Introduction

Pulmonary infection is a major cause of mortality and morbidity (1), particularly in patients who are critically ill (2,3). The innate immune response plays a critical role in host defense. This concerted host reaction to bacteria is influenced by the complex interplay between genetics, epigenetics and the environment and can be organ specific (4–7). Staphylococcus aureus is a leading cause of hospital-acquired and community-associated pneumonia and is typically associated with severe disease (8–10). Although many studies have led to a better understanding of the epidemiology, resistance mechanisms and potential virulence, there is still a paucity of knowledge about the mechanisms involved in the in vivo host immune response to S. aureus infections (11).

Lipoteichoic acid (LTA) and peptidoglycan (PGN) are components of the cell wall of Gram-positive bacteria, such as S. aureus, and can stimulate the generation of proinflammatory cytokines and activate leukocytes in vitro and in vivo (12–16). LTA and PGN can also induce lung inflammation in mice (16) and F344 rats (17). F344 rats have previously been shown to be arthritis resistant, whereas DA rats are susceptible to this inflammatory disease (18). Results of previous studies have demonstrated that neutrophil and macrophage migration into an inflammatory site, such as the synovial-like air pouch injected with carrageenan, is genetically regulated (19). Specifically, arthritis-susceptible DA rats have greater neutrophil and macrophage migration into the synovial inflammatory site than arthritis-resistant F344 rats (19). The difference in neutrophil migration in that model is regulated by the arthritis-severity quantitative trait loci (QTLs) Cia4 and Cia6 (19), located on rat chromosomes 7 and 8, respectively. Moreover, other arthritis QTLs, such as Cia3d and Cia5a, have also been shown to regulate joint inflammatory responses (18,20).

Neutrophils and macrophages are critical to the pathogenesis of acute lung injury (21,22), rheumatoid arthritis (23,24) and other inflammatory diseases. Cytokines, produced by neutrophils and macrophages such as tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF) and high mobility group box 1 (HMGB1) mediate arthritis damage and acute lung injury (25–30). These data suggest that arthritis and acute lung injury share some inflammatory mechanisms. However, it remains unclear whether the genetic differences between DA and F344 rats, in particular in arthritis QTLs, are associated with differences in the regulation of lung inflammation.

In the present study we used a pneumonia model in which lung inflammation was induced by Gram-positive bacterial components. We examined the differences in pulmonary inflammation between DA and F344 rats and investigated whether these differences are regulated by arthritis QTLs and what other factors may influence differences in lung inflammation.

Materials and Methods

Reagents

LTA and PGN from S. aureus (Sigma-Aldrich, St Louis, MO, USA) were dissolved in 0.9% saline to the target concentration, stored at −20°C, and used within 60 d.

Animals

All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research.

Arthritis-resistant F344 and arthritis-susceptible DA rats were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA). DA.F344(Cia3d), DA.F344(Cia4), DA.F344(Cia5a) and DA.F344(Cia6) congenics were generated as previously described (18,20). Briefly, arthritis QTLs were introgressed from F344 into DA background rats through 8 to 10 genotype-guided backcrosses followed by at least 5 intercrosses. All the animals were housed in a pathogen-free environment, under standard light and dark cycles, with free access to food and water.

In Vivo Experimental Protocol

Rats were males 8- to 14-wks old and were F344 (n = 7), DA (n = 7), or the congenic strains DA.F344(Cia3d), DA.F344(Cia4), DA.F344(Cia5a) and DA.F344(Cia6) (n = 5/congenic strain) with body weights of 201–305 g. Rats were anesthetized with isoflurane. The tracheas were surgically exposed and LTA 10 µg/kg and PGN 33 µg/kg (a dose that we have shown previously to induce lung inflammation [31]) were instilled intratracheally. The animals were allowed to recover from anesthesia and have access to food and water ad libitum. After 18 h, the rats were euthanized and the lungs were lavaged twice with saline (0.9% w/v, 35 mL/kg/lavage). The bronchoalveolar lavage fluid (BALF) from each lavage was collected into separate tubes. The tubes were centrifuged and the supernatant stored at −80°C. Only the supernatant from the first tube was used for measuring chemokines, cytokines and protein content, whereas BALF from both tubes was used for cell counting. After lavage, lung tissue was removed and immediately frozen in liquid nitrogen for myeloperoxidase (MPO), keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) quantification. Control F344 rats and DA rats (n = 5/group) were instilled with the same volume of 0.9% saline and then treated as described above.

In Vitro Alveolar Macrophage Chemokine Response to LTA and PGN

For the in vitro studies, F344 and DA rats (n = 5/group) were euthanized without prior instillation. The lungs were lavaged 5× with saline (0.9% w/v, 35 mL/kg/lavage). The BALF was centrifuged, and cells were mixed with 0.5 mL 0.2% saline for 10 s to lyse any erythrocytes. The cells were then resuspended in 10 mL Hanks’ Balanced Salt Solution (HBSS) and recentrifuged, and the macrophage pellet was collected. The resulting macrophages were 99% pure, as determined by HEMA 3 Stain Set stain (Fisher Scientific, Kalamazoo, MI, USA) (Figure 1A, B), and were 98% viable, based on trypan blue exclusion. The cells were resuspended at 2.5 × 105 cells/mL in RPMI 1640 with 1% fetal bovine serum and 1% penicillin/streptomycin.

Figure 1
figure 1

The BALF cell populations. (A) and (B) show cells directly lavaged from naïve F344 and DA rats. These macrophages were used in the in vitro studies. (C) and (D) were lavaged from F344 and DA rats receiving saline instillation, and show a predominance of macrophages. (E) and (F) show cells lavaged from lungs F344 and DA, respectively, 18 h after instillation of LTA/PGN, and show the influx of neutrophils.

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Macrophages from each animal were distributed to three different wells at 1 mL/well in 24-well low-attachment cell culture plates (Corning Costar, Cambridge, MA, USA) and cultured in medium, as described above, to which LTA 0.5 µg/mL + PGN 1.65 µg/mL, LTA 1.0 µg/mL + PGN 3.3 µg/mL or an equal volume of 0.9% saline (control) was added, for 6 h at 37°C in 5% CO2. The cell culture medium was then centrifuged and the supernatant stored at −80°C for the chemokine assay.

In Vitro Alveolar Macrophage Mitogen-Activated Protein Kinase Activation Study

To explore the differences in mitogen-activated protein kinase (MAPK) activation between these two strains, alveolar macrophages (AMs) lavaged from F344 and DA strains were stimulated by LTA and PGN in vitro (LTA 1.0 µg/mL + PGN 3.3 µg/mL). The whole cell lysates were collected at different time points (0, 1 and 6 h). The MAPK pathway markers (phosphorylated extracellular signal-regulated kinases 1 and 2 [ERK1/2] and P38) were determined in the cell lysates by Western blot. In addition, total ERK1/2 and total P38 protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control were determined by stripping the same membrane after the phosphorylated forms had been assessed. Toll-like receptor 2 (TLR2) levels in AMs from F344 and DA were also determined by Western blot. Antibodies used for Western blots (phosphor-P38, total-P38, phosphor-ERK1/2 and GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, USA). Total ERK1/2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TLR2 antibody was purchased from Abcam (Cambridge, MA, USA).

In Vitro MAPK Inhibition Study

To further detect the role of MAPK ERK1/2 and P38 pathways in the regulation of chemokine production, the effects of MAPK inhibition on KC and MIP-2 production were examined. AMs were prepared by using the method described above. The specific inhibitors for p38 (SB-203580; EMD Chemicals, Gibbstown, NJ, USA) and ERK1/2 (PD-98059; Calbiochem, San Diego, CA, USA) were added to the AMs of F344 and DA rats 30 min prior to LTA/PGN stimulation (LTA 1.0 µg/mL + PGN 3.3 µg/mL). After 6 h, cell culture medium was collected. KC and MIP-2 in the medium were measured by using an ELISA method as described below. To exclude the possibility that the inhibitors may have caused cell damage, a cytotoxicity study was performed at the same inhibitor doses and contact time, and the culture medium was assessed for lactate dehydrogenase (LDH) activity (Takara Bio, Shiga, Japan).

BALF Total Cell and Differential Cell Counts

BALF cells were mixed with 0.5 mL of 0.2% saline for 10 s to lyse any residual erythrocytes, and the cells were then re-suspended in 10 mL HBSS. The total cell number retrieved in the BALF was determined with a hemocytometer. Slides were prepared using a Shandon cytocentrifuge (Shandon Scientific, London, UK), and cells were stained with an HEMA 3 Stain Set (Fisher Scientific) for differential cell counting. Counts were made on at least 200 cells per slide by an observer blinded to the grouping.

Cytokine and Chemokine Assays

TNF-α, KC and MIP-2 were measured by enzyme-linked immunosorbent assay (ELISA) using commercial assay kits (TNF-α: R&D Systems, Minneapolis, MN, USA; KC and MIP-2: Antigenix America, Huntington Station, NY, USA) according to manufacturers’ instructions.

MPO Activity Measurement

MPO was measured by using a commercially available assay kit (CytoStore, Calgary, Alberta, Canada) according to the manufacturer’s instructions. Briefly, lung tissues were weighed and sample lysis buffer was added at 12 mL per gram of tissue, followed by tissue homogenization. Supernatant from each sample homogenate was collected. Tissue sample supernatant (20 µL) was combined with 200 µL of the mixture of chromogen and H2O2. Absorbance at 450 nm was measured immediately after addition of chromogen, and again after a 60-sec interval. The absorbance changes during the 60-sec interval were used as a measure of the MPO activity in the sample.

Protein Content Assay

BALF protein concentration was assayed by using the Coomassie (Bradford) Protein Assay Kit (Pierce Company, Rockford, IL, USA). The total protein content was calculated as the product of concentration and BALF volume.

Statistics

Data are presented as mean ± SD. Student t test or one-way ANOVA was used to evaluate the statistical significance of the results. Differences with a P value <0.05 were considered significant.

Results

F344 Rats Have Higher BALF and Lung Tissue Neutrophil Infiltration following LTA and PGN Instillation Than DA Rats

Both F344 and DA rats instilled with LTA and PGN showed significant increases in total BALF cell number compared with controls instilled with saline (Figure 2A). However, the cell recruitment into the alveolar space was eightfold greater in F344 rats than in DA rats (mean ± SD: F344 7.9 ± 2.6 × 106; DA 1.0 ± 0.4 × 106; P < 0.001) (Figure 2A). The increase was predominantly due to neutrophil influx (BALF neutrophil counts: F344 7.0 ± 2.6 × 106 versus DA 6.6 ± 4.5 × 105; P < 0.001) (Figure 2B and Figure 1E, F). Nevertheless, macrophage numbers in the BALF did not change significantly following LTA/PGN instillation in either strain. To determine whether DA neutrophils had been recruited to the lung, but had not crossed into the alveolar space, lung tissue was assessed for neutrophil content based on postlavage MPO assay. However, the lung tissue MPO activity was significantly higher in F344 rats compared with DA rats (P < 0.05) (Figure 2D). This demonstrates that the overall recruitment of neutrophils to the lung following LTA/PGN instillation was significantly different in these two strains.

Figure 2
figure 2

Differential cellular inflammatory reponse in the lung between F344 and DA rats. Following intratracheal instillation of LTA/PGN, both strains (saline control: n = 5/strain; LTA/PGN: n = 7/strain) show increased total numbers of cells in the BALF (A). This increased cell number is predominately due to a neutrophil influx to the lung (B). Although F344 rats had more macrophages in the BALF than DA rats following either saline or LTA/PGN instillation, there was no significant change in the number of macrophages in either strain owing to LTA/PGN (C). MPO was used as an indicator of accumulation of neutrophils within the lung tissue. F344 rats had higher MPO activity in lung tissue than DA rats, following LTA/PGN instillation (D). Bars indicate mean ± SD. **P < 0.01 versus F344 treated by LTA/PGN; *P < 0.05 versus F344 treated with LTA/PGN; ##P < 0.01 versus DA treated with LTA/PGN; #P < 0.05 versus DA treated with LTA/PGN.

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Inflammatory Mediators in BALF Higher in F344 than DA Rats Following LTA/PGN Stimulation

Concentrations of TNF-α in BALF and MIP-2 in both BALF and lung tissue following LTA/PGN instillation were significantly higher in samples from F344 rats than in the equivalent samples from DA rats under the same conditions (Figure 3), suggesting increased inflammatory responses in F344 compared with DA. In addition, the total protein in the BALF from F344 rats was higher than from DA rats following challenge, indicating increased tissue damage.

Figure 3
figure 3

Lung responses to LTA/PGN instillation. F344 rats showed greater inflammatory responses and lung injury to LTA/PGN than DA rats. Following LTA/PGN instillation, there was increased accumulation of cytokines in lung alveolar (A, B, C) and tissue (E, F) samples from both strains. Accumulation of MIP2 (B), TNF-α (C) and protein (D) in the BALF, and MIP-2 in lung tissue (F) were significantly higher in F344 rats, indicating a more robust response and increased damage. Bars indicate mean ± SD. **P < 0.01 versus F344 treated with LTA/PGN; *P < 0.05 versus F344 treated with LTA/PGN; ##P < 0.01 versus DA treated with LTA/PGN; #P < 0.05 versus DA treated with LTA/PGN; ND, not detectable.

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Higher Baseline Numbers of AMs in BALF, and Enhanced In Vitro AM Response in F344 Compared with DA

We next examined the number of AMs in naïve (untreated) rats. The baseline number of AMs in the BALF was significantly higher in F344 than DA (mean ± SD cell counts: 15.4 ± 5.2 × 105 versus 8.2 ± 2.3 × 105; P = 0.022, Figure 4C).

Figure 4
figure 4

LTA/PGN stimulated accumulation of chemokines in culture medium of isolated primary macrophages. In the absence of LTA/PGN, KC and MIP-2 accumulation was very low or nondetectable. LTA/PGN induced a dramatic accumulation of KC and MIP-2 in AM culture medium. AMs isolated from F344 rats accumulated significantly more of the neutrophil chemokines KC and MIP-2 per cell than macrophages isolated from DA rats (A, B). Furthermore, the number of resident AMs retrieved from naïve F344 rats was significantly greater than that from naïve DA rats (C). Data represent individual studies performed on macrophages isolated from five rats from each strain. Bars indicate mean ± SD. *P < 0.05 versus F344 corresponding group. ND, nondetectable.

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AMs from each strain were adjusted to the same concentration (2.5 × 105 cells/mL) and stimulated with LTA and PGN in vitro. After 6 h, F344 AMs produced significantly higher concentrations of KC and MIP-2 than DA AMs (Figure 4A, B). These results demonstrate that F344 not only has increased baseline numbers of AMs, but also its AMs produce increased amounts of neutrophil chemoattractants.

The DA.F344(QTL) Congenic Strains Have Similar Inflammatory Response to LTA and PGN as Their Background DA Strain

In previous studies we have shown that the specific arthritis QTLs Cia4 and Cia6, located on rat chromosomes 7 and 8, respectively, mediate the difference between DA and F344 in neutrophil migration into a synovial-like cavity in response to carrageenan (19). Therefore, we assessed whether either of these QTLs, as well as two additional arthritis QTLs, Cia3d and Cia5a, might account for a genetically regulated difference in lung inflammation induced with LTA and PGN. Following instillation of LTA/PGN, all four congenic strains (DA.F344[Cia3d], DA.F344[Cia4], DA.F344[Cia5a] and DA.F344[Cia6]) developed lung inflammatory responses similar to their DA background, including BALF total cell number, BALF neutrophil counts, BALF macrophage counts, BALF protein content and lung tissue MPO activity (Figure 5). These results suggest that these arthritis and inflammation QTLs do not explain the differences between lung inflammation detected in F344 and DA rats.

Figure 5
figure 5

Differential LTA/PGN-induced lung inflammation between F344, DA, and their congenic strains. Following LTA/PGN challenge, the total number of cells (A), number of neutrophils (B), number of macrophages (C) and total protein (E) in the BALF, and MPO activity in the lung tissue (D) were all significantly higher in samples from F344 than DA rats. There was no statistical difference between any of the four congenic strains and their background DA strain (n = 5/congenic strain). Bars indicate mean ± SD. **P < 0.01 versus F344; *P < 0.05 versus F344.

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Differential MAPK Pathway Responses to TLR2 Stimulation in AMs from F344 and DA Strains

To further explore the mechanism responsible for the strain differences in KC and MIP-2 release by AMs after LTA + PGN stimulation, we focused on the TLR2-MAPK pathway, which is considered to be a major pathway in the regulation of chemokine production during TLR2 stimulation (32,33).

There were similar levels of TLR2 in the AMs from these two strains (P = 0.69). Furthermore, there were no significant differences between the strains, in baseline total ERK1/2 (P = 0.30) and total p38 (P = 0.36). However, the activated forms, that is, the phosphorylated p38 and phosphorylated ERK1/2 levels were significantly higher in the AMs from F344 rats, after LTA + PGN stimulation (Figure 6). Similar results were achieved from three independent repeats. For F344 versus DA at 1-h and 6-h time points, the p-/t-p38 ratios were 1.47 ± 0.20-fold and 1.83 ± 0.52-fold higher (P = 0.015 and 0.049, respectively) and the p-/t-ERK1/2 ratios were 3.09 ± 1.24-fold and 2.59 ± 0.33-fold higher (P = 0.044 and 0.001). These results indicate that different activation (phosphorylation) of the MAPK pathway, but not the total TLR2 expression or total MAPK member expression, is associated with the different chemokine production between these two strains in this AM in vitro LTA + PGN stimulation model.

Figure 6
figure 6

Differential MAPK pathway response to TLR2 stimulation in AMs from F344 and DA strains. AMs from F344 and DA rats were treated with LTA and PGN (LTA 1.0 µg/mL + PGN 3.3 µg/mL) in vitro. Cell lysates were obtained at different time points. Basal level of TLR2 (A), and total P38 and ERK1/2 expressions (B, C) were similar in the two strains. However, the phosphorylation of both P38 and ERK1/2 were higher in the F344 strain (B, C). Similar results were achieved from three independent repeats. The p-P38/t-P38 ratios in F344 rats were 1.47 ± 0.20- and 1.83 ± 0.52-fold higher than DA rats after 1 and 6 h of stimulation, with P = 0.015 and P = 0.049, respectively. Similarly, the p-/t-ERK1/2 ratios in F344 are 3.09 ± 1.24 and 2.59 ± 0.33 fold higher than DA after 1 and 6 h of stimulation, P = 0.044 and 0.001.

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Effects of MAPK Inhibition on AM Chemokine Production

To further examine the role of MAPK pathway in the regulation of chemokine production, effects of MAPK inhibition on KC and MIP-2 production were assessed. In our pilot study, both p38 inhibitor SB-203580 and ERK1/2 inhibitor PD-98059 showed a dose-dependent inhibition of LTA/PGN stimulated KC and MIP-2 production. The minimum doses that can produce maximum inhibitory effects (SB-203850 20 µmol/L and PD-98059 50 µmol/L) were then chosen and used for subsequent experiments. Results showed that both SB-203850 and PD-98059 decreased LTA/PGN-induced KC and MIP-2 production (Figure 7A, B). In addition, if the two inhibitors were used in combination, this led to further inhibition of KC and MIP-2 production, compared with the LTA/PGN-stimulated group (Figure 7A, B). The cytotoxicity study showed that both the single and combined administrations did not increase cytotoxicity (LDH release) compared with the control group (Figure 7C).

Figure 7
figure 7

P38 and ERK1/2 pathway inhibitors inhibit LTA/PGN-induced KC and MIP-2 production. AMs were lavaged from naïve F344 or DA rats and cultured in vitro. Prior to the LTA/PGN stimulation, the ERK1/2 inhibitor P38 inhibitor SB203580 (20 µmol/L), PD98059 (50 µmol/L), or both of them were added and incubated with AMs for 30 min. Then LTA/PGN stimulation (LTA 1.0 µg/mL + PGN 3.3 µg/mL) was added. KC and MIP-2 production in culture medium were measured after 6 h of LTA/PGN stimulation. Blockade of both ERK1/2 and P38 phosphorylation blocked the majority of KC and MIP-2 production (A, B). The reduction in chemokine accumulation was not due to cytotoxicity as assessed by LDH accumulation in the culture medium (C). NC, negative control (culture medium only); PC, positive control (cell treated by 1% Triton X-100). Data represent individual studies performed on AMs isolated from six rats from each strain. Bars indicate mean ± SD. **P < 0.01 versus F344 LTA/PGN group; ##P < 0.01 versus DA LTA/PGN group.

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Discussion

Lung inflammation and pneumonias are major and common causes of morbidity and mortality, particularly in cases associated with S. aureus (10). Neutrophils have a central role in acute lung inflammation (34,35), but the local factors regulating neutrophil influx into the lung remain incompletely understood. In this study, we used a model of acute lung inflammation induced by LTA and PGN, components of the Gram-positive bacteria cell membrane that activate macrophages via TLR2 (16,36,37), to explore the factors regulating neutrophil influx into the lung. We demonstrated that the autoimmunity- and arthritis-susceptible DA rats and the resistant F344 rats developed acute pulmonary inflammation induced by LTA and PGN, as indicated by increased numbers of neutrophils and high levels of inflammatory mediators in the BALF. Unexpectedly, F344 rats had significantly higher numbers of BALF neutrophils and macrophages, and their macrophages produced increased levels of neutrophil chemotactic factors such as KC and MIP-2. These observations differ from those previously described in studies of acute inflammation in the synovial-like air pouch using carrageenan challenge (19,20), raising the possibility of tissue-specific regulation of inflammatory responses, with different cell types (for example, AMs versus synovial macrophages and synovial fibroblasts) implicated in the tissue responses to injury. Specifically, these two studies used different stimuli. LTA/PGN stimulate TLR2 to induce the inflammatory responses. Our results indicate that LTA/PGN stimulate the TLR2-MAPK-chemokine pathway leading to increased chemokine production and finally increased neutrophil infiltration. However, carrageenan is a high molecular weight sulfated polygalactan, stimulating inflammatory responses through different pathways than LTA/PGN. It has been reported that carrageenan induces IL-8 through a distinct Bcl10 pathway(38). Reactive oxygen species (39), lipoxygenase (40,41), and TLR4 (42) may also be involved in carrageenan-induced inflammation. In addition, these two studies explored inflammation at the different tissue sites. The lung has its own specific inflammatory cell population distinct from the joints. In the current study we found that lung residential cells, AMs, are particularly associated with the different inflammation in these two strains. It has been reported that macrophages from different sites have different inflammatory responses (43). We believe that the combination of different inflammatory stimuli activating different cell-signaling pathways at different stimulatory sites may account for the strain differences observed between this study and previous synovial-like air pouch studies (19,20).

Our observations demonstrate that neutrophil migration into the lung, and AM activation in response to bacterial products, are different between these two strains. In an attempt to determine the location of the trait-regulatory genes, we tested four congenic strains generated between DA and F344 (DA.F344[QTL] congenics) and known to regulate arthritis (Cia3, Cia4, Cia5a and Cia6) and acute inflammatory responses in the synovial-like air pouch model injected with carrageenan (Cia4 and Cia6) (19). However, all four congenics had BALF findings consistent with their background genome (DA) and excluding a contribution by the introgressed F344 intervals, which suggests that the differences in lung inflammation were not due to genes contained within these chromosomal intervals carried by these congenic strains.

Our previous studies with F344 and DA rats demonstrated that the two strains have similar peripheral blood neutrophil numbers and similar neutrophil chemotaxis in response to chemoattractants (19). These findings suggest that neutrophil responses themselves do not cause the differences in the BALF cell numbers. To explore the reason why more neutrophils migrate to lung in F344 rats, we focused on differences within AMs. The AM is a resident lung-specific cell population and has been reported to play a key role in acute lung injury caused by sepsis, hemorrhage, mechanical ventilation and aspiration (44–48). AMs are one of the first cell types to encounter lung pathogens, or in this study, the instilled bacterial products LTA and PGN. Our results demonstrate that there are differences in both the number of cells present, and in the quality of the AM response between the DA and F344 strains. BALF from F344 rats contained approximately 50% more AMs than from the DA strain. In addition to the higher numbers of AMs in the F344 strain, these cells also showed higher KC and MIP-2 production per cell following LTA and PGN during in vitro stimulation. KC and MIP-2, (also known as CXCL1 and CXCL2), belong to the CXC chemokine family and are major neutrophil chemoattractants acting via the neutrophil surface receptor CXCR2 (49,50). Neutralization of CXC chemokines (49) or their receptor (45,51–55) results in reduction of neutrophil recruitment into the lung following a variety of stimuli. AMs are a major source of chemokines in the alveolar space and respond directly to bacterial products such as bacterial lipopolysaccharide and gram-positive cell wall products such as LTA (50) and toxins such as enterotoxin A (56). In our study, LTA and PGN stimulation resulted in a dose-dependent elevation of KC and MIP-2 production, with F344 AMs producing approximately 50% more MIP-2 and 100% more KC than DA AMs. The significantly increased production of chemokines by F344 AMs may explain, at least partially, the different lung neutrophil influx detected between the two strains.

To further explore the mechanism involved in increased chemokine production by F344 AMs, we focused on the major chemokine production pathway during LTA + PGN stimulation, that is, the TLR2-MAPK pathway (32,33). Our results show that there are no differences in the basal expression of TLR2 and total MAPK members (p38 and ERK1/2) between the two strains. However, the phosphorylation of p38 and ERK1/2 was significantly higher in the F344 group after LTA + PGN stimulation. Furthermore, our results also showed that both p38 and ERK1/2 inhibitors can inhibit LTA/PGN-induced KC and MIP-2 production. If both of these two MAPK pathways are inhibited at the same time, a substantial inhibition of chemokines is achieved, which includes almost 100% of stimulated MIP-2 production and 75% of stimulated KC production. This observation indicates that the p38 and ERK1/2 pathways are two major cell-signaling pathways in LTA/PGN-induced KC and MIP-2 accumulation. The differences in MAPK pathway activation in these two strains can, at least partially, explain the differences in KC and MIP-2 production by AMs following LTA/PGN stimulation.

In conclusion, we have shown that there are significant differences in the regulation of pulmonary inflammation induced by bacterial products in the two different strains of rats. In particular, the differential regulation can be seen in neutrophil infiltration to the alveolar spaces. These differences are associated with different AM phenotypes and, specifically, differences in MAPK signaling pathways. The data presented should lead to a better understanding of the compartmentalization of the lung inflammatory response and tissue-specific regulation of the immune responses. Further study of these particular influences on the inflammatory processes may lead to more effective treatment of acute lung inflammation and injury.

Disclosure

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.