UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
RAGE and the innate immune response in infection and inflammation
van Zoelen, M.A.D.Publication date 2009
Link to publication
Citation for published version (APA):
van Zoelen, M. A. D. (2009). RAGE and the innate immune response in infection and inflammation.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
15
C
ha
p
te
r
Endogenous MCP-1 promotes lung
inflammation induced by LPS and
LTA
Marieke A.D. van Zoelen1,2, Marleen I. Verstege2, Christian Draing5, Regina de Beer1,2,
Cornelis van ’t Veer1,2, Sandrine Florquin3, Paul Bresser4, Jaring S. van der Zee4, Anje A. te
Velde2, Sonja von Aulock5 and Tom van der Poll1,2
Submitted
1Center for Infection and Immunity Amsterdam (CINIMA), 2Center for Experimental and Molecular Medicine
(CEMM), 3Department of Pathology, 4Department of Pulmonology, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands
Abstract
Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of pro-inflammatory cytokines.
We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall respectively.
Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6 h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like
cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro.
Compared to Wt mice, MCP-1-/- mice demonstrated lower TNF-α levels and a diminished
neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation.
After intrapulmonary delivery of LPS MCP-1-/- mice had decreased interleukin-6 and KC
concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation.
These data suggest that MCP-1 is a pro-inflammatory mediator during pulmonary inflammation induced by either LPS or LTA.
Introduction
Chemokines are members of the supergene family of small inducible peptides which attract leukocytes. Defined by a tetra cysteine motif, chemokines are divided into four families according to the configurations of the cysteine residues at their amino terminus; of these CXC and CC chemokines represent the largest groups [1,2]. Monocyte chemoattractant protein (MCP)-1 (CCL2) is a CC chemokine with pleiotropic activities [3]. MCP-1 primarily attracts monocytes and memory T cells, but during severe bacterial infection may also contribute to neutrophil recruitment [4,5]. In addition, MCP-1 has been found to exert anti-inflammatory effects during murine endotoxemia: anti-MCP-1 treatment increased LPS-induced lethality concurrently enhancing pro-inflammatory cytokine release, whereas recombinant MCP-1 protected against death and reduced pro-inflammatory cytokine levels [6].
Several studies have examined the role of MCP-1 in the regulation of lung inflammation during bacterial pneumonia. In a model of pneumonia caused by the gram-negative
respiratory pathogen Pseudomonas (P.) aeruginosa passive immunization against
MCP-1 resulted in increased neutrophil influx in the lungs and enhanced lung injury without an effect on the recruitment of macrophages [7]. Using wild type (Wt) mice
treated with an anti-MCP-1 antibody and mcp-1 gene deficient (MCP-1-/-) mice, we
and others demonstrated that endogenous MCP-1 does not play a role of significance in the lung inflammation accompanying pneumonia caused by the gram-positive pathogen
Streptococcus (S.) pneumoniae [8,9].
Intact bacteria express a variety of proteins and polysaccharides that can trigger immune responses via multiple receptors and pathways. We were interested to evaluate the contribution of MCP-1 in lung inflammation induced by two biologically relevant bacterial cell wall components: LPS, a major constituent of the outer cell wall of gram-negative bacteria and the predominant inducer of inflammatory responses to these pathogens [10], and lipoteichoic acid (LTA), an important pro-inflammatory molecule in the cell wall of gram-positive bacteria [11]. Considering that LPS induces signal transduction predominantly via Toll-like receptor (TLR)4, whereas LTA triggers cells mainly via TLR2 [12,13], we thereby sought to obtain insight into the influence of
Materials and Methods
Human study
Human healthy volunteers and design
Details of this study have been published previously [14]. In brief, 16 nonsmoking males
(age 22.5 ± 0.8 yrs) subjects inhaled either saline (n = 8) or LPS (n = 8; from Escherichia coli
O26:B6, 100 µg; Sigma-Aldrich, St. Louis, MO) using a large-volume reservoir delivery system. They were part of a larger study investigating the effects of inhaled salmeterol on lung inflammation induced by LPS of which the results have been reported earlier [14]; the subjects analyzed here represent the saline and LPS control groups that did not receive salmeterol. Bronchoalveolar lavage (BAL) was performed 6 h post-challenge in a standardized fashion according to the guidelines of the American Thoracic society, using a flexible fiberoptic videobronchoscope. Eight successive 20-ml aliquots of prewarmed 0.9% NaCl were instilled in a subsegment of the right middle lobe and each aliquot was aspirated immediately with low suction. BAL fluid (BALF) was immediately centrifuged
for 10 minutes at 250 x g at 4 ºC. The supernatant was collected and stored at -80 °C
until assayed. MCP-1 was measured using a commercially available ELISA according to the manufacturer’s recommendations (R&D systems, Abingdon, United Kingdom). The study was approved by the Institutional Ethics and Research Committees and written informed consent was obtained from all subjects.
Mouse studies
LPS and LTA preparations
LPS was from Escherichia coli O111:B4 (Sigma Aldrich, St. Louis, MO). LTA was
prepared from Staphylococcus aureus as described before [15]. The LPS contamination
of the LTA preparation was < 1 EU/mg as determined by the Limulus amoebocyte lysate
assay (Charles River, Charleston, SC). Cellular effects of this LTA preparation and dose were totally TLR2 dependent, as determined by TLR2 deficient murine cells, confirming that LPS contamination does not play a role in cell activation by the LTA preparation used (data not shown).
Stimulation of murine alveolar macrophages
The murine alveolar macrophage cell line MH-S (American Type Culture Collection, Rockville, MD) was grown in RPMI 1640 (Bio Whittaker, Verviers, Belgium) supplemented 2 mM L-glutamine, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin (GibcoBRL, Life Technologies, Rockville, MD) and 50 µM β-mercaptoethanol (Sigma, Aldrich, St.
de Rijn, The Netherlands) at a density of 1 x 105 cells/ml. Following overnight culture at
37 ºC in 5% CO2, adherent cells were washed twice and stimulated with LPS (0.1 µg/ml,
4 h), LTA (20 μg/ml, 24 h) or control (medium). Supernatants were collected and stored at -20 ºC until assayed.
Mice and design
Specific pathogen free 8-10 wks old C57BL/6 female Wt mice were purchased from Charles
Rivers (Maastricht, The Netherlands). MCP-1-/- mice, backcrossed to a C57BL/6 genetic
background, were obtained from Jackson Laboratory (Bar Harbor, Maine) and bred in the animal facility of the Academic Medical Center in Amsterdam, the Netherlands. All experiments were approved by the Animal Care and Use Committee of the University of Amsterdam (Amsterdam, the Netherlands). Mice were anesthetized by inhalation of isoflurane (Abott Laboratories LTD., Kent, UK) and LPS (10 μg per mouse) or LTA (100 μg per mouse) diluted in 50 μl sterile saline was instilled intranasally. MCP-1 levels in Wt mice after LPS or LTA were compared with control mice that received 50 μl of sterile saline only. Bronchoalveolar lavage, cell counts and differentials, assays, preparation of lung homogenates and histology were performed as described previously [16-18].
sRAGE measurement
For Western blotting of the soluble receptor for advanced glycation end-products (sRAGE), 10 µl of BALF was separated by 10% polyacrylamide SDS gel electrophoreses
and transferred to Immobilon P (Pharmacia, Piscataway, NJ ) polyvinylidene difluoride
membranes. Membranes were blocked in block buffer containing5% nonfat dry milk
proteins and 0.1% Tween 20 in 50 mM Tris,150 mM NaCl (pH 7.4; TBS), washed
with TBSand incubated overnight with primary antibodies (goat anti-mouse RAGE
polyclonal antibodies (Neuromics, Edina, MN) in block buffer at 4 °C.After washing
with TBS, membranes were probedwith peroxidase-labeled secondary Ab (Cell Signaling
Technology, Danvers, MA)for 1 hr at room temperature in TBS. After washingwith
TBS, membranes were incubated with Lumi-LightPlus Western Blotting Substrate (Roche,
Mijdrecht, The Netherlands) and positive bands were detectedusing a GeneGnome
Syngene Bio Imaging System (Syngene, Leusden, The Netherlands). Intensityof the bands
was quantified using the GeneTools software suppliedby Syngene.
Statistical analysis
Alldata are expressed asmean ± SEM. Differences between groups were analyzed by
Mann-Whitney U test. Values of p<0.05 were considered to represent a statistically significant difference.
Results
MCP-1 release in BALF of humans and mice challenged with LPS
To determine whether MCP-1 is released in the bronchoalveolar space upon intrapulmonary delivery of LPS, we first measured MCP-1 concentrations in BALF obtained from healthy humans 6 h after inhalation of nebulised LPS or saline. LPS inhalation elicited a strong rise in BALF MCP-1 levels (p<0.001 versus saline controls, fig. 1A). Similarly, intranasal administration of LPS to healthy Wt mice induced an increase in the pulmonary levels of MCP-1, both in BALF (p<0.001 versus saline controls, fig. 1B) and in lung homogenates (p=0.002 versus saline controls, fig. 1C). Our laboratory previously identified alveolar macrophages as a possible source for MCP-1 in humans exposed to LPS [14]. In line, murine MH-S alveolar macrophage-like cells released significant amounts of MCP-1
upon incubation with LPS in vitro (fig. 1D).
MCP-1
-/-mice demonstrate a diminished neutrophil influx upon LPS
instillation
MCP-1 has been implicated in the recruitment of different leukocyte subsets to sites of infection and inflammation [3-5]. To obtain insight into the role of endogenous MCP-1 in leukocyte influx during LPS-induced lung inflammation, we determined total leukocyte counts and differentials in BALF harvested 6 or 24 h after intranasal administration of
LPS (Table 1). Wt and MCP-1-/- mice showed similar total leukocyte counts in their
BALF upon intrapulmonary delivery of LPS. However, in MCP-1-/- mice the recruitment
Figure 1. LPS induces MCP-1 release in human and murine lungs. MCP-1 protein
concentrations in (A) BALF from healthy volunteers obtained 6 h after inhalation of saline or LPS (100 µg), (B) BALF and (C) lung homogenates of Wt mice 6 h after intranasal inoculation of saline or LPS (10 µg) and (D) culture supernatant from murine macrophage-like MH-S cells after stimulation (4 h) in the absence or presence of LPS (0.1 µg/ml). The data are mean ± SEM (n = 7 to 8 for the human and mouse experiments and n = 4 for the in
vitro experiment). * p<0.05 versus controls; ***
of neutrophils was significantly inhibited 6 h after LPS (p<0.05 versus Wt mice). At 24 h, the neutrophil counts did not differ any more between the two mouse strains. In line, MCP-1 deficiency inhibited degranulation of neutrophils entering the bronchoalveolar space at 6 h but not as 24 h, as reflected by a decrease in the concentration of the neutrophil degranulation product MPO in cell-free BALF (table 1, p<0.05 versus Wt mice). Since CXC chemokines play an important role in the attraction of neutrophils to the lung
during inflammation [19-21], we measured KC and MIP-2 in BALF. At 6 h after LPS
administration, MCP-1-/- mice had lower KC levels in their BALF (p<0.05 versus Wt
mice, fig. 2A); at 24 h BALF KC levels were very low and not different between groups
anymore. In contrast, MIP-2 concentrations were higher in MCP-1-/- mice, significantly
so at 24 h (p<0.001 versus Wt mice, fig. 2B).
Figure 2. Chemokine and cytokine pulmonary concentrations of Wt and MCP-1-/- mice 6 and
24 h after LPS. KC (A), MIP-2 (B), TNF-α (A) , IL-6 (B) and IL-10 (C) in BALF from Wt (open bars)
and MCP-1-/- mice (solid bars) 6 and 24 h after intranasal instillation of LPS (10 µg). The data are
mean ± SEM (n = 7-11 mice per group per time point). * p<0.05 versus Wt mice; ** p<0.01 versus Wt mice; *** p<0.001 versus Wt mice.
Table 1. Total leukocyte counts, differentials and MPO levels in BALF after LPS Wt MCP-1-/- Wt MCP-1 6 h 24 h Cells (x 105/ml) Total cells 2.72 ± 0.36 2.07 ± 0.19 11.57 ± 2.11 9.96 ± 1.22 Neutrophils 2.38 ± 0.36 1.51 ± 0.19* 10.83 ± 2.00 9.02 ±1.16 Macrophages 0.29 ± 0.07 0.38 ± 0.10 0.58 ± 0.11 0.80 ± 0.14 MPO (ng/ml) 366 ± 90.0 116 ± 58** 2088 ± 306.1 2590 ± 246.3
Definition of abbreviations: MPO, myeloperoxidase; BALF, bronchoalveolar lavage fluid;
Wt, wild type, MCP-1, monocyte chemoattractant protein-1.
6 or 24 h after LPS instillation, mice were killed and BALF was prepared. Data are mean ± SEM (n = 7-11 per group). * p<0.05 versus Wt mice; ** p<0.01 versus Wt mice.
Impact of MCP-1 deficiency on LPS-induced cytokine release
Cytokines play an important role in the regulation of lung inflammation. Since MCP-1 has been shown to exert anti-inflammatory effects during (systemic) endotoxemia [6], we were interested in the influence of MCP-1 deficiency on LPS-induced cytokine release in the bronchoalveolar compartment. Therefore, we measured the concentrations of tumor necrosis factor (TNF)-α (a prototypic pro-inflammatory cytokine), IL-6 (a cytokine with mixed pro- and anti-inflammatory properties) and IL-10 (a prototypic anti-inflammatory
cytokine) in BALF from Wt and MCP-1-/- mice. At 6 h, levels of TNF-α and IL-6 (fig.
2C and D) were decreased in MCP-1-/- mice, while levels of IL-10 (fig. 2E) were increased
at that time point (all p<0.05 versus Wt mice). At 24 h, TNF-α and IL-6 levels did not
differ anymore between Wt and MCP-1-/- mice. Remarkably, at this time point IL-10
concentrations were lower in MCP-1-/-mice (p<0.01 versus Wt mice).
MCP-1
-/-mice have less severe lung inflammation upon LPS
administration
To further investigate the role of endogenous MCP-1 during LPS induced lung
inflammation we analyzed lung tissue slides obtained from Wt (Fig. 3A) and MCP-1-/-
mice (fig. 3B) 24 h after LPS administration. On histopathological examination the lungs showed endothelialitis, perivascular inflammation and interstitial inflammation. Lung
inflammation in MCP-1-/- mice was less profound compared with Wt mice, as reflected
by lower semi-quantitative histology scores (determined according to the scoring system described in the Materials and Methods section) (p<0.05 versus Wt mice, fig. 3C).
sRAGE levels
sRAGE has been recently described as a marker of lung injury based on experimental
studies in rats and in patientswith ALI [22].To establish whether this model is associated
with increased levels of sRAGE in the bronchoalveolar space, we performed Western blot analysis on BALF samples from healthy control mice and mice intranasally challenged with LPS (post 24 h) and generated semi-quantitative data by densitometric evaluation. LPS administration elicited increased sRAGE concentration, as shown in fig. 4A and B
Figure 3. Lung inflammation of Wt and MCP-1-/- mice induced by LPS. Representative HE stainings of lung tissue from Wt (A) and MCP-1-/- mice (B) 24 h after intranasal administration
of LPS (10 µg). Magnification x10, inserts x40. Graphical representation of the degree of lung inflammation (C) from the Wt (open bars) and MCP-1-/- mice (solid bars), determined according to
the scoring system described in the Materials and Methods section. The data are mean ± SEM (n = 7-8 mice per group).
Figure 4. Bronchoalveolar sRAGE release during LPS induced lung inflammation. Western
blot was performed for soluble receptor for advanced glycation end products (sRAGE) (A) in BALF of healthy control mice and of mice intranasally administered with LPS (post 24 h). AU = arbitrary units. Semiquantitative data were generated by densitometric evaluation of healthy control mice (n = 4) versus LPS challenged mice (n = 7) and (B) and of Wt versus MCP-1-/- mice 24 h after LPS
(p<0.01 versus healthy control mice). Although MCP-1 deficiency tended to attenuate the release of sRAGE in BALF, the difference with Wt mice did not reach statistical significance (fig. 4C).
Role of MCP-1 in LTA-induced lung inflammation
Having established that endogenous MCP-1 plays a pro-inflammatory role in the early pulmonary response to LPS, we next wished to examine the contribution of MCP-1 to lung inflammation to LTA. First we established that intranasal administration of LTA resulted in a strong increase in the concentrations of MCP-1 in BALF and lung homogenates obtained from Wt mice (p<0.05 versus saline controls, fig. 5A and 5B). In
addition, MH-S alveolar macrophage-like cells released MCP-1 upon exposure to LTA in
vitro (fig. 5C). In line with the role of 1 in LPS-induced lung inflammation,
MCP-1-/- mice demonstrated a reduced early (i.e. 6 h) influx of neutrophils into BALF (table 2)
and no difference in total leukocyte counts compared to Wt mice. However, in contrast to the results obtained in the LPS model, MCP-1 deficiency did not impact on BALF MPO concentrations (table 2) or on the release of KC or MIP-2 after LTA challenge (data not
shown). Whereas the early (i.e. 6 h) TNF-α release into BALF was similar in both mouse
strains, MCP-1-/- mice displayed lower TNF-α concentrations 24 h after LTA instillation
(p<0.05 versus Wt mice, fig. 6A). IL-6 levels did not differ between Wt and MCP-1
-/-mice at either time point (fig. 6B). Of considerable interest, the biphasic effect of MCP-1 deficiency on IL-10 release in the LPS model was reproduced after LTA administration:
whereas MCP-1-/- mice had higher IL-10 levels in BALF at 6 h, their IL-10 levels were
lower at 24 h (p<0.001 versus Wt mice at both time points, fig. 6C).
Table 2. Total leukocyte counts, differentials and MPO levels in BALF after LTA
Wt MCP-1-/- Wt MCP-1-/- 6 h 24 h Cells (x 105/ml) Total cells 3.95 ± 0.36 2.97 ± 0.32 6.04 ± 0.60 6.14 ± 0.59 Neutrophils 3.64 ± 0.32 2.60 ± 0.27** 5.45 ± 0.59 5.58 ± 0.57 Macrophages 0.27 ± 0.04 0.27 ± 0.05 0.38 ± 0.03 0.44 ± 0.07 MPO (ng/ml) 376 ± 690.0 471 ± 81.1 290 ± 43.0 357 ± 46.7
Definition of abbreviations: MPO, myeloperoxidase; BALF, bronchoalveolar lavage fluid;
Wt, wild type, MCP-1, monocyte chemoattractant protein-1.
6 or 24 h after LTA instillation, mice were killed and BALF was prepared. Data are mean ± SEM (n = 7-9 per group). ** p<0.01 versus Wt mice.
Figure 6. Pulmonary cytokine levels from Wt and MCP-1-/- mice during LTA induced lung inflammation. Levels of TNF-α (A) , IL-6 (B) and IL-10 (C) in BALF from Wt (open bars) and MCP-1-/- mice (solid bars) 6 and 24 h after intranasal
instillation of LTA (100 µg). Data are mean ± SEM. N = 7-9
mice per group at each time point. * p<0.05 versus controls; *** p<0.001 versus controls.
Figure 5. LTA induces MCP-1 release in murine lungs. MCP-1 protein concentrations in (A)
BALF and lung homogenate (B) from Wt mice 6 h after intranasal inoculation of LTA (100 µg) and (C) in culture supernatant from murine macrophage-like MH-S cells after stimulation (24 h) with LTA (20μg/ml). The data are mean ± SEM (n = 6-8). * p<0.05 versus controls; ** p<0.01 versus controls.
Discussion
MCP-1 is a pleiotropic mediator that, as a CC chemokine, not only influences leukocyte trafficking, but also may exert anti-inflammatory effects during systemic endotoxemia by virtue of its capacity to inhibit the release of several pro-inflammatory cytokines. We here determined the role of MCP-1 in lung inflammation induced by LPS or LTA. We demonstrate that both LPS and LTA induce the release of MCP-1 in the bronchoalveolar
space. Although the phenotype of MCP-1-/- mice was somewhat different in the LPS and
LTA models, the overall effect of endogenous MCP-1 was pro-inflammatory, especially with regard to neutrophil influx and TNF-α release. Hence, our data suggest that MCP-1 is a pro-inflammatory mediator in lung inflammation induced by LPS and LTA.
Several studies have documented the local release of MCP-1, within the pulmonary compartment, during infectious and sterile lung injury [7-9,23,24]. We here report a rise in MCP-1 levels in BALF and whole lung homogenates of mice after intrapulmonary delivery of LPS, confirming earlier investigations [25-27]. In addition, inhalation of nebulised LPS was associated with an increase in BALF MCP-1 concentrations in healthy humans. To the best of our knowledge only one study examined the effect of LTA on pulmonary
MCP-1 production in vivo. In that study LTA elicited a transient rise in BALF MCP-1
levels with elevated concentrations 4 h after intratracheal LTA administration that had returned to baseline after 24 h [28]. Likely sources for MCP-1 are lung macrophages [29] (and the present study) as well as respiratory epithelial cells [9,29-31].
MCP-1-/- mice displayed a reduced neutrophil influx in BALF after either LPS or LTA
administration, especially after 6 h. Previously, treatment with an anti-MCP-1 antibody was reported not to influence neutrophil accumulation in lung tissue as determined by pulmonary MPO concentrations [5,26]. In these studies, LPS (from the same source as in the present investigation) was given intratracheally at a dose of 25 µg, whereas anti-MCP-1 was administered either intravenously [5] or intratracheally [26]. We here administered LPS intranasally at 10 µg and made use of mice with a targeted deletion of the mcp-1 gene. We consider it less likely that differences in the LPS dose and/or route
of administration can explain the different results in the two earlier studies by Speyer et al. [5,26] and our current investigation. The efficacy of the anti-MCP-1 antibody used in the former studies to neutralize MCP-1 activity was not reported [5,26], leaving the possibility open that not all endogenously active MCP-1 was eliminated. Although the
reduced KC levels in BALF of MCP-1-/- mice challenged with LPS could have contributed
to the diminished neutrophil influx, the local levels of MIP-2, another important CXC chemokine active on neutrophils, were higher in these genetically modified animals than
attenuated neutrophil influx in spite of the fact that they did not have altered KC or MIP-2 release in BALF. Of note, several lines of evidence indicate that neutrophils can respond to MCP-1. Deficiency or inhibition of CCR2, the receptor for MCP-1 and several other CC chemokines, resulted in a marked reduction in the neutrophil response after a lung challenge with MCP-1 and LPS [32]. Because circulating neutrophils are not attracted into the airspaces in response to MCP-1 alone [33], it is possible that reduced alveolar neutrophil recruitment after elimination of MCP-1 might be secondary to impaired (CCR2-dependent) cross-talk between monocytes and neutrophils and/or upregulation of CCR2 as a consequence of inflammation. Indeed, during septic peritonitis caused by cecal ligation and puncture, blood neutrophils were activated to express CCR2 and blockade of MCP-1 by administration of a specific antibody attenuated neutrophil accumulation in lungs in this model of severe infection [5]. Moreover, anti-MCP-1 treatment reduced peritoneal neutrophil recruitment during septic peritonitis [4]. While we focused on the early recruitment of neutrophils in response to purified bacterial antigens, a recent study reported on a possible late role for MCP-1 in the accumulation of neutrophils during gram-negative bacterial pneumonia: during respiratory tract infection by P. aeruginosa anti-MCP-1 treatment reduced the clearance of apoptotic neutrophils
from the lung by macrophages, resulting in increased neutrophil numbers and aggravation of lung tissue injury [7]. Hence, the influence of MCP-1 on neutrophil numbers during lung inflammation may depend on the phase and the duration of the inflammatory response, affecting both (early) influx and (late) clearance of these cells. Of note, MCP-1 deficiency attenuated LPS-induced MPO release in BALF, whereas it did not impact on LTA-induced MPO secretion. These findings indicate that (1) MPO concentrations in cell-free supernatant of BALF are not merely a reflection of neutrophil numbers but are representative of neutrophil degranulation and (2) that MCP-1 differentially influences
LPS- and LTA-induced exocytosis of neutrophils in the lungs in vivo. Further research is
warranted to establish the underlying mechanism by which MCP-1 impacts on neutrophils upon stimulation with LPS or LTA.
sRAGE concentrations in BALF have been identified as a marker for alveolar epithelial cell injury [22]. In accordance with a study in which rats received LPS intratracheally [22], we here showed that intrapulmonary delivery of LPS resulted in sRAGE release in BALF of mice. We here did not find effects of MCP-1 deficiency on sRAGE levels in the bronchoalveolar space, suggesting that the pro-inflammatory role of MCP-1 during LPS-induced lung inflammation is not associated with increased injury of the respiratory epithelium.
Several studies have implicated MCP-1 as an anti-inflammatory mediator during severe inflammation. During experimental peritonitis induced by either intraperitoneal LPS
injection or by cecal ligation and puncture anti-MCP-1 treatment increased circulating TNF-α levels, whereas IL-10 concentrations tended to decrease [6,34]. Conversely, administration of recombinant MCP-1 inhibited TNF-α release while concurrently enhancing IL-10 levels in response to intraperitoneal injection of LPS [6]. We here found an opposite role for MCP-1 during lung inflammation: anti-MCP-1 inhibited rather than enhanced TNF-α release, while potentiating IL-10 secretion. Although the underlying mechanism is unclear, these data suggest that MCP-1 may exert differential effects in the circulation and in the bronchoalveolar space.
In conclusion, we found that MCP-1 deficiency is associated with a reduced influx of neutrophils into BALF and an altered cytokine balance favouring an anti-inflammatory milieu (lower TNF-α and higher IL-10 levels) during lung inflammation caused by LPS or LTA. These data suggest that endogenous MCP-1 plays a pro-inflammatory role during the early phase of TLR4 and TLR2 dependent lung inflammation.
References
(1) Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol
2000; 18: 217-242.
(2) Coelho AL, Hogaboam CM, Kunkel SL. Chemokines provide the sustained inflammatory bridge between innate and acquired immunity. Cytokine Growth Factor Rev 2005; 16(6):
553-560.
(3) Daly C, Rollins BJ. Monocyte chemoattractant protein-1 (CCL2) in inflammatory disease and adaptive immunity: therapeutic opportunities and controversies. Microcirculation
2003; 10(3-4): 247-257.
(4) Matsukawa A, Hogaboam CM, Lukacs NW, Lincoln PM, Strieter RM, Kunkel SL. Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4. J Immunol 1999;
163(11): 6148-6154.
(5) Speyer CL, Gao H, Rancilio NJ, Neff TA, Huffnagle GB, Sarma JV, Ward PA. Novel chemokine responsiveness and mobilization of neutrophils during sepsis. Am J Pathol
2004; 165(6): 2187-2196.
(6) Zisman DA, Kunkel SL, Strieter RM, Tsai WC, Bucknell K, Wilkowski J, Standiford TJ. MCP-1 protects mice in lethal endotoxemia. J Clin Invest 1997; 99(12): 2832-2836.
(7) Amano H, Morimoto K, Senba M, Wang H, Ishida Y, Kumatori A, Yoshimine H, Oishi K, Mukaida N, Nagatake T. Essential contribution of monocyte chemoattractant protein-1/ C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia.
J Immunol 2004; 172(1): 398-409.
(8) Fillion I, Ouellet N, Simard M, Bergeron Y, Sato S, Bergeron MG. Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment. J Immunol 2001; 166(12): 7353-7361.
(9) Dessing MC, de Vos AF, Florquin S, van der Poll T. Monocyte chemoattractant protein 1 does not contribute to protective immunity against pneumococcal pneumonia. Infect Immun 2006; 74(12): 7021-7023.
(10) Beutler B, Rietschel ET. Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol 2003; 3(2): 169-176.
(11) Ginsburg I. Role of lipoteichoic acid in infection and inflammation. Lancet Infect Dis 2002;
2(3): 171-179.
(12) Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;
124(4): 783-801.
(13) Beutler B, Jiang Z, Georgel P, Crozat K, Croker B, Rutschmann S, Du X, Hoebe K. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006; 24: 353-389.
Bresser P, van der Poll T. Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers. Am J Respir Crit Care Med 2005; 172(7):
878-84.
(15) Morath S, Geyer A, Hartung T. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J Exp Med 2001; 193(3): 393-7.
(16) Rijneveld AW, Weijer S, Florquin S, Esmon CT, Meijers JC, Speelman P, Reitsma PH, Ten Cate H, van der Poll T. Thrombomodulin mutant mice with a strongly reduced capacity to generate activated protein C have an unaltered pulmonary immune response to respiratory pathogens and lipopolysaccharide. Blood 2004; 103(5): 1702-1709.
(17) Knapp S, Wieland CW, van ‘t Veer C, Takeuchi O, Akira S, Florquin S, van der Poll T. Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense. J Immunol 2004; 172(5):
3132-8.
(18) Wieland CW, Florquin S, Maris NA, Hoebe K, Beutler B, Takeda K, Akira S, van der Poll T. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung. J Immunol 2005; 175(9): 6042-6049.
(19) Schmal H, Shanley TP, Jones ML, Friedl HP, Ward PA. Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats. J Immunol 1996; 156(5):
1963-1972.
(20) Tsai WC, Strieter RM, Mehrad B, Newstead MW, Zeng X, Standiford TJ. CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia. Infect Immun 2000; 68(7): 4289-4296.
(21) Tsai WC, Strieter RM, Wilkowski JM, Bucknell KA, Burdick MD, Lira SA, Standiford TJ. Lung-specific transgenic expression of KC enhances resistance to Klebsiella pneumoniae in mice. J Immunol 1998; 161(5): 2435-2440.
(22) Uchida T, Shirasawa M, Ware LB, Kojima K, Hata Y, Makita K, Mednick G, Matthay ZA, Matthay MA. Receptor for advanced glycation end-products is a marker of type I cell injury in acute lung injury. Am J Respir Crit Care Med 2006; 173(9): 1008-1015.
(23) Raghavendran K, Davidson BA, Mullan BA, Hutson AD, Russo TA, Manderscheid PA, Woytash JA, Holm BA, Notter RH, Knight PR. Acid and particulate-induced aspiration lung injury in mice: importance of MCP-1. Am J Physiol Lung Cell Mol Physiol 2005;
289(1): L134-L143.
(24) Rosseau S, Hammerl P, Maus U, Walmrath HD, Schutte H, Grimminger F, Seeger W, Lohmeyer J. Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 2000; 279(1): L25-L35.
(25) Delayre-Orthez C, Becker J, Guenon I, Lagente V, Auwerx J, Frossard N, Pons F. PPARalpha downregulates airway inflammation induced by lipopolysaccharide in the mouse. Respir Res 2005; 6: 91.
(26) Speyer CL, Neff TA, Warner RL, Guo RF, Sarma JV, Riedemann NC, Murphy ME, Murphy HS, Ward PA. Regulatory effects of iNOS on acute lung inflammatory responses in mice. Am J Pathol 2003; 163(6): 2319-2328.
(27) Speyer CL, Rancilio NJ, McClintock SD, Crawford JD, Gao H, Sarma JV, Ward PA. Regulatory effects of estrogen on acute lung inflammation in mice. Am J Physiol Cell Physiol 2005; 288(4): C881-C890.
(28) Yamamoto S, Tin-Tin WS, Ahmed S, Kobayashi T, Fujimaki H. Effect of ultrafine carbon black particles on lipoteichoic acid-induced early pulmonary inflammation in BALB/c mice. Toxicol Appl Pharmacol 2006; 213(3): 256-266.
(29) Thorley AJ, Ford PA, Giembycz MA, Goldstraw P, Young A, Tetley TD. Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages. J Immunol 2007;
178(1): 463-473.
(30) Elizur A, Adair-Kirk TL, Kelley DG, Griffin GL, Demello DE, Senior RM. Clara Cells Impact the Pulmonary Innate Immune Response to LPS. Am J Physiol Lung Cell Mol Physiol 2007.
(31) Paine R, III, Rolfe MW, Standiford TJ, Burdick MD, Rollins BJ, Strieter RM. MCP-1 expression by rat type II alveolar epithelial cells in primary culture. J Immunol 1993;
150(10): 4561-4570.
(32) Maus U, von Grote K, Kuziel WA, Mack M, Miller EJ, Cihak J, Stangassinger M, Maus R, Schlondorff D, Seeger W, Lohmeyer J. The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice. Am J Respir Crit Care Med 2002;
166(3): 268-273.
(33) Maus U, Herold S, Muth H, Maus R, Ermert L, Ermert M, Weissmann N, Rosseau S, Seeger W, Grimminger F, Lohmeyer J. Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14. Am J Physiol Lung Cell Mol Physiol 2001; 280(1): L58-L68.
(34) Matsukawa A, Hogaboam CM, Lukacs NW, Lincoln PM, Strieter RM, Kunkel SL. Endogenous MCP-1 influences systemic cytokine balance in a murine model of acute septic peritonitis. Exp Mol Pathol 2000; 68(2): 77-84.
