In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury

Jean Louis Frossard1,2, Ashok K. Saluja1, Nicolas Mach3,4, Hong Sik Lee1, Lakshmi Bhagat1, Antoine Hadenque2, Laura Rubbia-Brandt5, Glenn Dranoff3, and Michael L. Steer1

1 Department of Surgery, Beth Israel Hospital Deaconess Medical Center, Harvard Medical School; 3 Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115; and Divisions of 2 Gastroenterology, 4 Oncology, and 5 Clinical Pathology, Geneva University Hospitals, 1211 Geneva 14, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12×) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.

granulocyte-macrophage colony-stimulating factor; caerulein; inflammation; adult respiratory distress syndrome; adhesion molecules; neutrophils


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE LUNG INJURY and the adult respiratory distress syndrome (ARDS) complicate many disease states and are central components of the systemic immune response syndrome (SIRS) (4, 17, 24). Although the mechanisms underlying these processes remain incompletely understood, the morphological and functional changes include sequestration of inflammatory cells within the lung (2, 7, 37), injury to the pulmonary microvascular surface (19), and extravasation of otherwise intravascular fluid across the microvascular endothelial barrier into the bronchoalveolar space (21). Acute lung injury and ARDS, along with other elements of SIRS, are frequently noted in patients with severe acute pancreatitis (30). Roughly 40% of patients who die during the early stages of severe pancreatitis succumb from lung injury and respiratory failure. Therefore a better understanding of the mechanism leading to lung injury is of importance.

Several models of acute pancreatitis in experimental animals have been shown to be accompanied by measurable lung injury (9, 14, 15, 37), and a number of recent studies have focused on identifying the factors that couple pancreatic inflammation to lung injury in those models (1, 2, 10, 26). In the current communication, we have utilized the secretagogue (caerulein)-induced model of acute pancreatitis (20) in mice to examine the role played by granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating acute lung injury in this model.

GM-CSF is a cytokine that promotes the growth of myeloid progenitor cells and the activation of mature neutrophils, eosinophils, and monocytes (23, 34). It also enhances complement- and antibody-mediated phagocytosis. GM-CSF as well as other proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin-1 (IL)-1, and chemokines, including macrophage inflammatory protein (MIP)-1alpha and MIP-2, have been reported to increase during numerous lung inflammatory conditions.

GM-CSF is synthesized and secreted by a wide variety of activated cells, including T lymphocytes, macrophages, lung epithelial cells, and cytokine-activated endothelial cells (8, 32). It has been shown to increase neutrophil chemotaxis, upregulate the intercellular adhesion molecule CD11b/CD18 and enhance the functional activity of mature cells (12, 29, 35). GM-CSF is secreted by lung epithelial cells, and GM-CSF injections, to humans, have been shown to induce 1) an increased neutrophil count, 2) neutrophil activation, 3) neutrophil degranulation, and 4) the release of IL-8 (33) as well as MIP-2, both cytokines known to play a key role in acute lung injury (9). On the basis of these observations and the fact that GM-CSF is secreted by lung epithelial cells, we postulate that it might mediate lung injury in diseases such as acute pancreatitis and that it might carry out this role both directly and indirectly by regulating release of other, downstream, proinflammatory factors such as MIP-2.

In this study, we have found that GM-CSF and MIP-2 are upregulated in the lung during caerulein-induced acute pancreatitis. We have compared the severity of the pancreatic and lung injury noted when pancreatitis is induced in control animals to the severity of these injuries when pancreatitis is induced in animals that are genetically incapable of generating GM-CSF [GM-CSF-deficient (GM-CSF -/-) mice]. We report that the absence of GM-CSF does not significantly reduce the severity of caerulein-induced pancreatitis, but it markedly reduces the severity of pancreatitis-associated lung injury in this model. We have also noted that the rise in lung levels of MIP-2, the mouse neutrophil chemoattractant mediator that seems to be comparable to IL-8 in primates (16), is blunted when pancreatitis is induced in GM-CSF -/- animals and that the severity of pancreatitis-associated lung injury can be reduced by administration of anti-MIP-2 antibodies. These observations lead us to conclude that GM-CSF plays an important role in regulating the severity of pancreatitis-associated lung injury but not in regulating the severity of pancreatitis in the secretagogue-induced model. We suggest that GM-CSF may regulate the severity of lung injury directly by increasing inflammatory cell infiltration into the lung and indirectly by modifying the magnitude with which other inflammatory mediators such as MIP-2 (i.e., IL-8) are released from neutrophils during pancreatitis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Homozygous GM-CSF -/- and wild-type Black 6 GM-CSF-sufficient mice (GM-CSF +/+) were provided by Dr. G. Dranoff. Animal genotyping was done before each experiment. Thus each GM-CSF -/- mouse was shown to be null for GM-CSF before the start of each set of experiments. We generated the GM-CSF -/- animals by gene-targeting techniques in embryonal stem cells (23). They were shown to have normal baseline and stressed hematopoiesis, i.e., the number of circulating hematopoietic cells and the progenitor cell levels in hematopoietic organs were similar to those in GM-CSF +/+ mice. Because GM-CSF -/- mice are known to develop surfactant accumulation in the alveolar space mimicking pulmonary alveolar proteinosis at older ages, we used mice 4 wk of age or younger in our studies. At that age, no histological lung abnormalities are detectable. Mice were bred and housed in standard cages in a climate-controlled room with an ambient temperature of 23 ± 2°C and a 12:12-h light-dark cycle. They were fed standard laboratory chow, given water ad libitum, and randomly assigned to experimental groups. All animals were fasted overnight before each experiment, but water was not withheld. Caerulein, the decapeptide analog of the pancreatic secretagogue cholecystokinin, was purchased from Research Plus (Bayonne, NJ). Monoclonal antibodies to MIP-2 were obtained from R&D Systems (Abingdon, UK). All other chemicals and reagents were from sources noted or previously reported (28, 36).

Induction of pancreatitis. Randomly chosen male and female GM-CSF +/+ and GM-CSF -/- mice weighing 20-22 g were given hourly (12×) intraperitoneal injections (0.2 ml) containing caerulein at a concentration calculated to deliver a dose of 50 µg/kg with each injection. Control animals received 0.2-ml hourly injections of saline. For most determinations, two sets of experiments were performed. In the first, the animals were killed by administration of a lethal dose of pentobarbital 1 h after receiving the final caerulein or saline injection (i.e., 12 h after the first injection of caerulein or saline), whereas, in the second set of experiments, the mice were killed either 1 or 24 h after the start of caerulein administration.

Anti-MIP-2 treatment. For experiments using anti-MIP2 antibody, 60 min before the start of caerulein administration, caerulein-treated animals received a single intravenous injection containing 5 mg/kg anti-MIP-2 antibody dissolved in 0.2 ml of saline (pH 7.4) as previously reported by others (8).

Quantitation of pancreatitis severity. We evaluated the severity of pancreatitis by quantitating serum amylase activity, sequestration of neutrophils within the pancreas [i.e., pancreas myeloperoxidase (MPO) activity], pancreatic edema (i.e., pancreatic water content), and morphological evidence of pancreatic acinar cell necrosis. Serum amylase activity was measured, in samples obtained at the time of death, as described by Pierre et al. (27) using 4,6-ethylidine (G1)-p-nitrophenyl (G1)-D-maltoheptaside (Sigma, St. Louis, MO) as the substrate. Pancreas MPO activity was measured as previously described (10) and expressed as a function of tissue wet weight. We quantitated pancreatic water content by weighing freshly obtained blotted samples and then reweighing those samples after dessication (95°C, 12 h). The difference between wet and dry weight (i.e., water content) was calculated and expressed as a percentage of tissue wet weight. For morphological examination of the pancreas, samples obtained at the time of death were immediately fixed, embedded in paraffin, and sectioned (5 µm). They were stained with hematoxylin-eosin and examined by an experienced morphologist who was not aware of the sample identity. The extent of acinar cell necrosis was quantitated by computer-assisted morphometry as previously described (1) and expressed as a percentage of total acinar tissue.

Quantitation of lung injury severity. We evaluated the severity of pancreatitis-associated lung injury by quantitating lung water content as described above for pancreas water content, measuring the sequestration of neutrophils within the lung (i.e., lung MPO activity), evaluating capillary-alveolar membrane thickness, and characterizing pulmonary microvascular permeability (i.e., leakage of intravenously administered FITC-labeled albumin into the bronchoalveolar space). Lung MPO activity was measured, on samples obtained at the time of death, as described in Induction of pancreatitis and previously (13). MPO activity was expressed as a function of lung wet weight. For estimation of capillary-alveolar membrane thickness, we distended the lung at the time of death by instilling 4% neutral buffered formalin at a hydrostatic pressure of 20 cmH2O. Portions of the formalin-distended lungs were harvested, fixed, paraffin-embedded, sectioned (5 µm), stained with hematoxylin-eosin, and examined by a morphologist who was not aware of the sample identity. Capillary-alveolar membrane thickness was visually estimated on a scale ranging from 0 to 4, with 0 being the thinnest and 4 the thickest as observed in the course of these experiments. Leakage of intravenously administered FITC-labeled albumin into the bronchoalveolar space was evaluated as previously described (30). The ratio of FITC fluorescence in bronchoalveolar lavage fluid and blood was calculated and expressed as the permeability index.

GM-CSF immunolocalization within pancreas and lung. Immunolocalization of GM-CSF was characterized by conventional light microscopy on cryostat sections from pancreas and lung samples taken from GM-CSF +/+ mice given either saline or caerulein injections. We performed immunohistochemical staining using primary antibodies to mouse GM-CSF (rat anti-mouse GM-CSF, dilution 1:50; Pharmingen) diluted in BSA-PBS. Briefly, frozen sections were mounted on silane-coated glass slides and pretreated with levamisole to block endogenous alkaline phosphatase activity. Negative control sections were incubated in BSA-PBS (without primary antibody) and showed no immunostaining. After incubation for 1 h at room temperature with the diluted primary antibodies, the sections were sequentially treated with secondary antibody (rabbit anti-rat IgG 1:100 in BSA-PBS) for 30 min, and reactions were revealed by the alkaline phosphatase complex method (Dako). Sections were counterstained with Mayer's hematoxylin.

Other assays. Circulating leukocytes were quantitated with blood samples obtained 13 h after the start of saline or caerulein administration. For these measurements, total and differential cell counts were made with Giemsa-stained samples. We measured GM-CSF and MIP-2 levels in pancreas, serum, and lung with commercially available ELISA kits according to the manufacturers' instructions (R&D Systems, Abingdon, UK).

Analysis of data. The results reported in this communication represent means ± SE values obtained from multiple determinations in three or more experiments for each set. In Figs. 2-4 and 6, vertical bars denote SE values, and the absence of such bars indicates that the SE is too small to illustrate. The significance of changes was evaluated by Student's t-test when data consisted of only two groups or by ANOVA when three or more groups were compared. For comparison of capillary-alveolar thickness measurements, Wilcoxon's nonparametric test was done using SPSS software package.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of secretagogue-induced pancreatitis on GM-CSF levels. The levels of GM-CSF in pancreas, serum, and lung of GM-CSF +/+ animals 12 h after the start of caerulein administration are shown in Table 1. Within 12 h of the start of caerulein administration, pancreas GM-CSF levels are slightly, but significantly, elevated. GM-CSF levels in serum are elevated to an even greater extent, whereas those in lung exceed even those noted in serum.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   GM-CSF levels in pancreas (pg/µg DNA), lung (pg/µg DNA), and serum (pg/ml) during caerulein-induced pancreatitis

The identity of pancreas and lung cells expressing GM-CSF during caerulein-induced pancreatitis was evaluated by immunolocalization using anti-GM-CSF antibody (Fig. 1). GM-CSF was not detected in either tissue when the primary antibody was omitted (not shown). In pancreas sections from control animals, GM-CSF was detected at very low levels in the inflammatory cells but not acinar cells (Fig. 1A). After 12 h of caerulein administration, the number of immunostained inflammatory cells increased, but GM-CSF was still undetectable in the acinar cells (Fig. 1B). Intense immunoreactivity for GM-CSF was observed within bronchial epithelial cells and macrophages 12 h after caerulein administration (Fig. 1D) compared with controls (Fig. 1C).


View larger version (112K):
[in this window]
[in a new window]
 
Fig. 1.   Granulocyte-macrophage colony-stimulating factor (GM-CSF) immunolocalization within pancreas and lung. Cryostat sections were treated as described in MATERIALS AND METHODS. The presence of GM-CSF in the pancreas from either control animals or animals receiving caerulein was not detected in the acinar cells (A), but there were some inflammatory cells positive for GM-CSF (arrows, B). Bronchial epithelial cells and macrophages in the lungs from control animals (C) were positive for GM-CSF. In animals given caerulein, the number of immunopositive cells as well as the staining intensity for GM-CSF increased significantly compared with controls (D). Magnification ×400. Pictures are representative of at least 3 experiments.

Effects of supramaximal secretagogue stimulation on circulating leukocytes in GM-CSF +/+ and GM-CSF -/- animals. Total circulating leukocytes in both GM-CSF +/+ and GM-CSF -/- animals were ~2,450 ± 212 cells/mm3 12 and 24 h after the first saline injection (Fig. 2). Twelve hours after the start of caerulein administration, total circulating leukocyte counts had increased more than threefold in the GM-CSF +/+ group. A nearly identical rise was noted at this time in the GM-CSF -/- group. The rise in circulating leukocyte count in both groups was similar and attributable to an increase in circulating neutrophil and nonneutrophil leukocytes (Fig. 2). Twelve hours later, i.e., 12 h after administration of the last dose of caerulein, the total circulating leukocyte count in both the GM-CSF +/+ and GM-CSF -/- groups had fallen to a similar extent toward, but not reaching, the precaerulein level (not shown).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Total peripheral white blood cell count and differential count during pancreatitis. Mice were given 12 hourly injections of caerulein (50 µg/kg ip) or saline and killed 1 h later. White blood cell counts and neutrophil counts were determined manually, using Giemsa-stained smears. There was no difference when GM-CSF-sufficient (GM-CSF +/+) mice were compared with GM-CSF-deficient (GM-CSF -/-) mice. Open bars, total white blood cell count; solid bars, neutrophil counts.

Effects of GM-CSF deletion on the severity of caerulein-induced pancreatitis. The severity of caerulein-induced pancreatitis was evaluated 12 h after the start of caerulein administration (Fig. 3). At this time, a marked and similar increase in serum amylase activity, pancreatic water content, and extent of acinar cell necrosis was noted in both the GM-CSF +/+ and GM-CSF -/- groups. Pancreas MPO activity was also markedly increased in both groups, but, in this case, the rise noted in the GM-CSF -/- group was significantly less than that noted in the GM-CSF +/+ group.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of GM-CSF deficiency on the severity of secretagogue-induced pancreatitis. Mice were given 12 hourly injections of caerulein (50 µg/kg ip) or saline (control) and killed 1 h later. Serum amylase activity (A), pancreas water content (B), pancreas myeloperoxidase (MPO, C) and acinar cell necrosis (D) were quantitated as described in MATERIALS AND METHODS. Hatched bars, wild-type GM-CSF +/+ animals; crosshatched bars, GM-CSF -/- animals. Results shown are means ± SE for >= 18 animals in each group. * P < 0.01 when GM-CSF -/- animals were compared with GM-CSF +/+ animals.

Effects of GM-CSF deletion on the severity of pancreatitis-associated lung injury. Guice et al. (14) have previously noted that, in the secretagogue-induced model of pancreatitis, pancreatic injury precedes the development of lung injury. For that reason, we evaluated the effect of GM-CSF deletion on the severity of pancreatitis-associated lung injury both 12 and 24 h after the start of caerulein administration (Fig. 4). We evaluated lung injury by quantitating neutrophil sequestration in the lung (i.e., lung MPO activity) and pulmonary microvascular permeability (i.e., leakage of intravenously administered FITC-labeled albumin into the bronchoalveolar fluid). In the GM-CSF +/+ group, lung MPO activity was increased at 12 and 24 h after the start of caerulein administration, and this rise in lung MPO activity was markedly blunted in the GM-CSF -/- group at both times. Pulmonary microvascular permeability was also markedly increased both 12 and 24 h after the start of caerulein administration in the GM-CSF +/+ group, and, at both times, a marked blunting of this response was noted in the GM-CSF -/- group. Capillary-alveolar membrane thickness was slightly increased 12 h after the start of caerulein administration, and this parameter of lung injury was more markedly increased 24 h after the start of caerulein administration (Figs. 4 and 5). At this later time, the changes noted in the GM-CSF +/+ group are markedly reduced in the GM-CSF -/- group.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of GM-CSF deficiency on the severity of secretagogue-induced pancreatitis-associated lung injury. Animal groups and bar description are as described in Fig. 3 legend. Lung MPO (A), lung permeability index (B), lung water content (C) and alveolar thickness (D) were quantitated as described in MATERIALS AND METHODS. FITC-labeled albumin was administered intravenously 2 h before animals were killed, and leakage of FITC-labeled albumin into the bronchoalveolar space was quantitated as described in Quantitation of lung injury severity. Results shown are means ± SE for >= 10 animals in each group. * P < 0.01 when GM-CSF -/- animals were compared with GM-CSF +/+ animals.



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 5.   Lung morphology. GM-CSF +/+ mice were given either saline (A) or caerulein (B and C) for 12 h. They were killed 12 (B) or 24 h (C) after the start of caerulein administration. Identical studies were performed on GM-CSF -/- mice given either saline (D) or caerulein (E and F), which were killed 12 (E) or 24 h (F) after the start of caerulein administration. Alveolar hemorrhage was more pronounced 12 h after the start of caerulein in GM-CSF +/+ mice (B) than in GM-CSF -/- mice (E), and thickening of the alveolar membrane was dramatically reduced in -/- mice at either 12 (E) or 24 h (F) compared with GM-CSF +/+ mice (B and C). Magnification is ×150 for lung.

Effect of GM-CSF deletion on MIP-2 levels. Twelve hours after the start of caerulein administration, pancreas, serum, and lung MIP-2 levels in GM-CSF +/+ animals were markedly elevated (Table 2). A similar rise in pancreas MIP-2 level was observed in the GM-CSF -/- group, but the rise in both serum and lung MIP-2 level was markedly blunted in the GM-CSF -/- group compared with that observed in the GM-CSF +/+ animals.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   MIP-2 levels in pancreas (pg/mg protein), lung (pg/mg protein), and serum (pg/ml) during caerulein-induced pancreatitis

Effects of anti-MIP2 treatment on caerulein-induced lung injury. As shown in Fig. 6, anti-MIP-2 antibody administration in mice receiving caerulein resulted in a significant decrease of all parameters that characterize the severity of pancreatitis-associated lung injury.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of anti-macrophage inflammatory protein (MIP)-2 treatment on the severity of caerulein-induced lung injury. One hour before the start of caerulein administration [12 hourly injections of caerulein (50 µg/kg ip) or saline (control)], mice received an intravenous injection of a solution containing either 5 mg/kg of rat monoclonal anti-mouse MIP-2 antibody or a nonimmune rat IgG. Severity of lung injury was assessed as described in MATERIALS AND METHODS. Results shown are means ± SE for >= 7 animals in each group. * P < 0.05 when animals receiving anti-MIP-2 antibody were compared with animals receiving a nonimmune IgG solution. Open bars, control animal; solid bars, animals receiving caerulein; gray bars, animals that had been previously given anti-MIP-2 antibody receiving caerulein. Lung MPO (A), lung permeability index (B), lung water content (C), and alveolar thickness (D) were quantitated as described in MATERIALS AND METHODS. FITC-labeled albumin was administered intravenously 2 h before animals were killed, and leakage of FITC-labeled albumin into the bronchoalveolar space was quantitated as described in Quantitation of lung injury severity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Acute lung injury, ARDS, and respiratory failure are clinically important complications of severe pancreatitis. They account for significant morbidity and mortality in patients with this disease (31). Recent studies, most of which have employed experimental models of pancreatitis, have indicated that a number of inflammatory factors regulate the coupling of pancreatic to lung injury (9, 26). Those studies have shown that neutrophils (2, 10), T lymphocytes (10, 22), adhesion molecules (10), platelet-activating factor (18), TNF-alpha (13, 14), substance P (1), and chemokines acting via the CCR-1 receptor (13) are each involved in this process.

The currently reported studies have examined the role of GM-CSF in regulating the severity of pancreatitis-associated lung injury. For these studies, the secretagogue-induced model of pancreatitis in mice was used, and genetically modified animals unable to generate GM-CSF were employed to evaluate the role of GM-CSF.

GM-CSF is a proinflammatory cytokine that is synthesized by several types of cells, including monocytes, T lymphocytes (5, 10, 34), lung epithelial cells (23), activated eosinophils (25), and cytokine-activated endothelial cells in vitro (8). It promotes the growth and maturation of granulocyte and monocyte progenitors and the activation of mature neutrophils, eosinophils, and monocytes (34). It primes neutrophils, making them more receptive to activation by secondary stimuli (3, 33). GM-CSF has been shown to increase neutrophil chemotaxis, upregulate the expression of intracellular adhesion molecule CD11b/CD18 (22), and enhance the functional activity of mature cells (35). In humans, injection of GM-CSF has been shown to activate and degranulate neutrophils and to cause release of IL-8 (33).

The studies reported in this communication indicate that pancreas and serum GM-CSF levels are moderately increased during caerulein-induced pancreatitis. Of perhaps more importance, however, is our observation that lung GM-CSF levels are even more markedly increased during caerulein-induced necrotizing pancreatitis (Table 1).

As shown in Fig. 3, caerulein administration to both GM-CSF +/+ and GM-CSF -/- mice results in severe pancreatitis, which is characterized by marked and similar increases in serum amylase levels, pancreatic edema, and acinar cell necrosis in both animal groups. Pancreas MPO activity, an index of the extent of neutrophil sequestration in the pancreas, is also increased in both groups, but, in this case, the increase noted in the GM-CSF -/- group is less marked than that noted in the GM-CSF +/+ group. The significance of this isolated difference among the various parameters used to quantitate the severity of pancreatitis is not known, but it may reflect the recognized importance of GM-CSF in the process of neutrophil activation and the previously reported observation that neutrophil-independent events play pivotal roles in regulating the severity of pancreatic injury (10, 13).

In contrast to its marginal effect on the pancreatic injury in the caerulein model, the lack of GM-CSF in the GM-CSF -/- group of mice markedly reduces the severity of pancreatitis-associated lung injury. Twelve hours after the start of caerulein administration, the increase in lung MPO activity, as well as the increase in pulmonary microvascular permeability that occurs in the GM-CSF +/+ group, is markedly depressed in the GM-CSF -/- group of animals. The protection against increased microvascular permeability that accompanies GM-CSF deletion persists for at least an additional 12 h, and 24 h after the start of caerulein administration, GM-CSF -/- mice have much less capillary-alveolar membrane thickening than do the GM-CSF +/+ animals.

Together, these observations indicate that GM-CSF plays an important proinflammatory role in regulating the severity of pancreatitis-associated lung injury but that it does not play a critical role in regulating the severity of the pancreatic injury. This action of GM-CSF does not appear to reflect its known ability to regulate the growth and maturation of inflammatory cells systemically, since circulating leukocyte and neutrophils counts are similar in GM-CSF +/+ and GM-CSF -/- animals both before and after caerulein administration has induced pancreatitis-associated lung injury.

It is tempting, therefore, to speculate that GM-CSF exerts its proinflammatory effect on pancreatitis-associated lung injury by upregulating inflammatory cell function rather than number. Support for this speculation comes from our findings that the rise in lung MIP-2 levels seen during pancreatitis-associated lung injury in GM-CSF +/+ animals is substantially blunted in GM-CSF -/- mice and that the severity of pancreatitis-associated acute lung injury in GM-CSF +/+ animals can be reduced by administration of anti-MIP-2 antibodies. MIP-2 is believed by most observers to represent the mouse equivalent of IL-8, a potent neutrophil chemotactic cytokine, but the equivalence of MIP-2 and IL-8 is still speculative. However, our studies using anti-MIP-2 antibodies now provide strong evidence for the central role played by MIP-2 in translating pancreatic inflammation to the lung. Further support for our suggestion that GM-CSF promotes pancreatitis-associated lung injury via its effect on neutrophil function comes from recent observations, by others, indicating that GM-CSF levels rise in other models of lung injury (5) and that GM-CSF exerts a potent antiapoptotic effect on neutrophils in vitro in areas of inflammation (25). Presumably, the delay in neutrophil apoptosis induced by local generation of GM-CSF might favor persistence and worsening of the local (i.e., pulmonary in this case) inflammatory process, a feature recently suggested by O'Neill et al. (25) in neutrophils from patients with acute pancreatitis. Regardless of the mechanisms responsible for the proinflammatory effects of GM-CSF in pancreatitis-associated lung injury, the observations reported in this communication suggest that therapies designed to reduce or abort the rise in lung GM-CSF or prevent its signaling during pancreatitis may prove beneficial in reducing the severity of pancreatitis-associated lung injury.

One might speculate that the effects of GM-CSF deficiency noted in our studies might be due to the lifelong absence of GM-CSF in our knockout mice and not the absence of an acute GM-CSF-mediated response. The use of knockout mice devoid of active pro- or anti-inflammatory mediators allows examination of the effects of a specific cytokine without the drawbacks induced by pharmacological manipulations (26), but one must keep in mind that the specific mutation has been present in the mouse from the time of its conception. This may result in phenotypic changes due to the mutation itself, but it may also result in changes caused by adaptation to and compensation for the mutation (26). This latter possibility would seem unlikely, however, in our studies, in view of our observation that deletion of GM-CSF alters neither lung morphology nor circulating leukocyte levels at 3 mo of age.


    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grants DK-31396 (M. L. Steer) and RO1 CA-74886 (G. Dranoff) and by the Cancer Research Institute/Partridge Foundation (G. Dranoff). J. L. Frossard was financially supported by Swiss National Science Foundation Grant 32-63618.00.


    FOOTNOTES

Address for reprint requests and other correspondence: M. L. Steer, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: msteer{at}caregroup.harvard.edu).

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

May 3, 2002;10.1152/ajplung.00413.2001

Received 23 October 2001; accepted in final form 4 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bhatia, M, Saluja AK, Hofbauer B, Frossard JL, Lee HS, Castagliuolo I, Wang CC, Gerard N, Pothoulakis C, and Steer ML. Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury. Proc Natl Acad Sci USA 95: 4760-4765, 1998[Abstract/Free Full Text].

2.   Bhatia, M, Saluja AK, Hofbauer B, Lee HS, Frossard JL, and Steer ML. The effects of neutrophil depletion on a completely noninvasive model of acute pancreatitis-associated lung injury. Int J Pancreatol 24: 77-83, 1998[ISI][Medline].

3.   Bittleman, DR, Erger RA, and Casale TB. Cytokines induce selective granulocyte chemotactic responses. Inflamm Res 45: 89-95, 1996[ISI][Medline].

4.   Bone, RC. Immunologic dissonance: a continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann Intern Med 125: 680-687, 1996[Abstract/Free Full Text].

5.   Clark, SC, and Kamen R. The human hematopoietic colony-stimulating factors. Science 236: 1229-1237, 1987[ISI][Medline].

6.   Deng, H, Mason N, and Auten RL. Lung inflammation in hyperoxia can be prevented by antichemokine treatment in newborn rats. Am J Respir Crit Care Med 162: 2316-2323, 2000[Abstract/Free Full Text].

7.   Feddersen, CO, Willemer S, Karges W, Puchner A, Adler G, and Wichert PV. Lung injury in acute experimental pancreatitis in rats. II. Functional studies. Int J Pancreatol 8: 323-331, 1991[ISI][Medline].

8.   Fibbe, WC, Daha MR, and Hiemstra PS. Interleukin1 and poly(rl)poly(rC) induce production of granulocyte CSF, macrophage CSF and granulocyte-macrophage CSF by human endothelial cells. Exp Hematol 17: 229-234, 1989[ISI][Medline].

9.  Frossard JL, Hadengue A, Spahr L, et al. Long term lung injury in mouse experimental pancreatitis. Crit Care Med. In press.

10.   Frossard, JL, Kwak B, Chanson M, Morel P, Hadengue A, and Mach F. CD40 ligand-deficient mice are protected against cerulein-induced acute pancreatitis and pancreatitis-associated lung injury. Gastroenterology 121: 184-194, 2001[ISI][Medline].

12.   Gasson, JC. Molecular physiology of granulocyte-macrophage colony stimulating factor. Blood 77: 1131-1145, 1991[ISI][Medline].

13.   Gerard, C, Frossard JL, Bhatia M, Saluja A, Gerard NP, Lu B, and Steer ML. Targeted disruption of the beta-chemokine receptor CCR1 protects against pancreatitis-associated lung injury. J Clin Invest 100: 2022-2027, 1997[Abstract/Free Full Text].

14.   Guice, KS, Oldham KT, Caty MG, Johnson KJ, and Ward PA. Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis. Ann Surg 210: 740-747, 1989[ISI][Medline].

15.   Guice, KS, Oldham KT, Johnson KJ, Kunkel RG, Morganroth ML, and Ward PA. Pancreatitis-induced acute lung injury. An ARDS model. Ann Surg 208: 71-77, 1988[ISI][Medline].

16.   Hang, L, Haraoka M, Agace WW, Leffler H, Burdick M, Strieter R, and Svanborg C. Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract. J Immunol 162: 3037-3044, 1999[Abstract/Free Full Text].

17.   Haveman, JW, Muller Kobold AC, Tervaert JW, van den Berg AP, Tulleken JE, Kallenberg CG, and The TH. The central role of monocytes in the pathogenesis of sepsis: consequences for immunomonitoring and treatment. Neth J Med 55: 132-141, 1999[ISI][Medline].

18.   Hofbauer, B, Saluja AK, Bhatia M, Frossard JL, Lee HS, Bhagat L, and Steer ML. Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis. Gastroenterology 115: 1238-1247, 1998[ISI][Medline].

19.   Holman, RG, and Maier R. Superoxide production by neutrophils in a model of adult respiratory distress syndrome. Arch Surg 123: 1491-1495, 1988[Abstract].

20.   Lampel, M, and Kern HF. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchow Arch A Pathol Anat Histol 373: 97-117, 1977.

21.   Muramaki, H, Nakao A, Kishimoto W, Nakano M, and Takagi H. Detection of O2-generation and neutrophil accumulation in rat lungs after acute necrotizing pancreatitis. Surgery 118: 547-54, 1995[ISI][Medline].

22.   Nagase, T, Ohga E, Sudo E, Katayama H, Uejima Y, Matsuse T, and Fukuchi Y. Intercellular adhesion molecule-1 mediates acid aspiration-induced lung injury. Am J Respir Crit Care Med 154: 504-510, 1996[Abstract].

23.   Nagata, M, Sedgwick JB, and Busse WW. Differential effects of granulocyte-macrophage colony-stimulating factor on eosinophil and neutrophil superoxide anion generation. J Immunol 155: 4948-4954, 1995[Abstract].

24.   Nomura, S, Kagawa H, Ozaki Y, Nagahama M, Yoshimura C, and Fukuhara S. Relationship between platelet activation and cytokines in systemic inflammatory response syndrome patients with hematological malignancies. Thromb Res 95: 205-213, 1999[ISI][Medline].

25.   O'Neill, S, O'Neill AJ, Conroy E, Brady HR, Fitzpatrick JM, and Watson RW. Altered caspase expression results in delayed neutrophil apoptosis in acute pancreatitis. J Leukoc Biol 68: 15-20, 2000[Abstract/Free Full Text].

26.   Pastor, CM, and Frossard JL. Are genetically modified mice useful for the understanding of acute pancreatitis? FASEB J 15: 893-897, 2001[Abstract/Free Full Text].

27.   Pierre, K, Tung K, and Nadj H. A new enzymatic kinetic method for determination of amylase. Clin Chem 22: 1219, 1972.

28.   Saluja, AK, Lu L, Yamaguchi Y, Hofbauer B, Runzi M, Dawra R, Bhatia M, and Steer ML. A cholecystokinin-releasing factor mediates ethanol-induced stimulation of rat pancreatic secretion. J Clin Invest 99: 506-512, 1997[Abstract/Free Full Text].

29.   Socinski, MA, Cannistra SA, Sullivan R, Elias A, Antman K, Schnipper L, and Griffin JD. Granulocyte-macrophage colony-stimulating factor induces expression of the CD11b surface adhesion molecule on human granulocytes in vivo. Blood 72: 691-697, 1988[Abstract].

30.   Steer, ML. Etiology and pathophysiology of acute pancreatitis. In The Pancreas: Biology, Pathobiology and Disease, edited by Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA.. New York: Raven, 1993, p. 581-592.

31.   Steer, ML, and Meldolesi J. Pathogenesis of acute pancreatitis. Annu Rev Med 39: 95-105, 1988[ISI][Medline].

32.   Strieter, RM, Kunkel SL, Showell HJ, Remick DG, Phan SH, Ward PA, and Marks RM. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS and IL-1 beta. Science 243: 1467-1469, 1989[ISI][Medline].

33.   Van Pelt, LJ, Huisman MV, Weening RS, von dem Borne AE, Roos D, and van Oers RH. A single dose of granulocyte-macrophage colony stimulating factor induces systemic interleukin-8 release and neutrophil activation in healthy volunteers. Blood 12: 5305-5313, 1996.

34.   Vliagoftis, H, Befus AD, Hollenberg MD, and Moqbel R. Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2. J Allergy Clin Immunol 107: 679-685, 2001[ISI][Medline].

35.   Wang, JM, Colella S, Allavena P, and Mantovani A. Chemotactic activity of human recombinant granulocyte-macrophage colony-stimulating factor. Immunology 60: 439-444, 1987[ISI][Medline].

36.   Yamanaka, K, Saluja AK, Brown GE, Yamaguchi Y, Hofbauer B, and Steer ML. Protective effects of prostaglandin E1 on acute lung injury of caerulein-induced acute pancreatitis in rats. Am J Physiol Gastrointest Liver Physiol 272: G23-G30, 1997[Abstract/Free Full Text].

37.   Willemer, S, Feddersen CO, Karges W, and Adler G. Lung injury in acute experimental pancreatitis in rats. I. Morphological studies. Int J Pancreatol 8: 305-321, 1991[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 283(3):L541-L548
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society