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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1445-1450, (2002)
© 2002 American Thoracic Society

Brief Communication

Differential Role of Interleukin-6 in Lung Inflammation Induced by Lipoteichoic Acid and Peptidoglycan from Staphylococcus aureus

Jaklien C. Leemans, Margriet J. B. M. Vervoordeldonk, Sandrine Florquin, Kok P. van Kessel and Tom van der Poll

Laboratory of Experimental Internal Medicine, Division of Clinical Immunology and Rheumatology, Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam; and Eijkman-Winkler Institute, Utrecht University, Utrecht, The Netherlands

Correspondence and requests for reprints should be addressed to Tom van der Poll, Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, G2-130, 1105 AZ Amsterdam, The Netherlands. E-mail: t.vanderpoll{at}AMC.UVA.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoteichoic acids (LTA) and peptidoglycans (PepG) are major components of the cell walls of gram-positive bacteria that trigger inflammatory responses in vitro. To study the in vivo effects of LTA and PepG from Staphylococcus aureus in lungs and to determine the role of interleukin (IL)-6 herein, these compounds were intranasally administered to IL-6 gene deficient (IL-6-/-) and wild type (IL-6+/+) mice. In IL-6+/+ mice, LTA and PepG induced acute pulmonary inflammation in a dose-dependent way, characterized by neutrophilic influx and IL-6 production in the bronchoalveolar lavage fluid. Endogenously produced IL-6 attenuated inflammation induced by 10 µg LTA, as reflected by enhanced neutrophil influx, and increased tumor necrosis factor-{alpha}, macrophage inflammatory protein-1-{alpha}, and cytokine-induced neutrophil chemoattractant (KC) release into bronchoalveolar lavage fluid of IL-6-/- mice, compared with IL-6+/+ mice. By contrast, pulmonary inflammation induced by 100 µg LTA was similar (neutrophil influx) or even tended to be attenuated (cytokine and chemokine release) in IL-6-/- mice. Endogenous IL-6 increased inflammation induced by PepG, as reflected by decreased neutrophil influx into lungs of IL-6-/- mice, compared with IL-6+/+ mice. These data suggest that IL-6 plays an anti-inflammatory role during LTA-induced pulmonary inflammation, which is dependent on the severity of the inflammatory challenge, and a proinflammatory role in peptidoglycan-induced acute lung inflammation. Thus, the contribution of IL-6 to lung inflammation may vary with the stimulus used.

Key Words: interleukin-6 • lipoteichoic acid • peptidoglycan • lung • anti-inflammatory


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of gram-positive infections has increased considerably over the past few years, and gram-positive organisms are now as common as gram-negative bacteria in causing sepsis (1–3). Staphylococcus aureus is the most frequently isolated gram-positive pathogen in nosocomial infections associated with severe complications (4). In addition, S. aureus accounts for 6–33% of bacterial isolates from patients with hospital-acquired pneumonia (5, 6). Gram-positive inflammation is presumed to be due to bacterial cell wall components, such as lipoteichoic acid (LTA) and peptidoglycan (PepG). LTA are phosphate-containing polymers that are considered to be surface antigens, as well as membrane components that mediate the attachment of certain bacteria to host cells (7, 8). PepG is a glycosyl macromolecule interlinked by peptide bridges, which is especially abundant in gram-positive bacteria, where it provides stress-resistance and determines the form of the bacterial cell wall. Both LTA (9, 10) and PepG (10–12) can induce inflammatory responses in vitro. Furthermore, intravenous administration of S. aureus LTA and PepG resulted in a systemic inflammatory response syndrome in rats (13). Knowledge of the in vivo effect of LTA or PepG from S. aureus within the lungs is highly limited. Such knowledge is important, considering the role of S. aureus in nosocomial pneumonia.

Several lines of evidence suggest that the pleiotropic cytokine interleukin (IL)-6 is involved in regulation of inflammatory responses during gram-positive bacterial infection. Elevated IL-6 concentrations were detected in plasma of patients with gram-positive sepsis (14) and in bronchoalveolar lavage fluid (BALF) of patients with pneumonia (15). In a murine model of pneumococcal pneumonia, this endogenously released IL-6 played an important role in antibacterial host defense, as reflected by enhanced bacterial outgrowth and increased mortality in IL-6 gene–deficient mice (16). The role of IL-6 in the pathophysiology of inflammation in general, and of inflammation in the pulmonary compartment in particular, has not been elucidated completely. Indeed IL-6 has been reported to have both proinflammatory and antiinflammatory effects (17, 18).

In the present study we sought to determine the acute pulmonary inflammatory response caused by local exposure to LTA or PepG from S. aureus and the regulatory role of IL-6 herein. For this purpose, IL-6 gene–deficient (IL-6-/-) and wild type (IL-6+/+) mice received LTA or PepG via the intranasal route.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
For experiments with LTA, 8- to 10-week-old female IL-6-/- mice on a BALB/c background were kindly donated by Dr. Manfred Kopf (Basel Institute of Immunology, Basel, Switzerland). IL-6+/+ BALB/c mice were purchased from Harlan Sprague Dawley Inc. (Horst, The Netherlands). At the time when the studies with PepG were done, IL-6-/- BALB/c mice were not available. For these experiments, 8- to 10-week-old female IL-6-/- mice on a C57Bl/6 background and normal C57Bl/6 wild type mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The Animal Care and Use Committee of the University of Amsterdam, The Netherlands, approved all experiments.

Materials
LTA from S. aureus was purchased from Sigma (St. Louis, Mo). PepG was prepared from S. aureus (a clinical isolate derived from a patient with catheter-related sepsis) according to the method of Peterson and colleagues (19) as described earlier (12). The amount of lipopolysaccharide (LPS) present in LTA and PepG was determined with the chromogenic Limulus Amoebocyte Lysate assay (Chromogenix, Mölndal, Sweden) and was, respectively, 4.15 pg LPS/mg LTA and below the detection limit (2.5 pg/ml). Hence, 100 µg LTA (the highest dose used in our experiments) contained less than 1 pg LPS. This LPS dose is not capable of eliciting an inflammatory reaction in the lung (data not shown).

Experimental Design
Briefly, mice were anesthetized by inhalation with isoflurane (Abbott Laboratories Ltd., Kent, UK), after which sterile saline, LTA, or PepG dissolved in saline was administered intranasally. After 4 hours, mice were anesthetized by intraperitoneal injection of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Roche, Mijdecht, the Netherlands) and killed by bleeding from the vena cava inferior, and lungs were lavaged.

Analysis of BALF
Bronchoalveolar lavage (BAL) and leukocyte differentiation was done as described previously (20, 21). BALF cytokines were measured by specific ELISAs according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The detection limits were 37 pg/ml (IL-6), 31 pg/ml (tumor necrosis factor-{alpha} [TNF{alpha}]), 8 pg/ml (IL-10), 8 pg/ml (IL-1{alpha}), 8 pg/ml (IL-1ß), 31 pg/ml (macrophage inflammatory protein [MIP]-1{alpha}), 12 pg/ml (KC), and 47 pg/ml (MIP-2).

Histologic Analyses
Lungs for histologic examination were harvested at 4 hours after infection, fixed in 4% formalin, and embedded in paraffin. Sections of 4 µm were stained with hematoxylin and eosin. Granulocyte staining was done with fluorescein isothiocyanate–labeled anti-mouse Ly-6-G mAb (Pharmingen, San Diego, CA) exactly as described previously (22). The inflammatory infiltrate was scored semiquantitatively, and the number of abscesses (well-defined inflammatory collections) was counted per 12 mm2 (five fields of x10).

Stimulation of Peritoneal Macrophages
Peritoneal macrophages from IL-6+/+ (C57Bl/6) and IL-6-/- mice (C57Bl/6 background) were isolated by washing the peritoneal cavity with RPMI 1640 (Bio Whittaker, Verviers, Belgium). Collected cells maintained in medium (RPMI 1640, 10% fetal calf serum, 1% antibiotic–antimycotic [GIBCO-BRL Life Technologies, Rockville, MD]) were allowed to adhere to 96-well tissue culture plates (1 x 104 cells) for 1 hour at 37° C, after which nonadherent cells were removed by rinsing the monolayer. Macrophages were incubated with medium containing either LTA or PepG.

Statistical Analysis
All values are expressed as mean ± SEM. Differences between groups were analyzed by Mann–Whitney U test. A p value of of 0.05 or less was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LTA Induces a Dose-Dependent Influx of Polymorphonuclear Cells and Release of IL-6 in BALF
To determine pulmonary inflammatory responses to LTA in vivo, IL-6+/+ mice were intranasally inoculated with either sterile saline (control mice) or 10, 100, or 150 µg LTA, and BAL was performed after 4 hours. LTA induced a dose-dependent increase in leukocytes in BALF. When compared with control mice, BALF leukocyte counts were 5-fold higher after administration of 10 µg LTA, 20-fold higher after instillation of 100 µg LTA, and 38-fold higher after inoculation with 150 µg LTA (Figure 1A) . The LTA-induced increase in the number of cells in BALF mainly resulted from influx of polymorphonuclear cells (PMN), although numbers of alveolar macrophages were also increased. Lymphocytes were present in low numbers and were similar in all groups. Inhalation of LTA was also associated with a dose-dependent release of IL-6 in BALF (Figure 1B), whereas IL-6 remained undetectable in BALF of saline-treated animals. In subsequent experiments, the role of IL-6 in LTA-induced lung inflammation was investigated after administration of either 10 or 100 µg LTA.



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  		Figure 1. (A) Cellular composition of BALF after administration of LTA. (B) Dose-dependent increase of IL-6 in BALF after inoculation of LTA. LTA or saline (control ) was administered intranasally. BALF was obtained after 4 hours. Data represent the mean ± SEM of four mice per group. *p < 0.05 versus control.

 
Inflammatory Responses in IL-6-/- and IL-6+/+ Mice After a Low Dose of LTA
To investigate the role of IL-6 in the pulmonary effects of LTA at a dose that caused mild lung inflammation, IL-6+/+ and IL-6-/- mice received 10 µg LTA intranasally, and BALF was obtained 4 hours later. The absence of IL-6 resulted in higher leukocyte counts in the BALF, which was mainly caused by a greater influx of PMN (Figure 2) .



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  		Figure 2. Effect of LTA on cellular composition and cytokine and chemokine concentrations of BALF from IL-6+/+ mice (open squares) and IL-6-/- mice (closed squares). 10 µg LTA (left panel ) or 100 µg LTA (right panel ) was given intranasally, and BALF was obtained 4 hours thereafter. Data represent the mean ± SEM of eight mice per group. *p < 0.05 versus IL-6+/+ mice. Note that cytokine and chemokine concentrations induced by 100 µg LTA were markedly higher than those induced by 10 µg LTA, which is why the Y-axes are different in the left and right panels.

 
In accordance with the cell counts in the BALF, the lungs of IL-6-/- mice (Figure 3A) treated with low dose LTA showed more diffuse and dense inflammatory infiltrates than did IL-6+/+ mice (Figure 3B) 4 hours after inoculation. The influx of PMNs was also increased in IL-6-/- (inset of Figure 3A) mice, compared with IL-6+/+ mice (inset of Figure 3B).



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  		Figure 3. Representative histologic sections of lungs of IL-6+/+ (B) and IL-6-/- (A) mice 4 hours after intranasal inoculation with 10 µg LTA. Hematoxylin and eosin staining, original magnification x50; inset, antigranulocyte immunostaining, original magnification x80.

 
To understand the impact of IL-6 deficiency on cytokine and chemokine responses, TNF-{alpha}, IL-1{alpha}, IL-1ß, IL-10, MIP-1{alpha}, KC, and MIP-2 were measured in BALF. Whereas LTA induced a TNF-{alpha} response in both groups of mice, BALF TNF-{alpha} concentrations were more than 5-fold higher in IL-6-/- mice than in IL-6+/+ mice (p = 0.002) (Figure 2). Further, in IL-6-/- animals, MIP-1{alpha} and KC concentrations were, respectively, 2.6-fold (p = 0.01) and 1.5-fold higher (p = 0.07) than in IL-6-/- mice, whereas MIP-2 concentrations were not different between the two groups (Figure 2). LTA did not induce a detectable release of IL-1{alpha}, IL-1ß, or IL-10 in the BALF of either mouse strain.

Inflammatory Responses in IL-6-/- and IL-6+/+ Mice After a High Dose of LTA
In contrast to pulmonary inflammation induced by 10 µg LTA, administration of 100 µg LTA was not associated with differences in leukocyte influx in IL-6-/- and IL-6+/+ mice (Figure 2). Furthermore, TNF-{alpha}, MIP-1{alpha}, and MIP-2 (p = 0.012) tended to be lower in IL-6-/- mice, whereas KC concentrations were similar in the BALF of both mice strains.

PePG Induces a Dose-Dependent Influx of PMN and Release of IL-6 in BALF
Next, we wished to determine the role of IL-6 in pulmonary inflammatory responses to PepG from S. aureus. To determine the dose of PepG that induced an immune response comparable to 10 µg LTA, IL-6+/+ mice were intranasally inoculated with either 50 or 180 µg PepG, and BAL was performed 4 hours later. Inoculation with PepG resulted in a dose-dependent increase in numbers of leukocytes and IL-6 concentrations in the BALF (Figure 4) . Leukocyte differentiation revealed that the increase in BALF cells resulted from an influx of PMN. Additional experiments using IL-6+/+ and IL-6-/- mice were performed with 50 µg PepG.



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  		Figure 4. (A) Cellular composition of BALF after administration of PepG. (B) Dose-dependent increase of IL-6 in BALF after inoculation of PepG. PepG or saline (control ) was administered intranasally. BALF was obtained after 4 hours. Data represent the mean ± SEM of four mice per group. *p < 0.05 versus control.

 
Inflammatory Responses in IL-6-/- and IL-6+/+ Mice After PepG
Having established that PepG induced an influx of PMN into lungs, we were interested in the role of IL-6 herein. Surprisingly, the number of recruited PMN in the lungs was markedly decreased in IL-6-/- mice, compared with IL-6+/+ mice at 4 hours after inoculation (p = 0.004) (Figure 5) .



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  		Figure 5. Effect of PepG on cellular composition and cytokine and chemokine concentrations of BALF from IL-6+/+ mice (open squares) and IL-6-/- mice (closed squares). PepG (50 µg) was given intranasally, and BALF was obtained 4 hours thereafter. Data represent the mean ± SEM of eight mice per group. *p < 0.05 versus IL-6+/+ mice.

 
Histopathologic analysis of the lungs of mice inoculated with PepG revealed numerous well-defined collections of PMN (abscesses) generally centered around small bronchi together with a slight interstitial inflammatory infiltrate. In line with the results of cell counts, IL-6-/- mice (Figure 6A) displayed fewer abscesses, mostly consisting of PMN, than did IL-6+/+ mice (Figure 6B) (13.1 ± 2.4 versus 17.4 ± 3.3 per 12 mm2, not statistically significant) (see insets of Figure 6).



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  		Figure 6. Representative histologic sections of lungs of IL-6+/+ (B) and IL-6-/- (A) mice after intranasal inoculation with 50 µg PepG. Hematoxylin and eosin staining, original magnification x50; insert, antigranulocyte immunostaining, original magnification x80.

 
To determine the role of IL-6 in PepG-induced cytokine and chemokine release in the lungs, the concentrations of TNF-{alpha}, MIP-1{alpha}, MIP-2, and KC were measured in the BALF of IL-6-/- and IL-6+/+ mice, 4 hours after PepG administration. No significant differences were found between IL-6+/+ and IL-6-/- mice (Figure 5).

Role of IL-6 in TNF-{alpha} Release by Isolated Macrophages
Having established that endogenous IL-6 inhibited TNF-{alpha} release in BALF in response to LTA but not to PepG, we were interested in the role of IL-6 in TNF-{alpha} production by isolated macrophages. To determine whether LTA and PepG of S. aureus can induce IL-6 release in vitro, mouse peritoneal IL-6+/+ macrophages were exposed to increasing concentrations of LTA and PepG for 4 hours, and the concentrations of IL-6 were evaluated in culture supernatants. As shown in Figures 7A and 7B , both LTA and PepG induced IL-6 in a dose-dependent manner. To investigate the role of IL-6 in macrophage responses to LTA and PepG, we examined the responsiveness of IL-6+/+ and IL-6-/- macrophages to increasing concentrations of these bacterial components. The production of TNF-{alpha} in response to S. aureus LTA was significantly higher in IL-6-/- mice at the two highest doses (Figure 7C). In contrast, IL-6+/+ and IL-6-/- macrophages produced comparable amounts of TNF-{alpha} in response to S. aureus PepG (Figure 7D).



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  		Figure 7. IL-6 production by isolated macrophages in response to LTA (A) and PepG (B). Effect of LTA (C) and PepG (D) on TNF-{alpha} release by isolated IL-6+/+ (open squares) and IL-6-/- (closed squares) macrophages. Stimulation was performed for 4 hours at 37° C with increasing concentrations of LTA (0, 1, 10, and 100 µg/ml) or PepG (0, 10, 100, and 1,000 µg/ml). Data represent the mean ± SEM of macrophages derived from six mice per group. (A and B) *p < 0.05 versus control. (C and D) *p < 0.05 versus IL-6+/+ macrophages.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
S. aureus is a major cause of hospital-acquired pneumonia (6). LTA and PepG are cell wall components of S. aureus that have proinflammatory activities in vitro. The present study reports on the in vivo effects of LTA and PepG from S. aureus in the lung. Intranasal administration of LTA or PepG led to enhanced leukocyte influx predominated by PMN. Increasing the dose clearly led to an increase in pulmonary inflammation. In addition, LTA and PepG induced elevated lung concentrations of IL-6 in a dose-dependent manner. Endogenous IL-6 was found to exert a negative feedback effect on PMN recruitment and the local release of TNF-{alpha}, MIP-1{alpha}, and KC at the lower, but not the higher, LTA dose tested. In contrast to its role in the pulmonary response to low dose LTA, endogenous IL-6 positively influenced PMN influx into the lungs after PepG instillation.

LTA can signal partially via the same mechanisms as LPS from gram-negative bacteria. CD14 functions as a ligand binding receptor of LPS (23) but was also shown to recognize LTA (24). In addition, the LPS signaling receptor toll-like receptor (TLR)4 is likely also involved in the recognition of LTA (25). Although data exist indicating that TLR2 can be a signal transducer for LTA from Bacillus subtilis, Streptococcus pyogenes, and Streptococcus sanguis (26), others have shown that TLR4, and not TLR2, is required for signal transduction of S. aureus LTA (25, 27, 28). TLR2 has been reported to be the signaling receptor for PepG from S. aureus (25, 29). The data of the current study suggest that intrapulmonary administration of LTA provokes an inflammatory response that is very similar to that induced by LPS. LPS administered to the pulmonary compartment caused a PMN influx and induction of TNF-{alpha} and IL-6 in the BALF of mice (30, 31). Likewise, in this study, intranasal administration of LTA resulted in an increase in PMN and elevated BALF concentrations of TNF-{alpha} and IL-6. Like LTA, the intranasal inoculation of PepG also resulted in increased PMN influx and cytokine concentrations, but pathologic analysis of the lungs of these mice revealed extensive differences when compared with lungs of LTA-inoculated mice. LTA-inoculation induced an interstitial inflammatory infiltrate in the lungs, whereas PepG exposure led to the formation of numerous abscesses generally centered around small bronchi and mostly composed of PMN.

Several studies have reported on the importance of IL-6 for inflammatory responses during bacterial pulmonary inflammation. High IL-6 concentrations were found in the BALF of patients with pneumonia (15) and in the lungs and plasma of mice infected with Streptococcus pneumoniae (16). Moreover, elevated IL-6 concentrations were found in mice during acute lung and systemic inflammation caused by LPS (32). IL-6 has proinflammatory as well as antiinflammatory properties. In a carrageenan-induced pleurisy model, endogenous IL-6 played a proinflammatory role, as reflected by reduced PMN infiltration and diminished lung injury in IL-6-/- mice (33). In contrast, IL-6 played an antiinflammatory role in an LPS-induced acute lung inflammation model (32). The data of the current study suggest that IL-6 plays an antiinflammatory role in lung inflammation caused by low dose LTA but that this antiinflammatory role is lost (PMN recruitment) or even is converted into a modest proinflammatory role (cytokine and chemokine release) during lung inflammation induced by high dose LTA. Interestingly, our findings demonstrate that IL-6 plays a proinflammatory role (PMN recruitment, abscess formation) in pulmonary inflammation induced by PepG of S. aureus. Together, these data suggest that the role of IL-6 in inflammation depends on the stimulus and/or the model of inflammation used. As mentioned earlier, LTA and PepG can signal via different receptors, which could be the basis for the pleiotropic characteristics of IL-6. During inflammation induced by LPS (32) and LTA (this study), bacterial components that signal at least in part via TLR4, IL-6 plays an antiinflammatory role. However, a proinflammatory role of IL-6 has been demonstrated in mice with a PepG-induced pulmonary inflammation (this study) and in mice with a nonseptic shock induced by zymosan (34). Interestingly, zymosan and PepG are both ligands for TLR2 (27).

TNF-{alpha} production by IL-6-/- macrophages was higher than that of IL-6+/+ macrophages after incubation with LTA. In addition, BALF TNF-{alpha} concentrations were higher in IL-6-/- than in IL-6+/+ mice after low dose LTA, suggesting that endogenous IL-6 inhibits TNF-{alpha} production after LTA stimulation. This is in line with observations that IL-6 inhibits transcription of the TNF-{alpha} gene (35). Moreover, recombinant IL-6 reduced TNF-{alpha} release in mice administered LPS intratracheally (36). It is furthermore known that TNF-{alpha} is an inducer of IL-6; therefore, the inhibitory effect of IL-6 on TNF-{alpha} production elicited by low dose LTA may be the negative arm of a regulatory circuit. In contrast, TNF-{alpha} concentrations in supernatants of PepG-stimulated IL-6-/- macrophages and BALF of PepG-inoculated IL-6-/- mice were not significantly different from those of IL-6+/+ macrophages and mice. In a model of carrageenan-induced pleurisy (33) and collagen-induced arthritis (37), IL-6-/- mice had even lower TNF-{alpha} concentrations than did IL-6+/+ mice. Together, these indicate that TNF-{alpha} production is regulated in a different way during LPS- (32) and low dose LTA-induced acute lung inflammation versus high dose LTA- and PepG-induced pulmonary inflammation.

Elevated concentrations of MIP-1{alpha} in the lung after low dose LTA could be a consequence of increased TNF-{alpha} concentrations. Exogenous TNF-{alpha} has been shown to be a potent stimulator for MIP-1{alpha} secretion by human PMN, and neutralizing anti–TNF-{alpha} antiserum partially blocked this expression (38). Interestingly, an IL-6 deficiency resulted in increased KC release in BALF without influencing MIP-2 concentrations after low dose LTA, suggesting that IL-6 influences the production of these chemokines differentially. Although a clear explanation for this finding is not available, a recent investigation also reported on differential expression of MIP-2 and KC during lung inflammation. Indeed, mice deficient for both type I TNF-{alpha} receptor and type I IL-1 receptor demonstrated reduced release of KC, but unaltered MIP-2 secretion into BALF, upon pulmonary exposure to Escherichia coli (39).

These data indicate that IL-6 plays a role in increasing PMN accumulation at sites of low dose LTA-induced lung inflammation, probably at least in part by increasing the local induction of cytokines/chemokines. Although MIP-2 concentrations were moderately decreased in IL-6-/- mice, alterations in cytokine/chemokine concentrations could not explain the reduced PMN recruitment after PepG administration.

We chose to investigate inflammatory responses in BALF at 4 hours after intranasal administration of LTA or PepG for several reasons. First, this time point seems most suitable for concurrently evaluating neutrophil influx and cytokine release (32, 40). Second, Xing and coworkers demonstrated that this time point is suitable for reliably studying the role of endogenous IL-6 in LPS-induced lung inflammation (32). Third, many previous studies investigated the regulation of LPS-induced pulmonary inflammation 3–6 hours after the challenge, allowing easy comparison with the present data (41–43).

In conclusion, we demonstrate that in vivo administration of LTA and PepG from S. aureus to the pulmonary compartment triggered acute lung inflammation, characterized by PMN influx and a strong induction of IL-6 and other cytokines and chemokines in BALF. The absence of IL-6 resulted in a more profound proinflammatory response at a LTA dose that caused relatively mild inflammation. At a higher LTA dose, however, IL-6 deficiency was not associated with antiinflammatory effects. The absence of IL-6 during PepG-induced pulmonary inflammation resulted in a reduced PMN infiltration. These data suggest that the role of IL-6 in lung inflammation induced by S. aureus LTA and PepG depends on the severity of the challenge and on the stimulus used.


   Acknowledgments
 
The authors wish to thank Joost Daalhuisen and Nike Claessen for their expert technical assistance.

Supported by a grant from The Netherlands Organization for Scientific Research (J. C. L.)

Received in original form June 13, 2001; accepted in final form March 22, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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