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Contribute To a Model of Acute Lung InjuryT Cell Pathways Involving CTLA4

David L. Perkins and Patricia W. FinnJen, Jan E. Schnitzer, Samir S. Makani, Nathan Parker, Takeshi Nakajima, Carlos Jose Suarez, Ko-Wei Lin, Kai Yu

http://www.jimmunol.org/content/184/10/5835doi: 10.4049/jimmunol.0903238April 2010;

2010; 184:5835-5841; Prepublished online 12J Immunol 

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The Journal of Immunology

T Cell Pathways Involving CTLA4 Contribute To a Model ofAcute Lung Injury

Takeshi Nakajima,*,1 Carlos Jose Suarez,*,1 Ko-Wei Lin,* Kai Yu Jen,*

Jan E. Schnitzer,† Samir S. Makani,* Nathan Parker,* David L. Perkins,‡ and

Patricia W. Finn*

Acute lung injury (ALI) is a frequent pulmonary complication in critically ill patients. We characterized a murine model of LPS-

induced ALI, focusing on Th cells. Following LPS administration, bronchoalveolar lavage lymphocytes, neutrophils, IL-6, TNF-a,

and albumin were increased. Analysis of LPS-induced T cells revealed increased Th cell-associated cytokines (IL-17A, -17F, and -22),

as well as increased expression of CD69 (a cell activation marker), Foxp3, and CTLA4 in CD4+ T cells. Administration of anti-

CTLA4 Ab decreased LPS-induced bronchoalveolar lavage albumin and IL-17A, while increasing CD4+Foxp3+ cell number and

Foxp3 expression in CD4+Foxp3+ cells. These data suggest that pulmonary LPS administration promotes CD4+ T cells and that T cell

pathways involving CTLA4 contribute to ALI. The Journal of Immunology, 2010, 184: 5835–5841.

Acute lung injury (ALI) is associated with acute respiratorydistress syndrome (ARDS), a major cause of severe re-spiratory failure with high morbidity and mortality in

critically ill patients (1, 2). The pathogenesis of ARDS remains illdefined, and the treatment of ARDS remains largely supportive.ALI models include characterization of LPS-induced pulmonaryimmune responses (3). LPS, a component of Gram-negative bac-teria, binds to a signal-transducing integral membrane protein,TLR4, which is expressed on APCs (e.g., alveolar macrophagesand lung dendritic cells). TLRs allow vertebrates to recognizea vast range of microbial products in a rapid innate immune re-sponse (4, 5). TLRs were also recently found to be expressed onT cells, suggesting a potential pathway by which LPS may directlyaffect T cell activity (6–8). Innate and adaptive immune responsesto invading microbes (e.g., Gram-negative bacteria) are tightlyinterwoven and engage the total immunological capability of thehost. Innate immunity is the first line of lung defense, and it in-cludes structural barriers, alveolar macrophages, neutrophils, NKcells, and dendritic cells. Adaptive immunity, which is promotedby innate immunity, is composed of Ag-specific lymphocytesthat eliminate or prevent pathogenic challenges. Lymphocytes,including T and B cells, are major cells of the adaptive immunesystem. T lymphocytes (CD4+ Th cells) have no cytotoxic orphagocytic activity and cannot kill infected cells or clear patho-gens, but they manage the immune response by directing other cellsto perform these tasks.

Experimental depletion of CD4+ T lymphocytes in mice withStaphylococcus aureus pleural empyema is associated with de-creased bacterial clearance (9). Sepsis induces a striking depletionof lymphocytes, leading to an inability of the host to combat theongoing source of infection and predisposing to secondary op-portunistic infections (10). Also, sepsis activates the remaininglymphocytes (11), and inhibition of lymphocyte apoptosis mayimprove sepsis outcomes (12). Infection is a precipitating factorfor ARDS, and T cells are considered to play an important role inhost defense for infection.Prior studies showed that lymphocytes, in addition to neutro-

phils, infiltrate the lung inALImodels (13–15). Recently, D’Alessioet al. (16) demonstrated that regulatory T cell (Treg) T cell subsetscontribute to the resolution of ALI. Analysis of additional T cellsubsets and T cell pathways in ALI remain ill defined. In thecurrent study, we characterized a model of LPS-induced ALI. Toelucidate which T lymphocytes contribute to ALI, we examinedparameters related to T cells, including T cell number, activity, andexpression of CTLA4 and Foxp3, which are T cell-dependentsuppressive molecules. Also, we tested whether the administrationof anti-CTLA4 Ab would impact ALI.

Materials and MethodsMice

Female BALB/c mice (8–10 wk old) were purchased from Harlan Breeders(Indianapolis, IN). T and B cell-deficient RAG knockout (KO) mice(BALB/c background) were purchased from The Jackson Laboratory (BarHarbor, ME). The mice were maintained according to the guidelines of theUniversity of California, San Diego Animal Care Program.

Materials

LPS from Escherichia coli 055:B5, purified by gel-filtration chromatog-raphy, was purchased from Sigma-Aldrich (St. Louis, MO). Mouse anti-CTLA4 (UC10-4F10-11) was purchased from Bio X Cell (West Lebanon,NH). Hamster IgG1, k isotype control (IgG, A19-3) was purchased fromBD Biosciences (San Jose, CA).

LPS-induced ALI

Mice were anesthetized with isoflurane (Minrad, Bethlehem, PA). Thetongue was gently extended, and the tip of an otoscope was introduced toreach the trachea. Once in the trachea, LPS (100 mg) in 50 ml PBS wasadministered through the cone of the otoscope. In the RAG KO miceexperiments, LPS was administered intratracheally (i.t.) to wild type (WT)

*Division of Pulmonary and Critical Medicine and ‡Division of Nephrology, De-partment of Medicine, University of California, San Diego, La Jolla, CA 92093; and†Proteogenomics Research Institute for Systems Medicine, San Diego, CA 92121

1T.N. and C.J.S. contributed equally to this work.

Received for publication October 2, 2009. Accepted for publication March 9, 2010.

This work was supported by National Institutes of Health Grant 5R01HL081663-05.

Address correspondence and reprint requests to Dr. Patricia W. Finn, Division ofPulmonary and Critical Medicine, Department of Medicine, University of California,San Diego, 9500 Gilman Drive 0643, La Jolla, CA 92093. E-mail address: pwfinn@ucsd.edu

Abbreviations used in this paper: ALI, acute lung injury; ARDS, acute respiratorydistress syndrome; BAL, bronchoalveolar lavage; i.t., intratracheally; KO, knockout;MFI, mean fluorescence intensity; Treg, regulatory T cell; WT, wild type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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or RAG KO mice, and the mice were harvested at day 2 after LPS ad-ministration. In the time-course experiments, LPS was administered i.t.to WT mice. PBS was administered to controls. Mice were harvested atindicated time points (day 1–6) after LPS or PBS administration.

Treatment with anti-CTLA4

Mice were injected i.p. with anti-CTLA4 (100 mg) in 200 ml PBS 1 d priorto i.t. administration of LPS. Hamster IgG1 (IgG, 100 mg) was adminis-tered to controls. Anti-CTLA4-treated mice were harvested at indicatedtime points (day 1–6) after LPS administration. IgG-treated mice wereharvested at days 2 and 4.

Bronchoalveolar lavage harvest and cell count

Bronchoalveolar lavage (BAL) fluid was obtained by cannulating the tra-chea and lavaging the lungs three times with 1 ml PBS containing 0.6 mMEDTA. BAL fluid cells were pelleted, and the supernatant was stored at280˚C until use.

BAL fluid cells were counted using a hemocytometer and resuspended inRPMI 1640 (5 3 105 cells/ml). Slides for differential cell counts wereprepared with Cytospin (Thermo Scientific, Waltham, MA) and then fixedand stained with Diff-Quick (Imeb, San Marcos, CA). For each sample, aninvestigator blinded to the treatment groups performed two counts of 100cells.

BAL albumin and cytokines

Albumin levels in BAL fluid were measured by ELISA (Alpco Diagnostics,Salem, NH). Samples of BAL fluid were aliquoted in duplicate into 96-wellplates (100 ml/well) precoated with Ab to specific murine albumin andassayed according to the manufacturer’s instructions. OD was measured at450 nm. Levels of TNF-a and IL-2, -4, -6, -13, and -17A in BAL fluidwere measured by a multiplexed immunoassay (Millipore, Bedford, MA),and IL-17F, -22, -23, and -27 were measured by a multiplexed immuno-assay (Biolegend, San Diego, CA). Mean fluorescence intensity (MFI) wasmeasured by Luminex 100 total system (Luminex, Austin, TX).

Preparation of lung homogenates

Following the BAL procedure, the lungs were perfused with 20 ml salineand isolated. The lungs were cut into small pieces with scissors in a petridish, finely chopped, and incubated at 37˚C for 60 min in 5 ml per lung ofa digestion mixture consisting of collagenase from Clostridium histo-

lyticum, type IV (1 mg/ml, Sigma-Aldrich) and DNase I from bovinepancreas (0.5 mg/ml, Sigma-Aldrich). Cell suspensions were obtained bypassing the homogenate through a 70-mm nylon Falcon cell strainer (BDBiosciences) to remove pieces of tissue and debris. The pellets were re-suspended in 5 ml 10 mM EDTA in PBS. After lysis of erythrocytes withammonium chloride potassium bicarbonate lysing buffer, cell pellets wereresuspended and washed in RPMI 1640 medium.

Flow cytometric analysis

Samples of lung homogenates were resuspended in staining buffer (PBScontaining 2% FBS and 0.05% sodium azide) to be 13 107 cells/ml; 100 mlprepared cells (13 106 cells) were added to each tube. After incubating for30 min at 4˚C with anti-mouse Abs (CD3 [PECy7], CD4 [FITC], CD8[allophycocyanin], CD25 [allophycocyanin], CD19 [PE], or CD69 [PE])or their respective immunoglobulin isotypes (eBioscience, San Diego,CA), samples were washed twice with staining buffer, fixed by Fixation/Permeabilization solution (eBioscience), and incubated overnight. Intra-cellular staining with PE-conjugated anti-mouse CTLA4 and Foxp3 Abswas performed using a Mouse T regulatory cell staining kit (eBioscience).Samples were run on a BD FACSCalibur flow cytometry system (BDBiosciences). Data yielded were analyzed on FlowJo v8 software (TreeStar, Ashland, Oregon).

Statistical analysis

Data are expressed as means 6 SEM. All parameters were evaluatedwith the two-tailed unpaired Student t test or compared by one-wayANOVA, followed by the Bonferroni test. A p value,0.05 was consideredsignificant.

ResultsLymphocytes are present and contribute to the severity of ALI

We analyzed a murine model of LPS-induced ALI in WT andlymphocyte-deficient (RAG KO, T and B cell deficient) mice.Compared with WT mice, RAG KO mice exhibited a significantdecrease in BAL neutrophils (Fig. 1A). The decrease in LPS-induced neutrophils in RAG KO mice suggests that lympho-cytes contribute to ALI. Upon further characterization of ALI inWT mice over 6 d, we analyzed increased pulmonary vascular

FIGURE 1. Analysis of pulmonary inflammatory responses in a model of LPS-induced ALI. A, LPS (100 mg) was administered i.t. to WT BALB/c mice

or RAG KO mice. BAL fluid was collected at day 2 after LPS administration, and cell counts were determined by differential staining for neutrophils and

macrophages. Data shown are geometric mean6 SEM (n = 3 per group). ‡p, 0.05; RAG KO versus WT. B, LPS (100 mg) or PBS was administered to WT

mice (BALB/c) i.t. BAL fluid was collected at the indicated days (D1–D6). Levels of albumin in the BAL fluid were determined by ELISA. C, Absolute

neutrophil count in the BAL fluid was determined by differential staining of cells. Levels of IL-6 (D) and TNF-a (E) in BAL fluid were determined by

a multiplexed immunoassay. Data are geometric mean 6 SEM (n = 3–6 per group). *LPS group versus respective PBS group (p , 0.05); †p , 0.05.

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permeability, defined by increased BAL levels of albumin (Fig.1B). BAL albumin production increased at day 1, peaked at day 4,and returned to baseline by day 6. BAL neutrophils in LPS-exposedmice also increased at day 1, but they exhibited more rapid ki-netics: peaking at day 2 and decreasing by day 4 (Fig. 1C). LPS-induced ALI is associated with proinflammatory cytokines (e.g.,IL-6 and TNF-a). LPS increased BAL IL-6, with similar kineticsto the neutrophil count (Fig. 1D). LPS also increased TNF-a,which decreased by day 2 (Fig. 1E). We next examined LPS-induced BAL lymphocytes. In contrast to BAL neutrophils, lym-phocytes peaked at day 4 and remained elevated at day 6 (Fig. 2A).We also analyzed lymphocyte numbers in lung tissue from LPS-exposed mice. The total number of CD19+ (B) cells, CD3+CD8+

(T cytotoxic) cells, and CD3+CD4+ (Th) cells was determined byflow cytometry (Fig. 2B). Each subset increased at day 4 followingLPS administration, which paralleled the increase in the numberof BAL lymphocytes.

LPS activates CD4+ T cells and induces Th17 cytokines

Based on the LPS-induced increase in Th cells and their establishedrole in inflammation, we focused on the analysis of Th cell-associated cytokines (i.e., IL-2, -4, -13, and -17A). IL-17A in-creased in LPS-exposed mice at day 4 (Fig. 3A), whereas IL-2, -4,and -13 were not modified (data not shown). We then analyzedother cytokines associated with Th17 cell response (i.e., IL-17F,

-22, -23, and -27). IL-17F and -22 increased at day 4, similar toIL-17A (Fig. 3B, 3C), whereas IL-23 and -27 were not detectedthroughout the time points analyzed (data not shown). To evaluatethe activities of CD4+ T cells, CD69, a marker of cell activation,was examined in the CD3+CD4+ cell population by flow cy-tometry. LPS administration increased CD69 expression on CD4+

cells, which was greatest at day 4, in parallel with increased IL-17A, -17F, and -22 (Fig. 3D, 3E).Thus, LPS exposure increased CD4+ cell number and activity.

To define the T cell population modified by LPS exposure, weanalyzed lung tissue for two markers expressed by T cells: CTLA4and Foxp3. LPS exposure increased MFI of CTLA4 expression(Fig. 4A, 4B). LPS exposure also increased Foxp3 expression,which was greatest at day 6 (Fig. 4C, 4D).

Administration of anti-CTLA4 and analysis of CD4+Foxp3+

cell activation

LPS exposure increased CTLA4 expression in CD4+ T cells. Toexamine the potential role of CTLA4 in ALI, we analyzed theimpact of anti-CTLA4. BAL albumin, increased at day 4 in LPS-exposed mice, was significantly decreased by anti-CTLA4 ad-ministration, suggesting that vascular permeability is reduced(Fig. 5A). Anti-CTLA4 significantly reduced LPS-induced BALIL-17A (Fig. 6A) without impacting neutrophil count, lymphocytenumber (Fig. 5B, 5C), IL-6, or TNF-a (data not shown). In-terestingly, the reduction in Th17 cells at day 4 by anti-CTLA4was counterbalanced by a significant increase in CD69 and Foxp3expression in CD4+ T cells at day 2 (Fig. 6B–D). We then focusedon CD4+Foxp3+ cells at day 2 following LPS administration.Absolute cell count and the ratio of CD4+Foxp3+ cells in the CD4+

cell population were increased by anti-CTLA4 (Fig. 7A). Incontrast, the number of CD4+Foxp32 cells was not significantlyincreased. Also, the ratio of CD4+Foxp32 cells in the CD4+ cellpopulation was decreased by anti-CTLA4 (Fig. 7B). Anti-CTLA4also increased LPS-induced Foxp3 in CD4+Foxp3+ cells (Fig. 7C,7D) and LPS-induced CD69 in CD4+CD25+ cells (Fig. 7E, 7F).

DiscussionALI is classically characterized as an innate response, predomi-nantly involving neutrophils. In a murine model of ALI, we focusedon the analysis of T cell-dependent pathways. Consistent with lunginjury models, LPS administration induces BAL neutrophils in WTmice. Lymphocyte-deficient mice were unable to increase neu-trophils in response to LPS, suggesting that T cells may contributeto ALI. LPS also increased a number of T cell-associated para-meters, including lymphocyte number and activity and IL-17Aproduction. Notably, LPS exposure, an Ag-independent pathway,also increased two molecules found primarily on T cells: CTLA4and Foxp3. Testing the functional role of CTLA4, anti-CTLA4administration to LPS-exposed mice resulted in decreased IL-17Aand albumin production. These findings suggest that CTLA4 andT cells may contribute to pulmonary inflammatory pathways inALI.We analyzed LPS-induced T cells over a 6-d time course, pro-

viding information on the early and late influx of cells and thesecretion of cytokines. As expected, LPS administration increasedBAL neutrophils and inflammatory cytokines (i.e., IL-6 and TNF-a)within 2 d (early phase). LPS administration increased lympho-cytes within 4–6 d (late phase). Other investigators showed thatLPS-induced TNF-a secretion in macrophages is decreased whenCD4+ T cells are depleted (17) and that gd T cells increase BALneutrophils by producing IL-17A (18), suggesting that lympho-cytes may facilitate neutrophil migration to the lungs. In our study,the kinetic analysis of neutrophils and lymphocytes supports the

FIGURE 2. LPS increases the number of lymphocytes in the BAL and

the lungs. LPS (100 mg) or PBS was administered to mice (BALB/c) i.t.

BAL fluid and lungs were harvested at the indicated days (D1–D6). Lungs

were digested as described in Materials and Methods. A, Absolute lym-

phocyte count in BAL fluid was determined by differential staining of

cells. B, Absolute counts of CD19+ cells, CD3+CD8+ (cytotoxic T) cells,

and CD3+CD4+ (Th) cells in lung tissue were determined by flow cy-

tometry. Data are geometric mean 6 SEM (n = 3–6 per group). *LPS

group versus respective PBS group (p , 0.05); †p , 0.05.

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concept that lymphocytes may play roles in addition to facilitatingneutrophil migration, perhaps in dampening inflammation in ALI.In a prior study, adrenaline attenuated ALI by diminishing therecruitment of CD4+ cells to lung (19). The acute phase of ALI is

characterized mainly by the influx of protein-rich edema fluid intothe air space as a consequence of increased permeability of thealveolar–capillary barrier (20). Following LPS exposure, albuminlevels peaked later than neutrophils, indicating that neutrophil

FIGURE 3. LPS activates CD4+ T cells. LPS (100 mg) or PBS was administered to mice (BALB/c) i.t. BAL fluid and lungs were harvested at the

indicated days (D1–D6). Lungs were digested as described in Materials and Methods. Levels of IL-17A (A), -17F (B), and -22 (C) in BAL fluid were

determined by a multiplexed immunoassay. Expression levels of CD69 in the CD3+CD4+ cell population in lung tissue were determined by flow cytometry;

representative bar graphs of CD69 expression (shaded areas represent isotype control) (D) and MFI for CD69 (E). Data are geometric mean6 SEM (n = 3–6

per group). *LPS group versus respective PBS group (p , 0.05); †p , 0.05.

FIGURE 4. LPS induces Th cell-dependent suppressive pathways. LPS (100 mg) or PBS was administered to mice (BALB/c) i.t. Lungs were harvested at

the indicated days (D1–D6). Lungs were digested as described inMaterials and Methods. Expression levels of CTLA4 (A) and Foxp3 (C) in the CD3+CD4+

cell population in lung tissue were determined by flow cytometry: representative graphs of CTLA4 and Foxp3 expression (shaded areas represent isotype

control). MFI for CTLA4 (B) and Foxp3 (D). Data are geometric mean 6 SEM (n = 3–6 per group). *LPS group versus respective PBS group (p , 0.05);#LPS group versus LPS D6 (p , 0.05); †p , 0.05.

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infiltration may not directly correspond with lung vascular per-meability. The time difference between the LPS-induced neutro-phil influx and the albumin increase may be due to several

possibilities. First, proteolytic enzymes released from neutrophils,such as elastase and free-oxygen radicals, require time to exerttheir effects. Second, the peak of tissue damage may be earlier thanthe peak of albumin levels. The kinetics of ALI indicate that lunginflammation peaks at day 2 (acute phase), with the resolutionprocess starting at day 4. Third, the severity of ALI may not bedetermined solely by neutrophils; different cell types may contributeto the lung damage. The expression of CD69, a cell-activationmarker, on T cells was increased by LPS throughout the time pointsanalyzed. Together, these data suggest that T cells may impact acuteand late phases of inflammation in ALI.Th17 cell-related cytokines (i.e., IL-17A, -17F, and -22) were

increased following LPS exposure. Interestingly, LPS-inducedTh17 cell-related cytokines increased in a pattern similar to in-creased lymphocyte number and CD69 expression. Other Th cell-related cytokines (i.e., IL-2, -4, and -13) were not increased (datanot shown), which implies that, among the subsets of Th cells, Th17cells may play a role in ALI. IL-17 is important for host defenseagainst infection, as well as neutrophil recruitment (21). Indeed,IL-17R–deficient mice succumb to infection (22). In contrast,elevated and prolonged expression of IL-17 is found in autoim-mune disease (23). In murine models of LPS-induced airway in-flammation, previous reports showed that neutralization of IL-17significantly reduced neutrophil infiltration (24–26). Recently,other investigators demonstrated that IL-17R antagonist KO miceshow reduced neutrophilic inflammation of ALI in the setting ofinfluenza infection-induced ALI (18). Together, these studies inALI suggest that IL-17 is increased at an early phase, inducesinflammatory cytokines, and affects neutrophil kinetics. In ourmodel, the time course for LPS-induced IL-17 was delayedcompared with the increase in neutrophil infiltration. IL-17 isprimarily considered a proinflammatory cytokine; however, in anallergic model, IL-17 suppresses eosinophil migration (27). Ina bleomycin-induced lung injury model, IL-17 produced by gdT cells controls resolution of inflammation (28). In a Helicobacterpylori-induced gastritis model, IL-17 exerts anti-inflammatoryeffects (29). Thus, IL-17 may play pathogenic and protective rolesin immune responses. Of note, the kinetics of LPS-induced IL-17family members and Foxp3 differ, with IL-17 secreted earlierthan Foxp3, which may impact their biological activities.The early stages of T cell activation are regulated by interaction

of costimulatory molecules CD80 and CD86 with their receptorCD28 on the T cell surface (30). Once activated, these T cellstransiently increase CTLA4 on their cell surface, interacting with

FIGURE 5. Anti-CTLA4 decreases LPS-induced increase in albumin.

Mice were injected i.p. with anti-CTLA4 (100 mg) or hamster IgG (100

mg) 1 d prior to i.t. administration of LPS (100 mg). D6 group was re-

injected i.p. with anti-CTLA4 (100 mg) on day 3. BAL fluid was collected

at the indicated days (D1–D6) after LPS exposure. A, Levels of albumin in

BAL were determined by ELISA. Neutrophil (B) and lymphocyte (C)

counts in BAL fluid were determined by differential staining of cells. Data

are geometric mean 6 SEM (n = 3–6 per group). †p , 0.05.

FIGURE 6. Anti-CTLA4 modifies LPS-induced cy-

tokine production and CD69 and Foxp3 expression in

CD4+ T cells. Mice were injected i.p. with anti-CTLA4

(100 mg) or hamster IgG (100 mg) 1 d prior to i.t.

administration of LPS (100 mg). D6 group was re-

injected i.p. with anti-CTLA4 (100 mg) on day 3. BAL

fluid and lungs were collected at the indicated days

(D1–D6) after the LPS exposure. Lungs were digested

as described in Materials and Methods. A, Levels of

IL-17A in BAL fluid were determined by a multiplexed

immunoassay. B, Expression of CD69 and Foxp3 in

the CD3+CD4+ cell population in lung tissue was de-

termined by flow cytometry: representative graphs of

CD69 and Foxp3 expression at day 2 (shaded areas

represent isotype control). MFI for CD69 (C) and

Foxp3 (D). Data are geometric mean 6 SEM (n = 3–6

per group). †p , 0.05.

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the same CD80 and CD86 costimulatory molecules but now in-hibiting cell-cycle progression and IL-2 production (31). Thus,CTLA4 signaling provides negative feedback to activated T cells,thereby dampening an immune response. In our model, LPS in-creased CTLA4 expression from days 1–6, indicating that Th cellswere activated and susceptible to downregulation from the early tothe late phase, concomitant with increased CD69 expression.Mattern et al. (32) demonstrated that monocyte-dependent acti-vation of human T cells by LPS requires costimulatory signals viaCD28 and/or CTLA4 but is not MHC restricted. TLRs are ex-pressed on T cells, and TLR ligands act directly upon T cells (6–8). Together, our findings in an ALI model suggest that T cellsmay be regulated, in part, by Ag-independent pathways.In vivo analysis of anti-CTLA4 administration resulted in de-

creased albumin and IL-17A levels, suggesting a beneficial effect inALI of manipulating a T cell pathway. Although BAL cell countswere not altered by aCTLA4 administration, the severity of ALImay not always be determined solely by neutrophils. Multiple cellsmay contribute to the lung damage, and CD4+ T cells may impactother cells (e.g., other lymphocyte subsets, macrophages, or epi-thelial cells). A specific subset of lymphocytes may act as anti-inflammatory cells, not proinflammatory cells: Tregs. D’Alessioet al. (16) recently demonstrated that Tregs play critical rolesduring the resolution phase in an ALI model. They showed thatTregs resolve lung inflammation by inducing TGF-b and neutro-phil apoptosis. The contribution of other T cell subsets, the impact

of T cells on the early phase in ALI, and the potential role ofCTLA4 were not examined. In our study, we focused on addi-tional T cell subsets and the early phase of ALI. We also analyzedanti-CTLA4, which may activate T effector cells, as well asTregs. Because Tregs constitutively express CTLA4 on their cellsurface (33), anti-CTLA4 may impair the suppressive function ofTregs. Prior studies in cancer patients showed that anti-CTLA4increases activated effector CD4 T cells and Tregs (34). Inhibit-ing CTLA4 signaling with anti-CTLA4 enhances Treg pro-liferation and overall Treg frequency (35). Notably, we de-monstrated that anti-CTLA4 increased LPS-induced Foxp3 inCD4+ cells, suggesting that the decrease in albumin levels byanti-CTLA4 may relate to Treg activation. Increased Treg acti-vation is supported by an increased absolute CD4+Foxp3 cellnumber but not CD4+Foxp32 (effector) cells in the anti-CTLA4-treated group. Further, LPS-induced Foxp3 expression in CD4+

Foxp3+ cells and LPS-induced CD69 expression in CD4+CD25+

cells were increased by anti-CTLA4. Our study suggests that anti-CTLA4 may activate Tregs more than T effector cells, perhapssuppressing inflammation or accelerating resolution. If so, theseverity of ALI may depend on the T effector cell/Treg balance.Also, anti-CTLA4 suppression of IL-17A synthesis is associatedwith decreased albumin. Tregs, which are activated by anti-CTLA4, may suppress Th17 cell activation. Thus, the Th17/Tregbalance may be important in ALI pathogenesis and warrantsfurther analysis.

FIGURE 7. Anti-CTLA4 increases LPS-induced Treg number and activation. Mice were injected i.p. with anti-CTLA4 (100 mg) or hamster IgG (100 mg)

1 d prior to i.t. administration of LPS (100 mg). Lungs were collected at day 2 after the LPS exposure and digested as described in Materials and Methods.

Absolute cell counts and ratio of CD4+Foxp3+ (Treg; A) and CD4+Foxp32 (T effector; B) cells in the CD4+ cell population were determined by flow

cytometry. Expression of Foxp3 (C) in the CD4+Foxp3 cell population and CD69 (E) in the CD4+CD25+ cell population in lung tissue was determined by

flow cytometry: representative graphs of CD69 and Foxp3 expression (shaded areas represent isotype control). MFI for Foxp3 (D) and CD69 (F). Data are

geometric mean 6 SEM (n = 3–6 per group). †p , 0.05.

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In summary, we focused on the analysis of T cells in a model ofALI. Notably, LPS induces increased number and activation ofT cells, as well as other T cell-associated markers (i.e., IL-17A,Foxp3, and CTLA4). Testing the functional role of CTLA4, anti-CTLA4 decreased LPS-induced albumin and IL-17A, while in-creasing the number of CD4+Foxp3+ cells and Foxp3 expression inCD4+Foxp3+ cells. These data are consistent with a role forCTLA4+ T cells in LPS-induced inflammation. The specificpathways by which T cells are activated in ALI remain to be de-termined. The potential therapeutic impact of manipulating T cellpathways involving CTLA4 in ALI merits further investigation.

AcknowledgmentsWe thank Koichiro Asano, Mark Fuster, David Rothstein, and Timothy

Bigby for critical review of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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