- Open Access
Endotoxin induced peritonitis elicits monocyte immigration into the lung: implications on alveolar space inflammatory responsiveness
© Steinmüller et al. 2006
- Received: 24 October 2005
- Accepted: 18 February 2006
- Published: 18 February 2006
Acute peritonitis developing in response to gram-negative bacterial infection is known to act as a trigger for the development of acute lung injury which is often complicated by the development of nosocomial pneumonia. We hypothesized that endotoxin-induced peritonitis provokes recruitment of monocytes into the lungs, which amplifies lung inflammatory responses to a second hit intra-alveolar challenge with endotoxin.
Serum and lavage cytokines as well as bronchoalveolar lavage fluid cells were analyzed at different time points after intraperitoneal or intratracheal application of LPS.
We observed that mice challenged with intraperitoneal endotoxin developed rapidly increasing serum and bronchoalveolar lavage fluid (BALF) cytokine and chemokine levels (TNFα, MIP-2, CCL2) and a nearly two-fold expansion of the alveolar macrophage population by 96 h, but this was not associated with the development of neutrophilic alveolitis. In contrast, expansion of the alveolar macrophage pool was not observed in CCR2-deficient mice and in wild-type mice systemically pretreated with the anti-CD18 antibody GAME-46. An intentional two-fold expansion of alveolar macrophage numbers by intratracheal CCL2 following intraperitoneal endotoxin did not exacerbate the development of acute lung inflammation in response to intratracheal endotoxin compared to mice challenged only with intratracheal endotoxin.
These data, taken together, show that intraperitoneal endotoxin triggers a CCR2-dependent de novo recruitment of monocytes into the lungs of mice but this does not result in an accentuation of neutrophilic lung inflammation. This finding represents a previously unrecognized novel inflammatory component of lung inflammation that results from endotoxin-induced peritonitis.
- Alveolar Macrophage
- Lung Permeability
- Acute Lung Inflammation
- Lung Inflammatory Response
- Neutrophilic Alveolitis
Overwhelming innate immune responses to systemic inflammation contribute to the clinical manifestation of sepsis and septic shock. Acute respiratory distress syndrome (ARDS) and multiorgan failure are frequent complications of severe sepsis that contribute to the morbidity and mortality of this critical illness . The molecular events eliciting sepsis-related complications such as acute lung injury have only been partially defined. For example, experimental animal models of endotoxin (lipopolysaccharide, LPS) induced acute peritonitis have characterized the kinetics of intraperitoneal LPS resorption and its rapid appearance within the vascular compartment within minutes to hours . In human studies, patients who develop ARDS have highly elevated serum and alveolar cytokine and chemokine levels that are associated with a massive accumulation of neutrophils within the lungs, expanded alveolar macrophage populations and increased lung injury scores [3, 4]. Key chemokines involved in this process of lung leukocyte invasion include the main monocyte chemoattractant CCL2 and the neutrophil chemoattractants MIP-2, KC and MIP-1α [4, 5].
Although expansion of alveolar macrophage populations in septic ARDS patients is a well described phenomenon, the underlying molecular events shaping this leukocytic response are not known. In particular, it is unclear whether such expanded alveolar macrophage pools in septic ARDS patients are functionally relevant in terms of aggravating lung inflammatory responses to secondary pneumonia, which complicate the later phase of septic ARDS. Previously published studies employing a two hit model of initial intraperitoneal LPS challenge to trigger sepsis-induced remote lung inflammatory responses followed by a secondary intratracheal LPS challenge to mimic the additional development of pneumonia have not addressed changes in alveolar macrophage pool sizes and related functional consequences . Clearly, both newly recruited alveolar monocytes and resident macrophages are potentially involved in the overall lung inflammatory response. We have recently demonstrated alveolar accumulations of CD14-positive mononuclear phagocytes and elevated BAL fluid CCL2 levels together with an expanded alveolar macrophage pool correlating with increased lung injury scores in patients with sepsis related ARDS . In addition, we recently showed in an animal model that intratracheal application of CCL2 plus LPS synergistically induced acute lung inflammation, indicating that monocytes that are recruited into the lungs of mice may both expand the alveolar macrophage pool and aggravate LPS-induced neutrophilic alveolitis and lung injury in mice . Thus, an expansion of alveolar macrophage pool sizes may be observed in both septic ARDS patients and in experimental animal models with ARDS-like inflammatory phenotypes [4, 6], and expanded alveolar macrophage pools may be involved in remote lung injury developing in response to systemic inflammation [4, 5]. Against this background, we hypothesized that LPS-induced peritonitis effects alveolar macrophage pool sizes by provoking the recruitment of circulating monocytes into the lungs, thereby amplifying lung inflammatory responses to a second hit intra-alveolar endotoxin challenge. We found that LPS-induced peritonitis was sufficient to elicit a robust twofold expansion of the alveolar macrophage pool without generating neutrophilic alveolitis. Using a two hit model of initial intraperitoneal plus intratracheal LPS application to mimic peritonitis-induced lung inflammation that is complicated by Gram-negative pneumonia, we found-contrarily to our initial hypothesis- that the observed alveolar macrophage expansion did not result in an aggravation of the overall lung inflammatory response to alveolar LPS challenge.
BALB/c female mice (18–22 g) were purchased from Charles River (Sulzfeld, Germany). CCR2-deficient mice were generated on a mixed C57BL/6 × 129/Ola genetic background by targeted disruption of the CCR2 gene, as described previously . The disrupted gene was backcrossed for six generations to wild-type BALB/c mice. Parent and offspring CCR2-/- mice on the BALB/c background were bred under specific pathogen free (SPF) conditions. Animals 8–12 weeks old were used for the described experiments. Each treatment group consisted of at least 5 mice, unless indicated otherwise. This animal study was approved by the local government committee.
Lipopolysaccharide (E. coli, serotype O111:B4) was purchased from Sigma (Deisenhofen, Germany). Murine recombinant CCL2 (JE/MCP-1) was purchased from Peprotech (Tebu, Offenbach, Germany). Function-blocking rat antimurine CD18 antibody (clone GAME-46) was purchased from BD Biosciences (Heidelberg, Germany). All reagents and antibodies were ascertained to be endotoxin-free by Limulus amoebocyte lysate (LAL) assay (Chromogenix, Mölndal, Sweden).
Intratracheal applications of the various inflammatory stimuli and recombinant proteins was done essentially as described elsewhere [5, 6, 8–11]. Briefly, tracheas of anaesthetized mice were surgically exposed, and an Abbocath catheter (Abbot, Wiesbaden, Germany) was inserted into the trachea. Subsequently, indicated concentrations of LPS or recombinant proteins (dissolved in total volumes of approximately 80 μl saline/0.1% endotoxin-free human serum albumin) were slowly instilled under stereomicroscopic control (Leica MS5, Wetzlar, Germany). Subsequently, the skin was sutured and mice were allowed to recover with free access to food and water.
Wild-type and CCR2-deficient mice (where indicated) were sedated with ketamine and received intraperitoneal (IP) injections of sterile LPS (50 μg/mouse) for various time intervals (0 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h). In a second set of experiments, mice received initial intraperitoneal LPS applications, and at various time points thereafter, additional intratracheal (IT) applications of LPS (1 μg/mouse) to mimic the frequently observed clinical complication of pneumonia developing subsequent to the onset of septic ARDS that is associated with peritonitis. In a third set of experiments, mice received initial intraperitoneal LPS applications and 48 h later, received a single intratracheal application of recombinant CCL2 (50 μg/mouse) to further increase alveolar monocyte accumulations within the lung. At another 48 h post CCL2 application (i.e., 96 h after initial IP LPS application), mice were challenged intratracheally with LPS (1 μg/mouse) for 24 h. In a fourth set of experiments, mice received intratracheal applications of both CCL2 (50 μg/mouse) plus LPS (1 μg/mouse), according to recently published protocols [6, 8, 9]. The dose of 1 μg LPS/mouse was chosen to induce an easily detectable neutrophilic alveolitis in mice that also allows monitoring of changes in magnitudes and durations of the neutrophilic responses depending on the various experimental settings . On the other hand, to elicit a robust monocytic response, we followed recently published experimental protocols and challenged mice intratracheally with 50 μg CCL2/mouse (where indicated). This strong CCL2 challenge has been described recently to establish a potent CCL2 chemokine gradient and subsequent selective monocyte recruitment towards the alveolar compartment [6, 8, 9]. For inhibition experiments, mice received intravenous injections of function-blocking anti-CD18 antibody GAME-46 (100 μg/mouse) 15 min prior to IP LPS application and every 24 h post IP LPS application. In initial experiments, repeated intraperitoneal GAME-46 antibody injections did not show any overt side effects and were well tolerated by the mice. Overall mortalities ranged below 10%.
Collection and analysis of blood samples and bronchoalveolar lavage
Mice were sacrificed and blood sample and bronchoalveolar lavage collection was done as recently outlined in detail [6, 8–10]. Briefly, mice were exposed to an overdose of isofluoran (Abbott, Wiesbaden, Germany) and blood was collected from the inferior vena cava. The bronchoalveolar lavage (BAL) fluid was obtained by cannulating the trachea with a shortened 21 G needle attached to a 1-ml insulin syringe, followed by repeated intratracheal instillations of 0.5 ml aliquots of PBS (pH 7,2, supplemented with 2 mM EDTA). Quantification of TNFα, MIP-2 and CCL2 proteins in BAL fluid and serum samples was performed using commercially available ELISA kits (lower detection limits, 2 pg/ml), as recommended by the manufacturer (R&D Systems, Wiesbaden, Germany). BAL cells were counted with a hemocytometer and quantitation of alveolar macrophages, alveolar recruited monocytes and neutrophils was done on differential cell counts of Pappenheim-stained cytocentrifuge preparations using overall morphological criteria, including differences in cell size and shape of nuclei and subsequent multiplication of those values with the respective total BAL cell counts, as recently described [10, 11].
In vivo lung permeability assay
For evaluation of IP LPS-induced lung permeability, mice received an intravenous injection of FITC-labeled human albumin (1 mg/mouse in 100 μl PBS; Sigma, Deisenhofen, Germany) 1 hour before death, as recently described [5, 9]. Undiluted BAL fluid samples and serum samples (diluted 1:10 and 1:100 in PBS, pH 7.4) were placed in a 96-well microtiter plate and fluorescence intensities were measured using a fluorescence spectrometer (Bio-Tek FL 880 microplate fluorescence reader) operating at 488 nm absorbance and 525 ± 20 nm emission wavelengths, respectively. The lung permeability index is defined as the ratio of fluorescence signals of undiluted BAL fluid samples to fluorescence signals of 1:10 diluted serum samples.
The study data are expressed as mean ± SEM. Significant differences between controls and treatment groups of serum and BAL fluid cytokine levels were calculated by one-factor ANOVA with posthoc tests by Dunnet. Significant differences in numbers of alveolar macrophages and PMN were calculated by two-way ANOVA with post-hoc tests using the Dunnet procedure. Differences were assumed to be significant when P values were < 0.01.
Thus, in sharp contrast to what we initially expected, these data lend support to the concept that monocytes recruited from inflamed vascular compartments into the lungs apparently do not aggravate neutrophilic alveolitis in response to secondary intra-alveolar LPS challenge, whereas alveolar monocytes recruited from non-inflamed vascular compartments significantly contribute to neutrophilic alveolitis upon intratracheal LPS application, the latter being consistent with recent reports [5, 6].
One possible explanation why the increased lung monocyte traffic leading to an expansion of the alveolar macrophage pool in response to intraperitoneal LPS did not aggravate the neutrophilic alveolitis upon subsequent intratracheal LPS application may involve changes in TLR expression profiles on circulating monocytes pre-exposed to systemic LPS (absorbed from the peritoneal cavity) prior to being recruited into the lungs [16, 17]. Such monocytes might exhibit a reduced potential to mount proinflammatory responses within the alveolar air space upon secondary challenge with locally applied LPS. Such attenuation of alveolar inflammatory responses might affect the lung host defense under conditions of systemic inflammation. This hypothesis of a dysregulated TLR gene expression pattern in LPS "pre-exposed" circulating monocytes is currently being addressed in our lab in more detail.
Together, the presented data for the first time show that intraperitoneal LPS application in mice elicits a remote lung inflammatory response reflected by a CCR2-dependent and β2 integrin mediated robust expansion of the alveolar macrophage pool. However, no evidence was found to demonstrate that the observed monocyte immigration and resulting alveolar macrophage expansion subsequent to systemic inflammation leads to an aggravated neutrophilic alveolitis developing in response to a "second hit" alveolar LPS application. This finding is opposite to the enhanced neutrophilic alveolitis observed in mice where monocytes were recruited from a non-inflamed vascular compartment to the lungs (ie, in response to intratracheal CCL2 plus LPS) to expand alveolar macrophage numbers. These data may contribute to a better understanding of the inflammatory capacity of mononuclear phagocytes in the lungs of septic ARDS patients.
We acknowledge the expert technical assistance of Regina Maus and Petra Janssen. This study has been supported by the German research foundation, grant SFB 547 "Cardiopulmonary Vascular System", and the network on community-acquired pneumonia, CAPNETZ. M Steinmüller is supported by a pre-doctoral fellowship by ALTANA Pharma.
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