inoculated with PMN from thermally injured SCIDbg mice (TI-PMN). Both SCIDbgN mice and those inoculated with PMN from unburned SCIDbg mice were resistant to the same infection. SCIDbgN mice were SCID-beige mice depleted of PMN. Among various immunocompetent cells, only intact M{phi} are present in these mice. SCIDbgMN mice were SCIDbgN mice depleted of M{phi}. Therefore, the role of TI-PMN on the antibacterial host defense against E. faecalis translocation was examined by focusing on the M{phi} activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Seven- to 11-wk-old, pathogen-free, male SCIDbg mice purchased from Taconic Farms were used in this study. These mice have been defined as immunodeficient mice without functional T, B, and NK cells. In some experiments, SCIDbg mice depleted of PMN (SCIDbgN mice) or PMN and M{phi} (SCIDbgMN mice) were used. SCIDbgN mice were SCIDbg mice treated with anti-Ly6G mAb (100 µg/mouse, i.p., every day for 5 days) plus whole body X-irradiation (4 Gy, 1 day before infection). Functional PMN were not recovered from SCIDbgN mice 1 to 7 days after the X-irradiation, even after they were exposed to pathogens (16). 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 PMN depletion. SCIDbgMN mice were SCIDbgN mice treated with carrageenan (0.4 mg/mouse, i.v., once daily for 5 days, starting 5 days before X-irradiation) and trypan blue (1 mg/mouse, i.p., 1 day before, and 1 and 3 days after X-irradiation) (16). Three to 7 days after the final treatment, no functional M{phi} were found in the reticuloendothelial systems of SCIDbgMN mice. All experiments with animals were performed according to protocols approved by The Institutional Animal Care and Use Committee of University of Texas Medical Branch at Galveston.

Bacteria, reagents, and media

E. faecalis (49757 strain) was purchased from The American Type Culture Collection. E. faecalis was grown in brain heart infusion broth for 18 h at 37°C in aerobic conditions. Murine rIL-12, rIL-10, and rCCL2 were purchased from BD Biosciences, and murine rCCL3, rCCL5, and rCCL17 were obtained from R&D Systems. mAbs directed against CCL2, CCL5, CCL17, and Ly6G (Gr-1) were obtained from eBioscience. For cultivation of various cell preparations, RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium) was used.

Burn injury

Thermally injured mice were created according to our previously reported protocol (17, 21). Thus, mice were anesthetized with pentobarbital (40 mg/kg, i.p.) and electric clippers were used to shave the hair on the back of each mouse from groin to axilla. The mice were then exposed to a gas flame for 9 s after pressing the window of the custom-made insulated mold (with a 4 x 5-cm window) firmly against the shaved back. A Bunsen burner equipped with a flame-dispersing cap was used as the source of the gas flame. This procedure consistently produced a third degree burn on ~25% of total body surface area for a 26-g mouse. Immediately after thermal injury, physiologic saline (1 ml per mouse, i.p.) was administered for fluid resuscitation. All mice remained alive >10 days after burn injury. Control mice had their back hair shaved but were not exposed to the gas flame. They also received physiologic saline (1 ml per mouse, i.p.). Buprenorphine (2 mg/kg) was given s.c. every 12 h during the postburn period. Control animals also received identical regimens of analgesics (buprenorphine) throughout the study period.

E. faecalis oral infection

Mice decontaminated by the oral administration of the antibiotic mixture were used in these experiments. For decontamination, mice were treated for 4 days with drinking water containing 4 mg/ml penicillin, streptomycin, and bacitracin (22). One day after decontamination, mice were exposed to flame burns, and then infected orally with 1 x 105 CFU/mouse of E. faecalis 4 h after burn injury. The development of infectious complications was evaluated by 1) growth of the bacteria in mesenteric lymph nodes and liver, and 2) the mortality rates of the test groups in comparison with the controls. To measure the quantity of bacteria, organ specimens (mesenteric lymph nodes and liver) were weighed and disrupted in 2 ml PBS using a Bruikman homogenizer. A serial 10-fold dilution of the homogenates was plated onto blood agar plates, and incubated for 24 h at 37°C. The colonies were counted and the number of bacteria per gram organ was determined. Because bacteria were not detected normally in mesenteric lymph nodes and liver, the presence of bacteria in these organs is considered to be evidence of translocation. To determine the percentage of survival, mice will be monitored twice a day for 5 days after infection.

Preparation of PMN and M{phi}

As previously described (16), PMN were isolated from whole peripheral blood of normal mice and thermally injured mice (mice 18 h after burn injury) using Ficoll-Hypaque and dextran sedimentations. In brief, peripheral blood was drawn from the heart of mice with a heparinized syringe. The peripheral blood was centrifuged with Ficoll-Hypaque, and precipitates were obtained as a PMN rich fraction. Then, precipitates were suspended in 1% dextran (T-500, Pharmacia) and kept for 1 h at room temperature to allow the sedimentation of erythrocytes. The resulting PMN fraction was further treated with a mixture of biotin-conjugated anti-CD3 (T cells), anti-F4/80 (monocytes/M{phi}), and anti-CD19 (B cells) mAbs for 30 min at 4°C. Then, these cells were suspended in MagCellect buffer (R&D Systems) and incubated with magnetic beads (Dynal) coated with streptavidin. The purity of PMN, isolated in this procedure, was >98% when measured morphologically (Wright-Giemsa/alkaline phosphatase stainings).

M{phi} were prepared from the peritoneal exudates and mesenteric lymph nodes of normal mice. Peritoneal exudates were obtained by injecting 4 ml of PBS and harvesting the fluids (16, 21), and single cell suspensions of mesenteric lymph nodes were obtained. These cells were adjusted to 5 x 106 cells/ml in MagCellect buffer (R&D Systems), and then M{phi} were isolated from these cell suspensions by positive selection using magnetic beads coated with anti-F4/80 mAb. Thus, the cell suspension was mixed with magnetic beads (Dynal) bearing anti-F4/80 mAb at a ratio of one cell to five beads for 30 min at 4°C. F4/80 positive cells were magnetically separated to the side of the tube, and the supernatant was eliminated. A M{phi}-enriched population (>97% pure as F4/80 positive cells) was consistently obtained using this technique.

Determination of M2M{phi}

M{phi} were considered to be M2M{phi} when they produced CCL17 (but not CCL5) and expressed mannose receptor (but not inducible NO synthase (iNOS)) mRNA (18, 21). For the chemokine production, M{phi} (1 x 106 cells/ml) were cultured for 48 h without any stimulation. Culture fluids harvested were assayed for chemokines using ELISA. mRNAs for mannose receptor and iNOS were analyzed by RT-PCR. Total RNA was extracted from M{phi} (1 x 106 cells) using RNA isolator, following the manufacturer’s recommendations. Within each experiment, each sample was normalized by the amount of isolated RNA. Then, this RNA was turned back into cDNA through the reverse transcription of mRNA. PCR was conducted using synthesized oligonucleotide primers from Sigma-Aldrich: mannose receptor, 5'-CCATCGAGACTGCTGCTGAG-3' (forward) and 5'-AGCCCTTGGGTTGAGGATCC-3' (reverse); iNOS, 5'-CCCTCCAGTGTCTGGGAGCA-3' (forward) and 5'-TGCTTGTCACCACCAGCAGT-3' (reverse) (21). Using a thermal cycler (GeneAmp PCR Sysyem 9600), 35 cycles of PCR were performed at 94°C for 15 s, 60°C for 15 s and 72°C for 20 s. The predicted products were run on 2% agarose gels containing ethidium bromide.

Characterization of TI-PMN

TI-PMN were tested for their abilities 1) to produce CCL2, CCL3, IL-10, and IL-12, and 2) to induce M1M{phi} or M2M{phi} from resident M{phi}. To determine the cytokine/chemokine-producing profile, PMN preparations with a cell density of 1 x 106 cells/ml were cultured with 0.0075% Staphylococcus aureus Ag (SAC; Calbiochem) for 18 h. The harvested culture fluids were assayed for CCL2, CCL3, IL-12, and IL-10 by ELISA.

The effect of TI-PMN on the M{phi} activation was tested in dual-chamber transwells (0.4 µm micropores; Corning) (21). In brief, the resident M{phi} suspension (1 x 106 cells/ml, lower chamber) was cultured with the PMN suspension (1 x 105 cells/ml, upper chamber) in a dual-chamber transwell. Eighteen hours after cultivation, the upper chamber was removed and M{phi} in the lower chamber were examined for M2M{phi} properties, as described above.

Statistical analysis

The results obtained were analyzed statistically using an ANOVA test. Survival curves were analyzed using the Kaplan-Meier test. All calculations were performed on a computer using the program Statview 4.5 from Brain Power. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Susceptibility of TI-PMN inoculated SCIDbgN mice to E. faecalis oral infection

Unburned SCIDbg mice and SCIDbg mice 18 h after burn injury (thermally injured SCIDbg mice) were infected orally with 105 CFU/mouse of E. faecalis. In the results, all unburned SCIDbg mice orally infected with E. faecalis survived, while 90% of the thermally injured SCIDbg mice died after the same infection (Fig. 1A). SCIDbgN mice (M{phi} function is intact) were shown to be resistant against infections, because all of these mice survived after orally infected with 105 CFU/mouse of the pathogen. In contrast, the antibacterial resistance of SCIDbgN mice decreased to the level observed in thermally injured SCIDbg mice when they were inoculated with TI-PMN (Fig. 1B). Because all of the SCIDbgN mice orally infected with E. faecalis survived after inoculation with PMN from unburned SCIDbg mice (normal PMN), these results indicate that TI-PMN suppress the host antibacterial resistance against E. faecalis infection in SCIDbgN mice. However, the host resistance of SCIDbgN mice against E. faecalis oral infection was not influenced by normal PMN. In the next series of experiments, the effect of TI-PMN and normal PMN against E. faecalis infection in SCIDbgMN mice (SCIDbg mice depleted of M{phi} and PMN) was examined. In the results, SCIDbgMN mice were not resistant to E. faecalis infection after inoculation with TI-PMN or normal PMN (Fig. 1C). These results indicate that TI-PMN decrease the host resistance against oral infection with E. faecalis through the modulation of M{phi} functions intactly remaining in SCIDbgN mice.


Figure 1
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  		FIGURE 1. PMN from thermally injured SCIDbg mice (TI-PMN) decreased the resistance of SCIDbgN mice orally infected with E. faecalis. A, SCIDbg mice 18 h after burn injury (10 mice, •) were infected orally with E. faecalis (105 CFU/mouse). Unburned SCIDbg mice (10 mice, {circ}) infected with the same pathogen served as a control. The difference of survival rates between thermally injured SCIDbg mice and unburned SCIDbg mice was p < 0.001 according to Kaplan-Meier log rank test. B, SCIDbgN mice were inoculated i.v. with 1 x 105 cells/mouse of normal PMN (12 mice, {circ}) or TI-PMN (12 mice, •). Two hours after PMN inoculation, these mice were infected orally with E. faecalis (105 CFU/mouse). As a basic control, SCIDbgN mice were infected with the pathogen in the same way (12 mice, {blacksquare}). The difference of survival rates between SCIDbgN mice inoculated with TI-PMN and those inoculated with normal PMN was p < 0.0001 according to Kaplan-Meier log rank test. C, SCIDbgMN mice were inoculated i.v. with 1 x 105 cells/mouse of normal PMN (12 mice, {circ}) or TI-PMN (12 mice, •). Two hours after inoculation, these mice were infected orally with E. faecalis (105 CFU/mouse). As a basic control, SCIDbgMN mice were infected with the pathogen in the same fashion (12 mice, {blacksquare}).

 
M{phi} cultured with TI-PMN failed to protect SCIDbgMN mice orally infected with E. faecalis

To determine the effect of TI-PMN or normal PMN on the antibacterial functions of M{phi}, peritoneal M{phi}, or mesenteric lymph node M{phi} from normal mice were transwell cultured with one of the each PMN preparation (1 x 106 cells/mouse) and adoptively transferred to SCIDbgMN mice. Then, these mice were infected orally with E. faecalis. In the results, all of the SCIDbgMN mice treated with media (a control group) died within 3 days of infection; however, all of the SCIDbgMN mice survived after inoculation with peritoneal M{phi} previously cultured with normal PMN in dual chamber transwells. All of the SCIDbgN mice, not additionally inoculated with M{phi} preparations, survived. In contrast, all of the SCIDbgMN mice inoculated with peritoneal M{phi} previously transwell cultured with TI-PMN died within 4 days of E. faecalis oral infection (Fig. 2). Similar results were obtained when mesenteric lymph node M{phi} were transwell cultured with TI-PMN. In addition, numbers of bacteria in mesenteric lymph nodes and liver taken from the above groups of mice 48 h after E. faecalis infection were determined by colony forming assay. Bacteria were isolated from mesenteric lymph nodes and liver of SCIDbgMN mice inoculated with peritoneal M{phi} previously transwell cultured with TI-PMN (mesenteric lymph nodes, 1 x 104 CFU/g; liver, 4 x 102 CFU/g). However, none of the bacteria were detected in the same organs of SCIDbgMN mice inoculated with M{phi} previously transwell cultured with normal PMN. These results indicate that M{phi} cultured with TI-PMN have no antibacterial activities against orally infected E. faecalis.


Figure 2
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  		FIGURE 2. M{phi} cultured with TI-PMN failed to protect SCIDbgMN mice orally infected with E. faecalis. Peritoneal M{phi} or mesenteric lymph node M{phi} (5 x 106 cells/ml, lower chamber) were cultured with TI-PMN (5 x 105 cells/ml, upper chamber) for 18 h in dual-chamber transwells. Cells harvested from the lower chamber were adoptively transferred i.v. to SCIDbgMN at a number of 1 x 106 cells/mouse (peritoneal M{phi}, 12 mice, •; mesenteric lymph node M{phi}, 12 mice, {blacktriangleup}). As a control, the same number of M{phi} that were cultured with normal PMN in dual chambers was transferred to SCIDbgMN mice (peritoneal M{phi}, 12 mice, {circ}; mesenteric lymph node M{phi}, 12 mice, {triangleup}). Two hours after inoculation, these mice were infected orally with E. faecalis (105 CFU/mouse). SCIDbgMN mice (10 mice, {square}) and SCIDbgN mice (10 mice, {blacksquare}) infected with E. faecalis in the same fashion served as additional controls. The difference of survival rates between SCIDbgMN mice inoculated with M{phi} that were cultured with TI-PMN and normal PMN was p < 0.0001 according to Kaplan-Meier log rank test.

 
TI-PMN were identified as PMN-II, and resident M{phi} influenced by TI-PMN converted to M2M{phi}

TI-PMN were tested for their cytokine/chemokine-producing profiles and surface Ag expressions. As shown in Fig. 3, TI-PMN produced CCL2 and IL-10 (biomarkers for PMN-II) into their culture fluids. However, IL-12 and CCL3 (biomarkers for PMN-I) were not produced by these PMN. Normal PMN did not produce CCL2 and IL-10. In the next experiments, the properties of M{phi} stimulated with TI-PMN were examined. Peritoneal M{phi} or mesenteric lymph node M{phi} from normal mice (lower chamber) were cultured with TI-PMN (upper chamber) for 18 h in a dual-chamber transwell. After removing the upper chamber, M{phi} in the lower chamber were examined for their abilities to produce CCL5 (a biomarker for M1M{phi}) and CCL17 (a biomarker for M2M{phi}). Peritoneal M{phi} transwell cultured with TI-PMN produced CCL17. However, CCL5 was not produced by these M{phi}. Peritoneal M{phi} transwell cultured with normal PMN produced neither CCL5 nor CCL17 (Fig. 4A). In addition, peritoneal M{phi} transwell cultured with TI-PMN expressed mannose receptor mRNA and did not express iNOS mRNA, while these M{phi} transwell cultured with normal PMN did not express either mRNA (Fig. 4B). Similar results were obtained when mesenteric lymph node M{phi} were transwell cultured with TI-PMN (Fig. 4, A and B). Furthermore, resident M{phi} were cultured with complete medium supplemented with the culture fluid (15%, v/v) of TI-PMN (2 x 106 cells/ml, stimulated with SAC for 18 h), and the generation of M2M{phi} was examined. M2M{phi} were generated from resident M{phi} cultures supplemented with the culture fluid of TI-PMN, while M2M{phi} were not generated from resident M{phi} stimulated with the culture fluids of normal PMN (Fig. 4C). These results indicate that soluble factors released from TI-PMN stimulate M{phi} conversion from resident M{phi} to M2M{phi}.


Figure 3
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  		FIGURE 3. Cytokine and chemokine-producing properties of TI-PMN. TI-PMN (1 x 106 cells/ml) were cultured with 0.0075% SAC for 18 h to determine their cytokine/chemokine-producing profiles. As a control, normal PMN were cultured under the same conditions. The amounts of CCL2, CCL3, IL-10, and IL-12 in their culture fluids were measured by ELISA.

 

Figure 4
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  		FIGURE 4. Properties of resident M{phi} after stimulation with TI-PMN. A, Peritoneal M{phi} or mesenteric lymph node M{phi} (1 x 106 cells/ml, lower chamber) were cultured with TI-PMN (1 x 105 cells/ml, upper chamber) for 18 h in double chamber transwells supplemented with 0.0075% SAC. As a control, M{phi} were transwell cultured with normal PMN in the same fashion. After removal of the upper chamber, M{phi} in the lower chamber were cultured for an additional 48 h, and the amounts of CCL5 (a biomarker for M1M{phi}) and CCL17 (a biomarker for M2M{phi}) in their culture fluids were measured using ELISA. B, The expression of iNOS and mannose receptor mRNAs by TI-PMN-stimulated M{phi} were analyzed using RT-PCR. C, Peritoneal M{phi} or mesenteric lymph node M{phi} were cultured with media supplemented with the culture fluids (15%, v/v) of TI-PMN (2 x 106 cells/ml, stimulated with SAC for 18 h). As a control, M{phi} were cultured with the culture fluids of normal PMN in the same fashion. Cells harvested 18 h after cultivation were cultured for an additional 48 h without any stimulation. The culture fluids obtained were assayed for CCL17.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
M{phi} located in the subepithelial lamina propria and mesenteric lymph nodes are the first cells that fight translocated bacteria. Resident M{phi} (M{phi} from unstimulated healthy individuals) are immunologically quiescent with low oxygen consumption and low levels of MHC class II gene expression. In the event of infection, resident M{phi} convert to M1M{phi} through the engagement of Toll-like receptors (23) or IFN receptors (24). M1M{phi} are actual effector cells in host antibacterial innate immunities (25, 26). These M{phi} exhibit 1) high oxygen consumption, 2) the ability to kill pathogens, 3) the ability to express iNOS, and 4) the ability to secrete NO, proinflammatory cytokines (IL-1, IL-6, and TNF-{alpha}), Th1 response-associated cytokines/chemokines (IFN-{gamma}, IL-12, IL-18, CCL3, and CCL5), and antimicrobial peptides (21, 27, 28). In contrast, M2M{phi} have been described to be activated by an alternative pathway involving Th2 cytokines (27). These M{phi} have been implicated in the negative regulation of both M1M{phi} and Th1 cell generation (27). M2M{phi} preferentially express mannose receptor, β-glucan receptors, and scavenger receptors, and produce arginase, IL-1 receptor antagonist, IL-10, and CCL17 (27). Recently, three different subtypes of M2M{phi} (M2aM{phi}, M2bM{phi}, and M2cM{phi}) were described (18). These subsets can be separated by their gene expression and chemokine profiles (18, 29, 30, 31). Thus, CCL17-producing M{phi} with FIZZ1 gene are identified as M2aM{phi}, CCL1-producing M{phi} with SPHK1 gene are classified as M2bM{phi}, and CXCL13-producing M{phi} with FIZZ1 gene are recognized as M2cM{phi} (18, 31). All of the M2M{phi} subtypes express the IL-10 gene (18). Unlike M2aM{phi} and M2cM{phi}, M2bM{phi} produce TNF-{alpha}, IL-1, and IL-6 (18, 31). Because Th2 cytokines are able to suppress the transcriptional activation of IFN-{gamma}- and LPS-responsive genes in M{phi} (32), resident M{phi} cannot convert to M1M{phi} in circumstances where M2M{phi} predominate.

In the present study, we examined the role of TI-PMN (PMN from thermally injured SCIDbg mice) on the development of sepsis stemming from burn-associated E. faecalis translocation, using SCIDbgN mice and SCIDbgMN mice. SCIDbgN mice were SCIDbg mice depleted of PMN, and SCIDbgMN mice were SCIDbgN mice depleted of M{phi}. SCIDbgN mice were shown to be resistant against oral E. faecalis infection, while SCIDbgMN mice were very susceptible to this infection (Fig. 1B). Also, M1M{phi} were isolated from SCIDbgN mice after oral infection with E. faecalis (17). Because functional T, B, and NK cells and PMN are not present in SCIDbgN mice, these results indicate that M1M{phi} are key effector cells for host defense against E. faecalis translocation (Fig. 1, B and C). In contrast, SCIDbgN mice were shown to be susceptible to E. faecalis infection after inoculation with TI-PMN (Fig. 1B). In a dual-chamber transwell, TI-PMN (upper chamber) stimulated M{phi} conversion from resident M{phi} (lower chamber) to M2M{phi} (Fig. 4). Also, M2M{phi} were isolated from SCIDbgN mice inoculated with TI-PMN (data not shown). M2M{phi} have been previously characterized as cells that are inhibitory on the generation of M1M{phi} (21). These results indicated that TI-PMN were cells responsible for the impaired resistance of thermally injured mice to E. faecalis oral infection by converting resident M{phi} to M2M{phi}. Subsequently, TI-PMN were characterized as PMN-II, because they produced IL-10 and CCL2 (Fig. 3). PMN-II have been described as IL-10 and CCL2-producing Gr-1+CD11b+CD11c–F4/80– cells with the ability to inhibit the generation of M1M{phi} (16). Also, PMN-II express TLR2, TLR4, TLR7, and TLR9 mRNAs (16).

M{phi} and PMN have been shown to accumulate in intestinal lymphoid tissues, such as the lamina propria and mesenteric lymph nodes, following gastrointestinal infections (33). We recently examined properties of F4/80+ M{phi} from lamina propria and mesenteric lymph nodes of mice 1 to 3 days after burn injury. These M{phi} produced CCL17 and expressed mannose receptor mRNA, while CCL17 production and mannose receptor expression were not shown by M{phi} isolated from unburned SCIDbg mice. These results indicate that M{phi} in lamina propria and mesenteric lymph nodes of thermally injured SCIDbg mice are M2M{phi}, while those from unburned SCIDbg mice are not. As mentioned above, M2M{phi} were generated from resident M{phi} in cultures with TI-PMN in dual-chamber transwells (Fig. 4, A and B). This indicates that the cell-to-cell contact between resident M{phi} and TI-PMN is not necessary when M2M{phi} were generated from resident M{phi} under the TI-PMN stimulation. In fact, M2M{phi} appeared in the resident M{phi} cultures supplemented with TI-PMN culture fluids (Fig. 4C). Because IL-10 and CCL2 were detected in the culture fluids of TI-PMN (Fig. 3), the role of these soluble factors on the TI-PMN-associated M2M{phi} generation was examined. In the results, M2M{phi} were not generated from resident M{phi} stimulated with the TI-PMN culture fluids that were previously treated with a mixture of anti-IL-10 and anti-CCL2 mAbs (data not shown). This indicates that IL-10 and CCL2 released from TI-PMN act as stimulators of M{phi} conversion from resident M{phi} to M2M{phi}.

TLR reactivity of M{phi} in mice 4–10 days after thermal injury has been widely described in many papers (34, 35). In response to TLR stimulation, these M{phi} produce IL-1, TNF-{alpha}, and IL-6 as well as NO. In these papers, however, the activated M{phi} are not classified as M1M{phi}. M{phi} with abilities to produce IL-12/CCL3, to kill bacteria/tumor cells, and to induce Th1 cells are specifically designated as M1M{phi} (31, 36). It has been reported that IL-1, TNF-{alpha}, IL-6, and NO are not only produced by M1M{phi}, but also produced by a subset of M2M{phi} (18). These facts indicate that NO- and proinflammatory cytokine-producing M{phi} detected in mice 4 to 10 days postburn injury may belong to M2bM{phi}. In this study, M2M{phi} generated from resident M{phi} under the TI-PMN stimulation will be classified with the M2aM{phi} subset, because CCL17 is detected in culture fluids of these M2M{phi} preparations. The incidence of infections remains high in patients 1 or more weeks after burn injury. In fact, Gram-negative and positive bacteria are frequently isolated from peripheral blood of patients 1 to 3 wk after burn injury (37, 38, 39), and a majority of these infections develop into sepsis. In our recent studies, M2aM{phi} and M2cM{phi} appeared in mice 1 to 3 days after burn injury, and then disappeared from the mice within 5 days of burn injury (Shigematsu, K., M. Kobayashi, D.N. Herndon and F. Suzuki, unpublished data). In contrast, M2bM{phi} appeared in mice ~1 wk after burn injury. These facts suggest that M2bM{phi} may play a role on the susceptibility of mice late after burn injury.

In our previous studies (40), PMN displaying PMN-II properties were found in the peripheral blood of burn patients (3rd degree, >40% total body surface area burns). When PMN from five healthy donors and eight burn patients were stimulated with SAC, seven of eight patient PMN produced CCL2 and IL-10 (CCL2, 639~54,782 pg/ml; IL-10, 183~13,541 pg/ml), while none of the healthy donor PMN produced these soluble factors (CCL2, < 30 pg/ml; IL-10, < 12 pg/ml). These results suggest that burn patients are carriers of PMN-II (or TI-PMN).

Apoptotic process of PMN from humans and rodents with severe burn injuries have been described to be inhibited (41, 42). It is possible that the prolongation of PMN in burned hosts may result in the long-term stimulation of M{phi} conversion from resident M{phi} to M2M{phi}. To determine whether PMN apoptosis contributed to the results shown in this study, SCIDbgN mice were inoculated i.v. with 1 x 106 cells/mouse of normal neutrophils (A) every 12 h or (B) once and exposed orally to 1 LD50 of E. faecalis. After the infection, 50% of group A and group B mice survived equally. Also, when SCIDbgN mice were inoculated i.v. with 1 x 106 cells/mouse of TI-PMN (C) every 12 h or (D) once, and infected with the same amount of E. faecalis, 100% of group C and group D mice died equally. Therefore, in our experimental model, the antibacterial resistance of mice is not influenced by apoptotic frequency of PMN.

The mechanism by which normal PMN differentiate into PMN-II (TI-PMN) remains unclear. However, prostaglandin E2 and catecholamines have abilities to induce immature myeloid cells (Gr-1+CD11b+ cells) (43). The increased levels of stress hormones (corticosteroids, catecholamines) and prostaglandin E2 have been demonstrated in the plasma of burn hosts (44, 45, 46, 47). PMN-II and Gr-1+CD11b+ immature myeloid cells have been demonstrated to be very similar to each other. CCL2 and IL-10, effector soluble factors of TI-PMN, have been detected in the culture fluids of Gr-1+CD11b+ immature myeloid cells. These descriptions suggest that prostaglandin E2 and stress hormones may be involved in the PMN conversion to PMN-II. Further studies are required to explore the role of prostaglandin E2 and the burn-associated PMN conversion from normal PMN to PMN-I.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Shriners of North American Grant 8840 (to F.S.). Back

2 Address correspondence and reprint requests to Dr. Fujio Suzuki, Department of Internal Medicine, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555. E-mail address: fsuzuki{at}utmb.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; M{phi}, macrophage; M1M{phi}, classically activated M{phi}; SCIDbg, SCID-beige mice; TI-PMN, PMN from thermally injured SCIDbg mice; M2M{phi}, alternatively activated M{phi}; SCIDbgN mice, SCIDbg mice depleted of PMN; SCIDbgMN mice, SCIDbgN mice depleted of M{phi}; iNOS, inducible NO synthase; SAC, Staphylococcus aureus Cowan I. Back

Received for publication July 13, 2007. Accepted for publication January 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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K. Shigematsu, A. Asai, M. Kobayashi, D. N. Herndon, and F. Suzuki
Enterococcus faecalis translocation in mice with severe burn injury: a pathogenic role of CCL2 and alternatively activated macrophages (M2aM{phi} and M2cM{phi})
J. Leukoc. Biol., October 1, 2009; 86(4): 999 - 1005.
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