Methylprednisolone improves lung mechanics and reduces theinflammatory response in pulmonary but not in extrapulmonarymild acute lung injury in mice*

Jose Henrique P. Leite-Junior, MD, PhD; Cristiane S.N.B. Garcia, PhD; Alba B. Souza-Fernandes, PhD;Pedro L. Silva, MSc; Debora S. Ornellas, PhD; Andrea P. Larangeira, MSc; Hugo C. Castro-Faria-Neto, MD, PhD;Marcelo M. Morales, MD, PhD; Elnara M. Negri, MD, PhD; Vera L. Capelozzi, MD, PhD; Walter A. Zin, MD, PhD;Paolo Pelosi, MD; Patricia T. Bozza, MD, PhD; Patricia R.M. Rocco, MD, PhD

Acute lung injury (ALI) and acuterespiratory distress syndrome(ARDS) are life-threateningforms of acute hypoxemic respi-

ratory failure, complicated by systemic in-flammation and tissue remodeling (1–3).The use of corticosteroids has been pro-posed in the treatment of ALI/ARDS (4)because of their anti-inflammatory proper-ties and potential effects on remodeling

process (5). Corticosteroids have been usedin three main situations: 1) prevention inhigh-risk patients (6–8); 2) early treatmentwith high dose, short course therapy (9);and 3) prolonged therapy in late-phase (4,10–13). Unfortunately, these trials failed toimprove survival of patients at high risk(6–8), early (9), or late-phase ALI/ARDS (4).

Some authors reported an increasednumber of myofibroblasts and cells produc-

ing procollagen types I and III in the earlycourse of ALI/ARDS, suggesting that theproliferative phase began much soonerthan previously described (14–19). Accord-ing to these observations, a recent experi-mental study (20) and small randomizedtrials (21, 22) suggested that steroids maybe useful in the management of ALI/ARDSpatients at an early phase.

There are many causes that triggerALI/ARDS and differences in the initialinsult combined with underlying condi-tions may result in the activation of dif-ferent inflammatory mechanisms (23,24). In this line, evidence indicates thatthe pathophysiology of early ALI/ARDSmay differ according to the type of pri-mary insult (23–28).

In the present study, we tested thehypothesis that methylprednisolone mayact differently on lung mechanics andhistology, inflammatory response, andtissue remodeling depending on the eti-ology of ALI.

Objective: Corticosteroids have been proposed to be effective inmodulating the inflammatory response and pulmonary tissue remodel-ing in acute lung injury (ALI). We hypothesized that steroid treatmentmight act differently in models of pulmonary (p) or extrapulmonary (exp)ALI with similar mechanical compromise.

Design: Prospective, randomized, controlled experimentalstudy.

Setting: University research laboratory.Subjects: One hundred twenty-eight BALB/c mice (20–25 g).Interventions: Mice were divided into six groups. In control animals

sterile saline solution was intratracheally (0.05 mL, Cp) or intraperitone-ally (0.5 mL, Cexp) injected, whereas ALI animals received Escherichiacoli lipopolysaccharide intratracheally (10 �g, ALIp) or intraperitoneally(125 �g, ALIexp). Six hours after lipopolysaccharide administration, ALIpand ALIexp animals were further randomized into subgroups receivingsaline (0.1 mL intravenously) or methylprednisolone (2 mg/kg intrave-nously, Mp and Mexp, respectively).

Measurements and Main Results: At 24 hrs, lung static elastance,resistive and viscoelastic pressures, lung morphometry, and collagenfiber content were similar in both ALI groups. KC, interleukin-6, andtransforming growth factor (TGF)-� levels in bronchoalveolar lavagefluid, as well as tumor necrosis factor (TNF)-�, migration inhibitoryfactor (MIF), interferon (IFN)-�, TGF-�1 and TGF-�2 messenger RNAexpression in lung tissue were higher in ALIp than in ALIexp animals.Methylprednisolone attenuated mechanical and morphometric changes,cytokine levels, and TNF-�, MIF, IFN�, and TGF-�2 messenger RNAexpression only in ALIp animals, but prevented any changes in collagenfiber content in both ALI groups.

Conclusions: Methylprednisolone is effective to inhibit fibrogen-esis independent of the etiology of ALI, but its ability to attenuateinflammatory responses and lung mechanical changes varies ac-cording to the cause of ALI. (Crit Care Med 2008; 36:2621–2628)

KEY WORDS: lung mechanics; collagen; electron microscopy;inflammation; cytokines

*See also p. 2700.From the Laboratory of Pulmonary Investigation (JHPL-J,

CSNBG, ABS-F, PLS, DSO, PRMR), Carlos Chagas FilhoInstitute of Biophysics, Federal University of Rio de Janeiro,Brazil; Laboratory of Immunopharmacology (APL, HCC-F-N,PTB), Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil;Laboratory of Cellular and Molecular Physiology (MMM),Carlos Chagas Filho Institute of Biophysics, Federal Universityof Rio de Janeiro, Rio de Janeiro, Brazil; Department ofPathology (EMN, VLC), School of Medicine, University of SaoPaulo, Sao Paulo, Brazil; Laboratory of Respiration Physiology(WAZ), Carlos Chagas Filho Institute of Biophysics, FederalUniversity of Rio de Janeiro, Rio de Janeiro, Brazil; andDepartment of Ambient, Health and Safety (PP), University ofInsubria, Varese, Italy.

Supported, in part, by Centers of Excellence Program(PRONEX-MCT and PRONEX-FAPERJ), Brazilian Councilfor Scientific and Technological Development (CNPq),Carlos Chagas Filho, Rio de Janeiro State Research Sup-porting Foundation (FAPERJ), Sao Paulo State ResearchSupporting Foundation (FAPESP).

The authors have not disclosed any potential con-flicts of interest.

For information regarding this article, E-mail:prmrocco@biof.ufrj.br

Copyright © 2008 by the Society of Critical CareMedicine and Lippincott Williams & Wilkins

DOI: 10.1097/CCM.0b013e3181847b43

2621Crit Care Med 2008 Vol. 36, No. 9

MATERIALS AND METHODS

Animal Preparation and ExperimentalProtocol. This study was approved by the Eth-ics Committee of the Carlos Chagas Filho In-stitute of Biophysics, Health Sciences Centre,Federal University of Rio de Janeiro. All ani-mals received humane care in compliancewith the “Principles of Laboratory AnimalCare” formulated by the National Society forMedical Research and the “Guide for the Careand Use of Laboratory Animals” prepared bythe National Academy of Sciences, Washing-ton, DC.

A total of 128 BALB/c mice (20–25 g) wererandomly assigned into six groups. In controlgroups, sterile saline solution (0.9% NaCl) wasinstilled intratracheally (0.05 mL, Cp, n � 20)or injected intraperitoneally (IP) (0.5 mL,Cexp, n � 20). In ALI groups, mice receivedEscherichia coli lipopolysaccharide (LPS, O55:B5, Sigma Chemical Co., St. Louis, MO) eitherintratracheally (10 �g diluted in 0.05 mL ofsaline/mouse, ALIp, n � 40) or intraperitone-ally (125 �g diluted in 0.5 mL of saline,ALIexp, n � 40). For intratracheal instillationmice were anesthetized with sevoflurane, a1-cm-long midline cervical incision was madeto expose the trachea, and LPS or saline wereinstilled using a bent 27-gauge tuberculinneedle. The cervical incision was closed with5.0 silk suture and the mice returned to theircage. The animals recovered rapidly after sur-gery. The dose of LPS was selected based onprevious studies demonstrating similar me-chanical and morphometrical compromise inALIp and ALIexp mice (27, 28). Six hours afterLPS administration ALIp and ALIexp animalswere further randomized into subgroups re-ceiving saline (0.1 mL intravenously [IV], n �20) or methylprednisolone (2 mg/kg diluted in0.1 mL of saline injected through tail vein, Mpand Mexp, respectively, n � 20) (Fig. 1). Allsubstances and surgical material were steril-ized.

Twenty-four hours after saline or LPS ad-ministration the animals were sedated (diaze-pam 1 mg IP), anesthetized (pentobarbital so-dium 20 mg/kg IP), tracheotomized, paralyzed(vecuronium bromide, 0.005 mg/kg IV), andventilated with a constant flow ventilator (Sa-may VR15; Universidad de la Republica,Mountevideo, Uruguay) with the following pa-rameters: frequency of 100 breaths/min, tidalvolume (VT) of 0.2 mL, and fraction of inspiredoxygen of 0.21. A positive end-expiratory pres-sure of 2 cm H2O was applied and the anteriorchest wall surgically removed. After a 10-minventilation period, lung mechanics were com-puted and at the end of the experiments (ap-proximately 30 mins), lungs were prepared forhistology.

A pneumotachograph was connected to thetracheal cannula for the measurements of airflow. VT was calculated by digital integrationof flow signal. The flow resistance of theequipment (Req), tracheal cannula included,amounted to 0.12 cm H2O/mL/s. Equipment

resistive pressure (Req.V�) was subtractedfrom pulmonary resistive pressure so that theresults represent intrinsic values. Trachealpressure (Ptr) was measured with a differentialpressure transducer SCIREQ (SC-24, Mon-treal, Quebec, Canada). All signals were fil-tered (100 Hz) and amplified in a 4-channelconditioner (SC-24, SCIREQ, Montreal, Que-bec, Canada). Flow and pressure signals werethen sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A, Data Transla-tion, Marlboro, MA), and stored on a micro-computer. All data were collected usingLABDAT software (RHT-InfoData, Montreal,Quebec, Canada).

Lung Mechanics. Lung mechanics and his-tology (light and electronic microscopy) werestudied in 36 mice (n � 6/group). Lung me-chanics were measured by the end-inflationocclusion method (29). In an open chest prep-aration, Ptr reflects transpulmonary pressure(PL). Briefly, after end-inspiratory occlusion,there is an initial fast drop in PL (�P1) fromthe preocclusion value down to an inflectionpoint (Pi), followed by a slow pressure decay(�P2), until a plateau is reached. This plateaucorresponds to the elastic recoil pressure ofthe lung (Pel). �P1 selectively reflects thepressure used to overcome the airway resis-tance. �P2 reproduces the pressure spent bystress relaxation, or viscoelastic properties ofthe lung, together with a small contribution ofpendelluft. Total pressure drop (�Ptot) isequal to the sum of �P1 and �P2. Lung static(Est) and dynamic (Edyn) elastances, and thedifference between dynamic and static elas-tance (�E) were determined. Lung mechanicsmeasurements were performed 10 times ineach animal. Values are presented as means �SEM for each group. All data were analyzedusing ANADAT data analysis software (RHT-InfoData).

Histologic Studies. The right lungs werequick-frozen by immersion in liquid nitrogen,

fixed with Carnoy’s solution and embedded inparaffin (30). Four micrometer-thick sliceswere cut and underwent hematoxylin-eosinstaining. Morphometric analysis was per-formed with an integrating eyepiece with acoherent system consisting of a grid with 100points and 50 lines (known length) coupled toa conventional light microscope (Axioplan,Zeiss, Oberkochen, Germany). The volumefraction of the lung occupied by hyperinflatedstructures (alveolar ducts, alveolar sacs or al-veoli wider than 120 �m) or collapsed alveoli(alveoli with rough or plicate walls) or normalpulmonary areas, and the amount of polymor-pho- and mononuclear cells and pulmonarytissue were determined by the point-countingtechnique (31), made across 10 random non-coincident microscopic fields at a magnifica-tion of �200 and �1000, respectively.

Collagen (picrosirius-polarization method[32]) and elastic fibers [Weigert’s resorcinfuchsin method, which allows the identifica-tion of elaunin and fully developed elastic fi-bers, and Weigert’s resorcin fuchsin methodmodified with oxidation, which allows theidentification of the three components of theelastic fiber system: elaunin, oxytalan, andfully developed elastic fibers (33)] were quan-tified in the alveolar septa. The area occupiedby fibers was determined by digital densito-metric recognition and divided by the lengthof each studied septum (19, 20).

Three slices of 2 � 2 � 2 mm were cutfrom three different segments of the left lungto obtain a stratified random sample. A speci-men was then fixed in 2.5% glutaraldehydeand phosphate buffer 0.1 M (pH 7.4), postfixedin 1% osmium tetroxide, dehydrated throughgraded ethanol solutions and embedded inEpon-Araldite. Ultrathin sections were inves-tigated using a transmission electron micro-scope (JEOL 1010 Transmission Electron Mi-croscope, Tokyo, Japan) after staining withuranyl acetate and lead citrate.

PULMONARY EXTRAPULMONARY

BALB/c mice

ALIp+Saline

Cexp(saline ip injected)

ALIexp(E. coli LPSinjection ip)

Cp(saline it instilled)

ALIp(E. coli LPSinstillation it)

ALIexp+Saline

PULMONARY EXTRAPULMONARY

ALIp+M (Mp)

BALB/c mice

ALIp+Saline

Cexp(saline ip injected)

ALIexp(E. coli LPSinjection ip)

Cp(saline it instilled)

ALIp(E. coli LPSinstillation it)

ALIexp+M (Mexp)ALIexp+Saline

Figure 1. A schematic flow chart of the design of the study. In pulmonary control (Cp) andextrapulmonary control (Cexp) groups, saline was intratracheally (it, 0.05 mL) instilled and intraperi-toneally (ip, 0.5 mL) injected, respectively. In pulmonary acute lung injury (ALIp) and extrapulmonaryacute lung injury (ALIexp) groups, mice received E. coli lipopolysaccharide (LPS) (10 �g, it and 125�g, ip, respectively). In pulmonary methylprednisolone (Mp) and extrapulmonary methylprednisolone(Mexp) groups, ALI animals were treated with methylprednisolone (2 mg/kg, intravenously) 6 hrs afterthe induction of ALI.

2622 Crit Care Med 2008 Vol. 36, No. 9

Ribonuclease Protection Assay. In eightother experimental groups (n � 4/group andtwo sham groups [n � 4]; total number � 32)were submitted to the aforementioned proto-cols to analyze messenger RNA (mRNA) ex-pression for murine cytokines by using ribo-nuclease protection assay (RPA). All protocolsfollowed the instructions for the RiboQuantMulti-Probe RNase Protection Assay System(BD-PharMingen, San Jose, CA). RNA was iso-lated from lung tissue and hybridized with amCK-3b Multi-Probe Template Set containingDNA templates for mouse mRNAs. The in vitrotranscription kit and a customized templateset (containing mouse lymphotoxin [LT]-�,[LT]-�, tumor necrosis factor TNF-�, inter-leukin [IL]-6, interferon [INF]-�, INF-�,transforming growth factor [TGF]-�1,TGF�-2, TGF�-3, migration inhibitory factor[MIF], and the housekeeping gene glyceralde-hyde-3-phosphate-dehydrogenase, and L32[ribosomal RNA]) were used to synthesize aradiolabeled probe set using [�-32P]UTP. Eachspecific hybridized product migrates accord-ing to its size, thereby allowing identificationof individual bands that were assigned to spe-cific mRNA products. After RNAse treatmentand purification, protected probes were run ona sequence gel, exposed to radiograph, anddeveloped. The quantity of each mRNA speciesin the original RNA sample was determined onthe basis of the signal intensity (by opticaldensitometry) given by the appropriatelysized, protected probe fragment band. Densityof each cytokine mRNA (TNF-�, MIF, INF-�,TGF-�1, and TGF-�2) is expressed relative tothat of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. These valueswere then related to ALIp group.

Evaluation of Bronchoalveolar LavageFluid (BALF). The remaining 60 animals (10mice/group) were submitted to the same pro-tocol previously described to obtain aliquots ofBALF. Amounts of IL-6, KC, and TGF-� werequantified by enzyme-linked immunosorbentassay on cell-free BALF according to manufac-turer’s protocol (Duo set, R & D Systems,Minneapolis, MN).

Statistical Analysis. SigmaStat 3.0 statisti-cal software package (Jandel Corporation, SanRaphael, CA) was used. Differences among thegroups were assessed by one-way analysis ofvariance followed by Tukey test when re-quired. p � 0.05 was considered significant.

RESULTS

The survival rate was 100% in bothcontrol and ALI groups, with or withoutmethylprednisolone.

During mechanical ventilation, therewas no significant difference in flow and VT

among the groups. Resistive (�P1) and vis-coelastic/inhomogeneous (�P2) pressures,�Ptot � �P1 �P2, and dynamic (Edyn)and static (Est) elastances, and differencebetween dynamic and static elastance (�E)

Figure 2. Lung static (Est) and dynamic (Edyn) elastances, and differences between dynamic andstatic elastance (�E) (upper panel). Total (�Ptot), resistive (�P1), and viscoelastic/inhomogeneous(�P2) pressure variations of the lung (lower panel). In pulmonary control (Cp) and extrapulmonarycontrol (Cexp) groups, saline was intratracheally (0.05 mL) instilled and intraperitoneally (0.5 mL)injected, respectively. In pulmonary acute lung injury (ALIp) and extrpulmonary acute lung injury(ALIexp) groups, mice received E. coli lipopolysaccharide (LPS) (10 �g, intratracheally and 125 �g,intraperitoneally, respectively). In pulmonary methylprednisolone (Mp) and extraplumonary meth-ylprednisolone (Mexp) groups, ALI animals were treated with methylprednisolone (2 mg/kg, intra-venously) 6 hrs after the induction of ALI. Values are means � SEM of six animals in each group (10determinations per animal). *Significantly different from Cp group (p � 0.05). **Significantlydifferent from Cexp group (p � 0.05).

Table 1. Morphometric parameters

Groups Normal Area (%) Alveolar Collapse (%) Total Cells (%) PMN (%) MN (%)

Cp 91.06 � 2.74 8.95 � 2.65 20.6 � 1.2 14.4 � 1.2 6.2 � 0.2ALIp 77.64 � 4.05a 22.36 � 4.05a 30.0 � 0.7a 23.1 � 1.0a 7.0 � 0.5Mp 88.58 � 1.14b 11.42 � 1.02b 22.2 � 0.9 14.7 � 1.1 7.5 � 0.7Cexp 92.11 � 0.27 7.18 � 0.52 21.2 � 0.8 16.3 � 0.9 4.9 � 0.3ALIexp 77.69 � 1.50c 22.19 � 1.32c 33.1 � 1.6c 29.6 � 1.9c 3.5 � 0.9Mexp 84.71 � 2.18c,d 14.50 � � 2.23c,d 19.5 � 0.8 12.7 � 0.2 6.0 � 0.8

Values are means (�SEM) of six animals in each group.All values were computed in 10 random, noncoincident fields per mice. Fractional areas of total

cells (total cellularity), PMN (polymorphonuclear), and MN (mononuclear cells). In pulmonary control(Cp) and extrapulmonary control (Cexp) groups, saline was instilled intratracheally (0.05 mL) andinjected intraperitoneally (0.5 mL), respectively. In pulmonary acute lung injury (ALIp) and extrapul-monary acute lung injury (ALIexp) groups, mice received E. coli lipopolysaccharide (10 �g, intratra-cheally and 125 �g, intraperitoneally, respectively). In pulmonary methylprednisolone (Mp) andextrapulmonary methylprednisolone (Mexp) groups, ALI animals were treated with methylpred-nisolone (2 mg/kg intravenously) 6 hrs after the induction of ALI.

aSignificantly different from Cp group (p � 0.05); bSignificantly different from ALIp group (p �0.05); cSignificantly different from Cexp group (p � 0.05); dSignificantly different from ALIexp group(p � 0.05).

2623Crit Care Med 2008 Vol. 36, No. 9

(Fig. 2) were affected by the same extent inALIp and ALIexp groups compared withcontrol. Methylprednisolone significantlyinhibited lung mechanical changes in-duced by Escherichia coli LPS in ALIp butnot in ALIexp (Fig. 2).

The fraction of area of alveolar col-lapse, total cell content, and the numberof polymorphonuclear cells were higherin ALI than in control groups indepen-dent of the etiology of the injury (Table1). Methylprednisolone reduced the frac-tion of area of alveolar collapse more sig-nificantly in ALIp than in ALIexp, and thetotal amount of cells decreased similarlyin both ALI groups (Table 1, Fig. 3).

Collagen fibers but not elastic fibers(elaunin, oxytalan, and fully developed elas-tic fibers) in the alveolar septa increasedsignificantly in both ALI groups. Methyl-

prednisolone prevented the increase in col-lagen fiber content in ALIp and ALIexpgroups, but did not affect elastic fiber con-tent (Table 2).

ALIp group showed cytoplasmatic de-generation of type II pneumocyte and inALIexp endothelial damage was present(Fig. 4A). In both ALI groups, the alveolarinterstitium was thickened because of in-creased amounts of extracellular matrixelements, such as type III collagen fibers(Fig. 4A and B). The use of methylpred-nisolone in ALIp group induced neutro-phil and fibroblast apoptosis (Fig. 4C),whereas in ALIexp group corticosteroidled to endothelial regeneration (Fig. 4D).

Although functional and morphologicpulmonary changes were similar inde-pendent of the etiology of ALI, ALIpyielded more pronounced cytokine re-

sponses, e.g., ALIp presented a three-foldincrease of IL-6 and TGF-� and doubleKC in the BALF compared with ALIexp(Fig. 5). Methylprednisolone minimizedthe increase of IL-6, KC, and TGF-� onlyin ALIp, but caused no modifications inALIexp (Fig. 5).

In ALIp, we consistently observed theup-regulation of TNF-�, MIF, IFN-�,TGF-�1 and TGF-�2 mRNA (Fig. 6). De-tailed quantitation by densitometry analysisshowed that lungs mRNA for TNF-�, MIF,IFN-�, and TGF-�2 were significantlyhigher in ALIp than ALIexp. Overall, theexpression of all ALIp-inducible cytokineswas inhibited in mice that were treatedwith methylprednisolone 6 hrs after ALIinduction. The expression of lung mRNAfor TNF-�, IFN-�, and TGF-�1 also in-creased in ALIexp, but methylprednisolonecaused a slight reduction in TGF-�1.

DISCUSSION

We found that one low-dose of meth-ylprednisolone given early in the courseof mild lung injury, prevented lung me-chanics and morphometric changes, anincrease in cytokine BALF levels, andTNF-�, MIF, IFN-�, TGF-�1 and TGF-�2mRNA expression in lung tissue in ALIpbut not in ALIexp group, although avoid-ing the increase in collagen fiber contentin both ALI groups.

In the present study, lung static anddynamic elastances, resistive and vis-coelastic/inhomogeneous pressures in-creased similarly in both ALI groups (Fig.2), which may have resulted from thesame amount of alveolar collapse andneutrophil infiltration (Table 2). Elasticfiber content was not different betweenALIp and ALIexp groups (Table 2). How-ever, both ALI groups presented differ-ences in collagen fibers, electron micros-copy, and inflammatory mediators’response. Electron microscopy in ALIpgroup showed an extensive injury of al-veolar epithelium, whereas endothelialcells were damaged in ALIexp group. Wealso observed that the levels of inflamma-tory cytokines (IL-6 and KC) and fibro-genic cytokines (TGF-�) were higher inALIp compared with ALIexp (Fig. 5).Therefore, in agreement with previousfindings (27) we observed that, althoughfunctional and morphologic pulmonarychanges were similar regardless of theunderlying cause of ALI, direct insultyielded more pronounced inflammatoryresponses compared with ALIexp. In thisline, our data confirm recent evidence

Figure 3. Representative photomicrographs of lung parenchyma from pulmonary control (Cp) andextrapulmonary control (Cexp), pulmonary acute lung injury (ALIp) and extrapulmonary acute lunginjury (ALIexp), and pulmonary methylprednisolone (Mp) and extrapulmonary methylprednisolone(Mexp). In Cp and Cexp groups, saline was instilled intratracheally and injected intraperitoneally,respectively. In ALIp and ALIexp groups, mice received E. coli lipopolysaccharide (10 �g, intratrache-ally and 125 �g, intraperitoneally, respectively). In Mp and Mexp groups, ALI animals were treated withmethylprednisolone (2 mg/kg, intravenously) 6 hrs after the induction of ALI. Photographs were takenat an original magnification of �200 from slides stained by hematoxylin-eosin.

2624 Crit Care Med 2008 Vol. 36, No. 9

suggesting that the inflammatory and fi-brotic processes seem to be separatelyregulated (34), thus offering the possibil-ity for early-directed treatments againstfibrosis independent of the effects on in-flammation.

Corticosteroids may play a relevantrole in modulating the inflammatory pro-cess, the immune response, the prolifer-ation of fibroblasts, and the collagen andelastin synthesis (35). Among differenttypes of corticosteroids, methylpred-nisolone may be better concentrated inthe lungs than others because it has a

larger volume of distribution, longermean residence time, and greater reten-tion in the epithelial lining fluid of thealveoli (36, 37). Clinical data related tothe possible effectiveness of corticoste-roids in early and late ARDS are contro-versial (4, 12, 35). The conflicting resultsof these studies could be attributed toheterogeneities of studied populations(37) with different degrees of inflamma-tion and lung remodeling (2, 12, 20).

We observed that methylprednisoloneprevented lung mechanical changes andreduced the amount of atelectasis as well

as the inflammatory response in ALIp,but not in ALIexp. However, methylpred-nisolone prevented any changes in colla-gen fiber content in both ALI groups.These modifications could be attributedto different effects of corticosteroids onthe inflammatory and early remodelingprocess (20, 38, 39) yielding less atelec-tasis, cellular influx, and fluid transuda-tion in ALIp group.

Methylprednisolone may act differ-ently when the primary damage is in thealveolar epithelium compared with vas-cular endothelium. Indeed, epithelialcells are likely to be important players inALIp as they may participate in leukocyterecruitment, production of chemokines,cytokines and growth factors, and arealso clear targets of corticosteroids inlung inflammation (40). In contrast toother cytokines, steroids have been re-ported to induce secretion of MIF in dif-ferent cells, and MIF has been shown tocounteract the anti-inflammatory effectsof steroids, with implications for humaninflammatory disease (41). MIF is aproinflammatory cytokine that plays amajor role in the pathogenesis of lunginflammation, including ARDS (42). In-terestingly, a significant inhibition ofMIF mRNA expression was observed inmethylprednisolone-treated animals withALIp. Similar inhibitory effects of ste-roids on MIF production have been re-ported in sepsis and ARDS (42, 43) andmay represent differences in MIF produc-tion according to the cell type analyzed,as recently suggested by Alourfi et al (44).

In our study, we observed that meth-ylprednisolone led to increased amountsof neutrophil apoptosis in the lung inALIp (Fig. 4). The role of neutrophil ap-optosis in the pathogenesis and/or reso-lution of lung inflammation has becomeincreasingly recognized (45). Engulfmentof apoptotic cells by phagocytes isthought not only to remove the dying cellfrom the tissues but also provide protec-tion from local damage resulting fromrelease or discharge of proinflammatorysubstances (46). Moreover, ingestion ofapoptotic cells actively suppresses theproduction of proinflammatory cyto-kines, resulting in accelerated resolu-tion of inflammation (47), and thismechanism may have contributed tothe observed steroid anti-inflammatoryeffects.

The present study has some limita-tions which need to be addressed: 1) Be-cause of the use of specific experimentalmodels of ALIp and ALIexp induced by

Figure 4. Electron microscopy of lung parenchyma in E. coli lipopolysaccharide instilled intratrache-ally (10 �g) (A) and injected intraperitoneally (125 �g) (B) mice. Type II cell was well preserved withtypical microvilli projecting from its surface (B). (C) and (D) acute lung injury animals were treatedwith methylprednisolone (2 mg/kg, intravenously) 6 hrs after the induction of acute lung injury. PII,types II pneumocytes; ALV, alveolar space; IE, interstitial edema, cap, capillary. Arrows indicateneutrophil apoptosis (C). Arrowheads indicate endothelial regeneration. Star represents type IIIcollagen fibers. Photomicrographs are representative of data obtained from lung sections derived fromsix animals.

Table 2. Collagen and elastic fibers in alveolar walls

GroupsCollagen Fiber

(�m2/�m)Elaunin Fiber

(�m2/�m)Fully Developed Elastic Fiber

(�m2/�m)Oxytalan Fiber

(�m2/�m)

Cp 0.016 � 0.01 0.208 � 0.01 0.399 � 0.03 0.191 � 0.03ALIp 0.049 � 0.01a 0.207 � 0.01 0.385 � 0.03 0.178 � 0.03Mp 0.020 � 0.02 0.222 � 0.02 0.355 � 0.02 0.133 � 0.03Cexp 0.017 � 0.01 0.209 � 0.02 0.351 � 0.01 0.142 � 0.01ALIexp 0.051 � 0.01b 0.185 � 0.02 0.359 � 0.02 0.174 � 0.03Mexp 0.020 � 0.01 0.233 � 0.01 0.352 � 0.02 0.119 � 0.02

Values are means (�SEM) of six animals in each group.In pulmonary control (Cp) and extrapulmonary control (Cexp) groups, saline was instilled intra-

tracheally (0.05 mL) and injected intraperitoneally (0.5 mL), respectively. In pulmonary acute lunginjury (ALIp) and extrapulmonary acute lung injury (ALIexp) groups, mice received E. coli lipopoly-saccharide (10 �g, intratracheally and 125 �g, intraperitoneally, respectively). In pulmonary meth-ylprednisolone (Mp) and extrapulmonary methylprednisolone (Mexp) groups, ALI animals were treatedwith methylprednisolone (2 mg/kg intravenously) 6 hrs after the induction of ALI.

aSignificantly different from Cp group (p � 0.05); bSignificantly different from Cexp group (p � 0.05).

2625Crit Care Med 2008 Vol. 36, No. 9

intratracheal and intraperitoneal LPS in-jection, we do not know if these resultscan be shifted to other experimentalmodels of ALI. 2) The degree of ALI in-

duced by intratracheal or intraperitonealadministration of LPS was mild as dem-onstrated by room air breathing and100% survival. However, a significant

lung injury was achieved as demonstratedby changes in lung mechanics, increasedamount of atelectasis, inflammatory cellsand mediators in the lung, and enhancedremodeling process. In addition, animalsbreathed spontaneously avoiding possibleside effects induced by prolonged me-chanical ventilation. 3) In clinical set-tings, there may exist differences in themechanical properties of the lung be-tween ALIp and ALIexp. In our study, LPSdose was titrated to induce similar me-chanical lung injury in both ALIp andALIexp groups and the effect of methyl-prednisolone was studied in standardizedconditions with comparable degrees ofseverity of lung injury. 4) Only one doseof methylprednisolone was used, andconsequently, we cannot exclude the pos-sibility that high dose of methylpred-nisolone may induce beneficial effects inALIexp. 5) An important overlap be-tween direct or indirect ALI may existin clinical practice because the patho-physiologic pathways are not alwayscompletely differentiated. 6) Our re-sults cannot directly be transferred toclinical practice, but require further in-vestigation. However, the present datamay improve our knowledge regardingthe appropriate use of corticosteroids inmild ALI.

CONCLUSIONS

Early administration of low-dosemethylprednisolone attenuated inflam-matory responses and lung mechanicalchanges in mild ALIp but not in ALIexp,although effectively inhibited fibrogen-esis independent of lung injury etiology.Thus, the etiology of ALI may play a rel-evant role in determining physiologic re-

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Figure 5. Methylprednisolone treatment in the concentrations of KC (interleukin [IL]-8 murine functional homolog), IL-6, and tumor necrosis factor(TGF)-� levels in the bronchoalveolar lavage fluid at 24 and 48 hrs. Values are means � SEM of 10 animals in each group. Pulmonary control (Cp) andextrapulmonary control (Cexp) groups � saline was instilled intratracheally (0.05 mL) and injected intraperitoneally (0.5 mL), respectively. Pulmonaryacute lung injury (ALIp) and extrapulmonary acute lung injury (ALIexp) groups, mice received E. coli lipopolysaccharide (10 �g, intratracheally and 125�g, intraperitoneally, respectively). In pulmonary methylprednisolone (Mp) and extrapulmonary methylprednisolone (Mexp) groups, ALI animals weretreated with methylprednisolone (2 mg/kg, intravenously) 6 hrs after the induction of ALI. *Significantly different from Cp (p � 0.05). **Significantlydifferent from Cexp group (p � 0.05). ‡Significantly different from ALIp (p � 0.05).

Figure 6. Expression of messenger RNA for murine cytokines investigated by RNase Protection Assay.(A) Representative result of four independent experiments using a mCK-3b Multi-Probe Template Set.(B) Each lane is an individual animal. Pulmonary control (Cp) and extrapulmonary control (Cexp)groups, saline was administered intratracheally and intraperitoneally, respectively. Sham group miceunderwent the same protocol as the control without saline injection. Pulmonary acute lung injury(ALIp) and extrapulmonary acute lung injury (ALIexp) groups, mice received E. coli lipopolysaccharide(10 �g, intratracheally and 125 �g, intraperitoneally, respectively). ALIp and ALIexp animals weretreated with methylprednisolone (pulmonary methylprednisolone [Mp] and extrapulmonary methyl-prednisolone [Mexp], respectively) 6 hrs after the induction of acute lung injury. (C) Correspondentbands for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene less exposed to radiograph wereused to normalize the experiments. (D–H) The bar graph presents the results (mean � SEM) of fourmice per group. Different letters indicate significant differences (p � 0.05) between data-points andsame letters indicate no significant difference between data-points. LT, lymphotoxin; TNF-�, tumornecrosis factor-�; INF-�, interferon-�; TGF-�, transforming growth factor-�; MIF, migration inhibi-tory factor.

2626 Crit Care Med 2008 Vol. 36, No. 9

sponse to corticosteroids in experimentallung injury.

ACKNOWLEDGMENTS

We express our gratitude to Mr. AntonioCarlos de Souza Quaresma for animal care;Mrs. Maria Margarida Teixeira Monteiro forher help with electron microscopy; andMrs. Patricia Maronas, Mrs. Jaqueline Limado Nascimento, and Mr. Andre Benedito daSilva for their skillful technical assistanceduring the experiments.

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