Extracellular Acidosis Induces Neutrophil Activation by a Mechanism Dependent on Activation of Phosphatidylinositol 3-Kinase/Akt and ERK Pathways

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3-Kinase/Akt and ERK PathwaysActivation of PhosphatidylinositolActivation by a Mechanism Dependent on Extracellular Acidosis Induces Neutrophil

and Jorge GeffnerÁlvarez, Gabriela Salamone, Tamara Tanos, Omar A. CosoCeballos, Juan Sabatté, Romina Gamberale, María Eugenia Diego Martínez, Mónica Vermeulen, Analía Trevani, Ana

http://www.jimmunol.org/content/176/2/1163doi: 10.4049/jimmunol.176.2.1163

2006; 176:1163-1171; ;J Immunol 

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Extracellular Acidosis Induces Neutrophil Activation by aMechanism Dependent on Activation of Phosphatidylinositol3-Kinase/Akt and ERK Pathways1

Diego Martınez,* Monica Vermeulen,* Analıa Trevani,* Ana Ceballos,* Juan Sabatte,*Romina Gamberale,* Marıa Eugenia Alvarez,* Gabriela Salamone,* Tamara Tanos,†

Omar A. Coso,† and Jorge Geffner2*

Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studiesaimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers humanneutrophil activation, inducing a transient increase in intracellular Ca2� concentration, a shape change response, the up-regu-lation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophilactivation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK,but not that of p38 and JNK MAPK. No degradation of I�B was observed, supporting the hypothesis that NF-�B is not activatedunder acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the inductionof Ca2� transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shapechange response without affecting the induction of Ca2� transients. We also found that acidosis not only induces a shape changeresponse and the induction of Ca2� transients in human neutrophils but also stimulates the endocytosis of FITC-OVA andFITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results supportthe notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERKpathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis wasassociated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway. The Journal ofImmunology, 2006, 176: 1163–1171.

T here is a large body of evidence showing that interstitialacidification is a common feature associated with thecourse of inflammatory reactions against pathogenic mi-

croorganisms in peripheral tissues, where extracellular pH valuesas low as 5.5–7.0 have been found (1–5). Similar observationswere made in peritoneal fluid in patients with intra-abdominal in-fection, as revealed by the analysis of drainage fluid following anemergency laparotomy (4, 5) in inflammatory exudates (1, 2) aswell as in the skin during the course of inflammatory reactions (3).Not only the inflammatory reactions against pathogens but alsoautoimmune processes such as rheumatoid arthritis and asthma areassociated with the development of acidic microenvironments ininjured tissues. The pH of the synovial fluid of compromised jointsin patients with rheumatoid arthritis is acidic (6.7–7.4), and aci-dosis appears to correlate not only with synovial fluid leukocytosis

but also with radiological joint destruction (6, 7). In contrast, ob-servations made in the lower airway of patients with acute asthmashowed that the values of pH found in airway vapor condensatesamples from asthmatic patients were more than one or two logorders lower than those in control subjects (8, 9). Acidosis has alsobeen shown to be associated with the development of solid tumors.Studies performed over the past 50 years in a variety of solidtumors have shown that tumor microenvironments are usuallymore acidic than the normal ones, with values of extracellular pHranging from 5.8 to 7.4, both in human and rodent malignanttissues (10–12).

Extracellular acidosis results from a complex array of factorssuch as the following: 1) the low oxygen tensions in areas of in-flammation (hypoxia) and the intense metabolic activity of inflam-matory cells, resulting in a switch to anaerobic glycolysis and thesubsequent accumulation of lactate (13–15); 2) the massive infil-tration of neutrophils and macrophages and the production of pro-tons during the activation of the respiratory burst (16, 17); and 3)the accumulation of short-chain fatty acids produced bybacteria (18, 19).

We have reported previously that extracellular acidosis inducesthe activation of both neutrophils (20) and dendritic cells (21, 22),supporting the idea that low pH values can be recognized by im-mune cells as a danger signal favoring the initiation of innate andadaptive immune response. Interestingly, previous studies havealso shown that acidosis activates the alternative pathway of com-plement (23–25), supporting the hypothesis that two of the mostimportant pathways of innate immunity are activated by protons.

How could neutrophils sense extracellular acidosis? We haveshown previously that acidification of the extracellular medium

*Institute of Hematologic Research, National Academy of Medicine and NationalReference Center for AIDS, Department of Microbiology, Buenos Aires UniversitySchool of Medicine, Buenos Aires, Argentina; and †Department of Physiology andMolecular Biology, Faculty of Exact and Natural Sciences, Buenos Aires University,Buenos Aires, Argentina

Received for publication June 1, 2005. Accepted for publication October 24, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Consejo Nacional de InvestigacionesCientıficas y Tecnicas, Buenos Aires University School of Medicine, Fundacion An-torchas, and Agencia Nacional de Promocion Cientıfica y Tecnologica, Argentina.2 Address correspondence and reprint request to Dr. Jorge Geffner, Departamento deInmunologıa, Instituto de Investigaciones Hematologicas, Academia Nacional de Me-dicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail address:[email protected]

The Journal of Immunology

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induces an abrupt drop in the intracellular pH of neutrophils (20).This response appears to be triggered by the rapid diffusion into thecell of CO2 originating from the reaction of protons with the bi-carbonate present in the culture medium, which, in turn, results inthe overproduction of intracellular protons. It is possible that thedrop in the intracellular pH may be able to trigger signaling path-ways leading to neutrophil activation. Supporting this hypothesis,previous studies focused on neutrophil chemotaxis have shownthat cytosolic acidification acts as a second messenger for the in-duction of neutrophil activation (26, 27). Alternatively, as ob-served for conventional agonists, the ability of extracellular pro-tons to activate neutrophils could be due to the interaction ofprotons with specific receptors expressed on the neutrophil surface.Supporting this possibility, recent reports have shown that extra-cellular protons can be effectively recognized by a subfamily of Gprotein-coupled receptors, namely GPR4 (28–30). This subfamilycomprises four receptors that share significant sequence homol-ogy: GPR4, ovarian cancer G protein-coupled receptor 1 (OGR1),T cell death-associated gene 8 (TDAG8), and G2A (from G2 ac-cumulation). Originally characterized by their ability to bind proin-flammatory lipids such as lysophosphatidylcholine, sphingo-sylphosphorylcholine, and the lysosphingolipid psychosine, recentobservations suggest that these receptors constitute a family ofproton-sensing G protein-coupled receptors (28–30). The originalobservations related to the ability of these receptors to recognizeprotons were conducted in osteosarcoma cells and primary humanosteoblast precursors and showed that acidosis triggered inositolphosphate formation (28). Subsequent studies performed in a va-riety of transfected cell lines cultured at low values of extracellularpH (6.0–7.0), found that OGR1 and G2A lead to the accumulationof inositolphosphate, whereas GPR4 and TDAG8 elicit cAMP for-mation (29, 30). The expression and function of these receptors inhuman leukocytes has not been defined.

In the present study, we show that the stimulation of humanneutrophils by extracellular acidosis is dependent on the activationof PI3K/Akt and ERK pathways. Moreover, using mouse perito-neal neutrophils, we found that acidosis not only stimulates endo-cytosis but also improves the presentation of extracellular Ags byneutrophils through MHC class I molecules (cross-presentation), apathway of exogenous Ag presentation usually restricted to den-dritic cells.

Materials and MethodsReagents

Dextran T-500 and Ficoll-Hypaque were obtained from Amersham Bio-sciences. OVA and dextran (40,000 Da) (Sigma-Aldrich) were conjugatedwith FITC as described (31). The OVA257–264 peptide was provided by Dr.S. Amigorena (Institut Curie, Paris, France). Sulfasalazine was from Sig-ma-Aldrich. The ERK inhibitor PD98059 and the JNK inhibitor SP600125were purchased from Biomol, and the PI3K inhibitors wortmannin andLY294002, the MEK inhibitor U0126, and the p38 MAPK inhibitorSB202190 were from Calbiochem. Fluo-3-acetoxymethyl ester (fluo-3-AM)3 was from Molecular Probes.

Preparation of human neutrophils

Blood samples were obtained from healthy donors by venipuncture of theforearm vein at the Instituto de Investigaciones Hematologicas blood bank.All of the reagents used in the isolation of neutrophils were adjusted to pH7.3. Neutrophils were isolated from heparinized human blood samples bydextran sedimentation and Ficoll-Hypaque gradient centrifugation as de-scribed (32). Contaminating erythrocytes were removed by hypotonic lysis.Unless otherwise stated, after washing the cell pellets (�96% of neutro-phils on May-Grunwald-Giemsa-stained cytopreparations) were resus-pended in RPMI 1640 medium (Invitrogen Life Technologies) supple-

mented with 1% heat-inactivated FCS, 50 U/ml penicillin, 50 �g/mlstreptomycin, and 0.1 mM nonessential amino acids (all from InvitrogenLife Technologies) (complete medium) previously adjusted to the desiredpH values.

Mice

Experiments were conducted using 2-mo-old virgin female C57BL/6 miceraised at the National Academy of Medicine, Buenos Aires, Argentina.They were housed six per cage and kept at 20 � 2°C under an automatic12-h light-dark schedule. Animal care was in accordance with institutionalguidelines.

Mouse exudate cells

C57BL/6 mice were injected i.p. with 2 ml of 5% casein (Sigma-Aldrich).The exudates were collected 6 h later by washing the peritoneal cavity with8 ml of cold PBS. Neutrophils were then purified by Percoll gradients(Amersham Biosciences) as described previously (33). These cells werefound to comprise �95% of neutrophils as estimated by Giemsa staining.

Acidification of the culture medium

Extracellular acidification was achieved by suspending cell pellets in com-plete medium previously adjusted to the desired pH values or by the ad-dition of a precalculated volume of isotonic HCl solution. Similar resultswere observed using both methods.

Flow cytometric measurements

Flow cytometric assays were performed in a FACScan argon laser flowcytometer (BD Immunocytometry System). Data were analyzed by usingCellQuest software (BD Biosciences).

Endocytosis of FITC-OVA and FITC-dextran

The analysis of the influence of extracellular acidosis on endocytosis wasassessed by using two fluorescent markers that differ in their chemicalcomposition: FITC-OVA, a protein with a molecular mass of �45,000 Da,and FITC-dextran, a polymer of glucose with a molecular mass of �40,000Da. Cells were suspended in complete medium adjusted to pH 7.3 or 6.5.FITC-OVA and FITC-dextran were added at final concentrations of 10and/or 100 �g/ml, and cells were incubated for 30 min at 37°C under 5 or7% CO2 for cultures performed at pH 7.3 or 6.5, respectively. The cellswere then washed three times with cold PBS containing 1% FCS and0.01% NaN3, and then analyzed on a FACS (BD Biosciences). The fluo-rescence background was determined by incubating cells with FITC-OVAor FITC-dextran at 4°C. In some experiments, we used the dye trypan blueto quench extracellular fluorescence as described (34, 35). In these exper-iments, endocytosis assays were performed as indicated previously, but theacquisition of samples was conducted in the presence of 200 �g/ml trypanblue. The efficacy of trypan blue to quench extracellular fluorescence wascontrolled in experiments in which neutrophils were stained with FITC-mAb directed to cell surface Ags (30 min at 4°C). Fluorescence intensitywas diminished by �90% when the acquisition of the samples was per-formed in the presence of trypan blue.

Calcium measurements

Changes in intracellular free calcium concentrations ([Ca2�]i) were mea-sured using fluo-3-AM as described previously (36). Neutrophils, sus-pended at a concentration of 5 � 106 cells/ml in complete medium, wereincubated with 4 �M fluo-3-AM for 30 min at 30°C. Then, cells werewashed three times with RPMI 1640 medium and suspended at 5 � 106

cells/ml in RPMI 1640 supplemented with 5% FCS. Aliquots of 50-�l eachwere then added to 450 �l of 37°C RPMI 1640 medium containing 5%FCS (pH 7.3). The prewarmed sample was immediately loaded onto theflow cytometer, and fluorescence was recorded for �25 s. Then, the me-dium was acidified by the addition of a predetermined volume of isotonicsolution of HCl to adjust its pH to 6.5, and the fluorescence was recordedduring an additional 100 s. A gate based on forward and side scatter wasused to exclude debris, whereas a time-based gate was used to divide theoriginal data file and separate cells according to the time at which theirfluorescence in an FL1 detector was measured. Measurements of fluores-cence in samples suspended at pH 7.3 were used to establish a marker ata FL1 fluorescence channel number greater than that exhibited by at least97% of these resting cells. This marker was then used to determine therelative percentage of activated cells. Cells that raised their [Ca2�]i tohigher levels than that shown by 97% of resting cells in response to pH 6.5were considered to be activated.

3 Abbreviations used in this paper: fluo-3-AM, fluo-3-acetoxymethyl ester; [Ca2�]i,intracellular free calcium concentration; FSC, forward light scatter.

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Neutrophil shape change

Cell pellets containing 2.5 � 106 neutrophils were suspended in 1 ml ofRPMI 1640 medium with 1% FCS adjusted previously to the desired pHvalue and incubated in a shaking water bath for 5 min at 37°C. The cellshape change was then evaluated by flow cytometry as described (37). Theshape change was measured as the shift in the forward light scatter param-eter. Results were expressed as mean forward scatter (FSC) values.

Ag presentation assay

Presentation of OVA257–264 epitope on Kb was detected using the T cellhybridoma B3Z, which carries a �-galactosidase construct driven byNF-AT elements from the IL-2 promoter (38). For Ag presentation assays,mouse peritoneal exudate neutrophils (�95% purity) were exposed to dif-ferent concentrations of OVA at 37°C for 3 h at pH 7.3 or 6.5. Cells werethen washed, suspended in complete medium at pH 7.3, and cultured in thepresence of the T cell hybridoma B3Z. After 18 h of culture, the cells werewashed with PBS, and a colorimetric assay using o-nitrophenyl-�-D-ga-lactopyranoside (Sigma-Aldrich) as a substrate was used to detect LacZactivity in B3Z lysates.

Western blotting

Abs directed to p38 MAPK (rabbit polyclonal), JNK-1 (rabbit polyclonal),phospho-JNK (T183/Y185, mouse monoclonal), and Akt-1 (goat poly-clonal) were obtained from Santa Cruz Biotechnology. Abs directedagainst ERK1/2 MAPK (rabbit polyclonal) (Promega), phospho-ERK1 andphospho-ERK2 (T202/Y204, mouse monoclonal), phospho-p38 MAPK(T180/Y182, rabbit polyclonal), phospho-Akt (S473, rabbit polyclonal)(Cell Signaling Technology), and I�B-� (BD Pharmingen) were also used.HRP-conjugated anti-mouse, anti-rabbit, or anti-goat IgG was from SantaCruz Biotechnology. Neutrophils suspended in complete medium (3 � 106

cells/300 �l) were prewarmed for 5 min at 37°C. The acidification of cellssuspended in medium at pH 7.3 was accomplished by the addition of aprecalculated volume of isotonic HCl solution. Cells were incubated at pH7.3 or 6.5 for different times (0–60 min) at 37°C. The reactions were

stopped at the times indicated by adding cold saline, and the samples werethen centrifuged. Cell pellets were resuspended in loading buffer (60 mMTris (pH 6.8), 2.3% SDS, 10% glycerol, 0.01% bromphenol blue, and 5%�-mercaptoethanol), boiled at 96°C for 5 min, and stored at �80°C. Sam-ples were then separated by SDS-PAGE (10 or 12%), transferred to poly-vinylidene difluoride membranes (Sigma-Aldrich), and then blocked with5% skimmed milk in PBS containing 0.05% Tween 20. Membranes werethen blotted with Abs against phospho-Akt, phospho-ERK, phospho-p38,phospho-JNK, or I�B-�, followed by HRP-conjugated anti-mouse or anti-rabbit IgG. Specific bands were developed by ECL (Amersham Bio-sciences). Membranes were stripped and reproved with Abs against Akt-1,ERK 1/2, p38, and JNK-1 MAPK to confirm that equal amounts of proteinwere present in each lane of the gel. Western blotting assays were allperformed in the absence of phosphatase inhibitors.

Neutrophil treatment with inhibitors of different signalingpathways

Cells were incubated with the inhibitors for 20 min at 37°C and pH 7.3 atthe concentrations shown. Then, cell functions were analyzed at pH 7.3 or6.5, as described above. In all of the experiments conducted with inhibitors,mock pretreatment was performed by incubating neutrophils with vehiclealone (DMSO) (maximum concentration 0.2%). At this concentration,DMSO did not exert any effect.

Statistical analysis

Student’s paired t test was used to determine the significance of differencesbetween means, and p � 0.05 was taken as indicating statistical significance.

ResultsExtracellular acidification activates PI3K and ERK but not p38MAPK, JNK, or NF-�B in human neutrophils

To determine the signaling mechanisms through which extracel-lular acidosis activates human neutrophils, Western blots of lysates

FIGURE 1. Acidosis activates PI3K and ERK in human neutrophils. Neutrophils (3 � 106 cells per 300 �l of complete medium) were prewarmed for5 min at 37°C, and acidification was accomplished by the addition of a precalculated volume of isotonic HCl solution. Cells were incubated at pH 7.3 or6.5 for different times at 37°C, and the samples were then analyzed by Western blotting as described in Materials and Methods. Pervanadate-treatedneutrophils (with 0.1 mM orthovanadate plus 0.3 mM H2O2 for 10 min at 37°C (pH 7.3)) were used as positive controls. Western blots are representativeof three to six experiments. The inhibitors of PI3K wortmannin and LY294002 were used at concentrations of 50 nM and 100 �M, respectively, whereasthe inhibitors of MEK, PD98059, and U0126, were used at 100 and 25 �M, respectively (A and B).

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from neutrophils cultured for different periods at pH 6.5 wereprobed with Abs against phosphorylated and total Akt, ERK, JNK,and p38 MAPK. Experiments were performed at pH 6.5, because,as described in the introduction (1–12), it represents a pH valuefrequently found at inflammatory sites. Control cells were culturedat pH 7.3 (neutral pH). The results (Fig. 1, A–D) show that acidosistriggers phosphorylation of Akt (a major target of PI3K) andERK1/2 but not of JNK or p38 MAPK. As expected, the phos-phorylation of Akt was prevented by the inhibitors of PI3K, wort-mannin and LY294002, supporting the notion that it was depen-dent on PI3K activity, whereas phosphorylation of ERK1/2 wassuppressed by the MEK inhibitors PD98059 and U0126 (Fig. 1, Aand B). We also analyzed the possible involvement of NF-�B inthe activation of neutrophils by acidosis. NF-�B activation usuallyrequires I�B phosphorylation and degradation in the cytoplasmand the subsequent translocation of NF-�B to the nucleus (39).Fig. 1E shows that acidosis does not trigger the degradation ofI�B, supporting the hypothesis that NF-�B is not activated underacidosis. Together, these results support the notion that extracel-lular acidosis activates neutrophils via two distinct signalingpathways.

Blockade of PI3K and ERK pathways inhibits neutrophilactivation triggered by extracellular acidosis

After appropriate stimulation, neutrophils undergo transientshape changes that can be detected by analyzing variations intheir light-scattering properties using flow cytometry or micro-

scopic examination. We have shown previously that neutrophilssuspended in culture medium adjusted to acidic pH values (6.5–7.0) underwent a rapid increase in the forward light-scatteringproperties (20). Fig. 2, A and B, shows a representative exper-iment in which the shape change response was evaluated bymicroscopic examination. To analyze the involvement of PI3Kand ERK pathways in the induction of this response, we usedthe PI3K inhibitors wortmannin and LY294002 and the MEKinhibitors PD98059 and U0126. Cells were cultured in the ab-sence or presence of these inhibitors at 37°C for 20 min at pH7.3. Then, cell suspensions were acidified to pH 6.5 by theaddition of a precalculated volume of an isotonic HCl solution.An equal volume of saline was added to control cells. After 5min at 37°C, cells were analyzed by flow cytometry. Fig. 2, C–I,shows that inhibitors of both the PI3K pathway and the ERKpathway significantly decreased the shape change response trig-gered by acidosis, with the effect of the PI3K inhibitors beingmore pronounced. Consistent with the results depicted in Fig. 1showing that acidosis does not trigger the activation of JNK,p38 MAPK, or NF-�B, we found that the specific inhibitors ofthese pathways, SB202190, SP600125, and sulfasalazine, usedat concentrations able to suppress their activation in humanneutrophils (40 – 42), did not inhibit the cell shape responsetriggered by pH 6.5.

We have also shown that acidosis triggers calcium mobiliza-tion in human neutrophils (20). Thus, we assessed the effect ofinhibitors of PI3K and MEK on this response. To this aim,

FIGURE 2. Involvement of PI3K and ERK signaling pathways in the shape change response of neutrophils induced by acidosis. A and B, Lightmicroscopic examination of neutrophils (2.5 � 106/ml) cultured for 5 min at 37°C at pH 7.3 (A) or 6.5 (B). C–I, Neutrophils (2.5 � 106/ml) were culturedfor 20 min at 37°C in complete medium at pH 7.3 with inhibitors of distinct signaling pathways. Cells were then cultured at pH 6.5 for 5 min, and thechanges in FSC values were analyzed by flow cytometry. C, Results are expressed as the mean of FSC values � SEM from 4 to 11 experiments. �, p �0.05 compared with untreated neutrophils cultured at pH 6.5. D–I, Representative experiments showing the distribution of FSC values observed forneutrophils cultured for 20 min at 37°C and pH 7.3 without inhibitors (D and E) or in the presence of 50 nM wortmannin (F), 100 �M LY294002 (G),100 �M PD98059 (H), or 25 �M U0126 25 (I), and then cultured at 37°C for 5 min at pH 7.3 (D) or 6.5 (E–I).

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neutrophils were loaded with fluo-3-AM and cultured in theabsence or presence of inhibitors for 20 min at 37°C at pH 7.3.The samples were then immediately loaded onto the flow cy-tometer, and fluorescence was recorded for �25 s. Then, themedium was acidified by the addition of a predetermined vol-ume of isotonic solution of HCl to adjust its pH to 6.5, and thefluorescence was recorded for an additional 100 s. Fig. 3A,which shows the dot plot data from a single donor, illustratesthe pattern of [Ca2�]i changes after extracellular acidification.A fraction of neutrophils exhibited a rapid increase in [Ca2�]i,followed by a gradual decrease to the resting [Ca2�]i levels�35– 45 s after acidification. As shown in Fig. 3, the inhibitorsof PI3K markedly diminished the fraction of neutrophilsthat exhibited an increase in [Ca2�]i levels in response toacidification, whereas the inhibitors of the MEK, JNK, p38MAPK, and NF-�B pathways did not mediate any inhibitoryeffect. As expected, no differences in fluorescence emissionwere observed in cells maintained at pH 7.3 throughout theexperiment.

Extracellular acidosis increases endocytosis by humanneutrophils; its dependence on the PI3K and ERK pathways

We have shown previously that acidosis improves the endocyticcapacity of mouse dendritic cells (21). Taking this finding intoaccount, to further characterize the impact of extracellular ac-idosis on neutrophil function we performed another set of ex-periments to establish whether acidosis was also able to stim-ulate endocytosis by human neutrophils. To this aim, we usedtwo markers, FITC-OVA and FITC-dextran. Cells were cul-tured in medium adjusted to pH 7.3 or 6.5 in the presence ofFITC-OVA or FITC-dextran (10 and 100 �g/ml) for 30 min at37°C. Then, cells were washed two times with saline, sus-

pended in culture medium at pH 7.3, and analyzed by flowcytometry. In agreement with the observations made in den-dritic cells (21), we found that the uptake of both markers byhuman neutrophils was markedly increased at pH 6.5 comparedwith pH 7.3 (Fig. 4), suggesting that the endocytosis of distinctcompounds may be favored at acidosis irrespective of theirchemical composition. To analyze whether the endocytic mark-ers were actually internalyzed by neutrophils and not merelyattached to the cell surface through nonspecific interactions, weperformed additional assays in which endocytosis of FITC-OVA was conducted as described above, but the acquisition ofsamples was performed in the presence of trypan blue (200�g/ml), a dye able to quench extracellular fluorescence (34, 35).The results obtained showed that trypan blue did not affect theincrease in fluorescence of neutrophils cultured with FITC-OVA (100 �g/ml) at pH 7.3 or 6.5, suggesting that the markeris actually internalized (percentage of increase was 596 � 54,mean � SEM, n � 7, p � 0.01 for pH 6.5 vs pH 7.3). Havingshown that acidosis increases endocytosis by human neutro-phils, we then analyzed the signaling pathways involved. Fig. 5shows that the inhibitors of PI3K and MEK significantly dimin-ished the stimulation of endocytosis triggered by acidosis,whereas the inhibitors of the JNK, p38 MAPK, and NF-�Bpathways did not mediate any inhibitory effect

Extracellular acidosis improves MHC class I-restricted Agpresentation by mouse peritoneal neutrophils

We have shown previously that extracellular acidosis not only in-creases endocytosis but also improves acquisition of extracellularAgs by dendritic cells for MHC class I-restricted presentation (21).This pathway, called cross-presentation, allows the display of ex-ogenous Ags in the context of MHC class I molecules (43, 44). To

FIGURE 3. Involvement of PI3K in the induction of calcium transients triggered by extracellular acidification. A–E, Dot plots showing fluo-3-AMfluorescence vs time from a single representative experiment (n � 4–7). Neutrophils were suspended in RPMI 1640 medium (2.5 � 106/ml) and culturedfor 20 min at 37°C in complete medium at pH 7.3 without inhibitors (A) or in the presence of 50 nM wortmannin (B), 100 �M LY294002 (C), 100 �MPD98059 100 (D), or 25 �M U0126 (E). Arrows indicate the addition of an isotonic HCl solution to each sample tube to adjust the extracellular pH to 6.5.F, Results are expressed as the percentage (mean � SE; n � 7) of the neutrophil population that underwent an increase in [Ca2�]i above the [Ca2�]i

exhibited by 97% of resting cells, which were considered as activated cells. �, p � 0.05 compared with untreated neutrophils cultured at pH 6.5.

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analyze whether extracellular acidosis was able to enhance Agdelivery into the MHC class I pathway in neutrophils, we usedinflammatory exudate cells (�95% of neutrophils) obtained fromthe peritoneal cavity of C57BL/6 mice 6 h after the injection ofcasein, as described in Materials and Methods. First, we analyzedwhether acidosis was able to enhance endocytosis by mouse peri-toneal neutrophils in a similar manner as that by human neutro-phils. Cells were cultured in medium adjusted to pH 7.3 or 6.5 inthe presence of FITC-OVA (100 �g/ml) for 30 min at 37°C. Then,cells were washed two times with saline, suspended in culturemedium at pH 7.3, and analyzed by flow cytometry. In agreementwith the results obtained in human neutrophils (Fig. 4A), we ob-served that the uptake of FITC-OVA by mouse peritoneal neutro-phils was markedly increased at pH 6.5 as compared with pH 7.3;percentage of increase was 378 � 57 (mean � SEM, n � 5). Toanalyze whether acidosis may improve the acquisition of Ags byneutrophils for MHC class I-restricted presentation, we studiedpresentation of OVA to a CD8� T cell hybridoma called B3Z,which carries a �-galactosidase construct driven by NF-AT ele-ments from the IL-2 promoter enabling the analysis of T cell ac-tivation by measuring �-galactosidase activity in cell lysates (38).Mouse peritoneal neutrophils were cultured with different concen-

trations of OVA for 3 h at 37°C at pH 7.3 or 6.5, and presentationof the OVA257–264-epitope/H-2Kb to B3Z cells was then evaluated.As shown in Fig. 6, extracellular acidosis enabled mouse perito-neal neutrophils to present OVA through a MHC class I-restrictedpathway. In fact, cells pulsed with OVA under neutral pH do notdisplay significant levels of Ag presentation. Consistent with thesignaling pathways involved in the stimulation of endocytosis byacidosis, we found that both wortmannin (50 nm) and PD98059(100 �M) significantly ( p � 0.01) prevented the stimulation ofcross-presentation triggered by acidosis (percentage of inhibitionwas 76 � 16 and 48 � 13, respectively, mean � SEM, n � 4).

FIGURE 4. Acidosis enhances endocytosis of FITC-OVA and FITC-dextran. Neutrophils (2.5 � 106/ml) were incubated for 30 min at 37°Cwith different concentrations of FITC-OVA (A) or FITC-dextran (B) at pH7.3 or 6.5, and the amount of ligand accumulated was measured by flowcytometry. The uptake of either FITC-OVA or FITC-dextran (100 �g/ml)by neutrophils after incubation for 30 min at 4°C is also shown. Results areexpressed as mean fluorescence intensity values and represent the mean �SEM of seven experiments. �, p � 0.05; ��, p � 0.01; compared withneutrophils cultured at pH 7.3. FIGURE 5. Involvement of PI3K and ERK signaling pathways in the

stimulation of endocytosis induced by extracellular acidosis. Neutrophilswere suspended in RPMI 1640 medium (2.5 � 106/ml) and cultured for 20min at 37°C at pH 7.3 with inhibitors of distinct signaling pathways. Then,cells were incubated with FITC-OVA (100 �g/ml) for 30 min at 37°C inculture medium adjusted to pH 6.5, and the amount of ligand accumulatedwas measured by flow cytometry. A, Histograms from a representativeexperiment are shown (n � 9). Wortmannin (W) was used at 50 nM andPD98059 (PD) at 100 �M. The uptake of FITC-OVA by neutrophils afterincubation at pH 7.3 for 30 min at 4°C is also shown. B, Results areexpressed as mean fluorescence intensity values and represent the mean �SEM of 4–11 experiments. �, p � 0.05 compared with untreated neutro-phils cultured at pH 6.5.

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Control experiments revealed that when neutrophils were fixedwith glutaraldehyde before the addition of OVA, no presentationwas observed (data not shown). We also determined whetheracidosis could modulate direct presentation of the OVA257–264

peptide. To this aim, neutrophils were cultured with the peptide(10 ng/ml) for 3 h at 37°C at pH 7.3 or 6.5, and MHC class Ipresentation was assessed as described in Materials and Meth-ods. We found no differences in the presentation of the peptidebetween neutrophils pulsed under neutral or acidic conditions;cell response measured as OD at 415 nm was 0.48 � 0.12 vs0.46 � 0.09 (pH 7.3 and 6.5, respectively, mean � SEM,n � 4).

DiscussionWe have reported previously that acidosis triggers human neutro-phil activation, inducing a transient increase in [Ca2�]i over theresting levels, a shape change response, the up-regulation of CD18expression, and a delay in the rate of apoptosis (20). In the presentstudy, we extend these observations and show that acidosis is alsoable to stimulate neutrophil endocytosis, enabling neutrophils tocross-present extracellular Ags.

Murine and human neutrophils express MHC class I mole-cules (45– 47). These molecules usually present peptide Agsderived from endogenously synthesized proteins that are de-graded in the cytosol by the proteosome. However, there is aspecialized pathway that allows the acquisition of extracellularAgs facilitating the generation of an MHC class I-restrictedimmune response. This pathway is called cross-presentation andallows display of exogenous Ags in the context of MHC classI molecules to stimulate CD8 T cells (43, 44). Although cross-presentation in vivo has been localized mainly to dendriticcells, multiple types of endocytic cells, including macrophages,B cells, keratinocytes, and L cells, can cross-present exogenousAgs in vitro with a low degree of efficiency (43, 44, 48 –51).

Our results support the notion that neutrophils, under the influ-ence of an acidic microenvironment, may also be able to take upextracellular Ags and present them through a MHC class-I re-stricted pathway. Because acute inflammation may occur simul-taneously with the recruitment of CD8� T cells during thecourse of the immune response against infectious agents, tu-mors, and allografts (52–54), our results support the notion thatextracellular acidosis may influence the development of adap-tive immunity, not only by activating dendritic cells (21) butalso by stimulating CD8� T cell responses in peripheral tissuesvia Ag cross-presentation mediated by neutrophils.

Previous studies have shown that low extracellular pH is ableto activate ERK2, JNK, and p38 MAPK in a variety of cell lines(55, 56). Moreover, decreasing extracellular pH from 7.4 to 6.1has shown to be capable to activate phospholipase C, leading toCa2� mobilization and the production of inositol triphosphatesin human fibroblast, endothelial, smooth muscle, and neuroblas-toma cells (57). Although the mechanism responsible for therecognition of extracellular protons by neutrophils remains tobe defined, our results demonstrate that neutrophil exposure toacidosis results in the activation of PI3K and ERK pathways.These pathways have shown to play a critical role in neutrophilfunction, because they are required for the activation of che-motaxis, phagocytosis, and the respiratory burst and are alsoinvolved in the control of neutrophil survival (58 – 60). More-over, we observed that the blocking of these pathways by spe-cific inhibitors prevented the activation of neutrophils by ex-tracellular acidosis. Whereas the inhibitors of PI3K exerted aprofound inhibitory effect on all the functions analyzed, theinhibition of MEK exerted a significant inhibition on the shapechange response and endocytosis without affecting the induc-tion of Ca2� transients triggered by acidosis.

Interestingly, Owen at al. (61) have recently shown that expo-sure of human neutrophils to extracellular acidosis results in amarked increase in the synthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The maximum in-crease was found at pH 5.4, but a significant enhancement was alsoobserved at pH 6.4, a value of extracellular pH similar to the onewe used throughout our study (pH 6.5). When analyzing the mech-anisms responsible for the stimulatory effect of acidosis, the au-thors found that pH 5.4 triggered the activation of ERK1, whereaspH 6.5 triggered the activation of both ERK1 and ERK2. Regard-ing p38 MAPK, the authors found that maximum activation wasinduced at pH 5.4, and low but significant levels of activation wereobserved at pH 6.5. This last result appears to be in disagreementwith our findings, because we observed no phosphorylation of p38MAPK in neutrophils cultured at pH 6.5. The reasons for thesediscrepant results are unknown. They could be related to the dif-ferent experimental conditions used in each study. For example,Owen et al. (61) conducted their experiments using neutrophilssuspended in a serum-free modified HBSS supplemented withTris, histidine, acetic acid, and lactic acid, whereas all of our ex-periments were performed in RPMI 1640 medium supplementedwith 1% heat-inactivated FCS.

It is widely appreciated that inflammatory responses in normalperipheral tissues as well as in tumors are usually associated withthe development of acidic microenvironments (1–12). In fact, ac-idosis appears to be a hallmark of inflammatory processes. Sur-prisingly, there are few studies directed at analyzing the effect ofextracellular acidosis on the immune response. We have reportedpreviously that extracellular acidosis induces the activation of neu-trophils (20) and dendritic cells (21), suggesting that acidosis acts

FIGURE 6. Acidosis improves the acquisition of OVA by neutrophilsfor MHC class I-restricted presentation. Inflammatory exudate cells(�95% of neutrophils) were obtained from the peritoneal cavity ofC57BL/6 mice 18 h after the injection of casein. Cells (1 � 106/ml) werecultured with different concentrations of OVA for 3 h at 37°C at pH 7.3 or6.5. The cells were then washed, suspended in complete medium at pH 7.3,and cultured for 18 h at 37°C in the presence of B3Z cells (1 � 106/ml),a T cell hybridoma specific for OVA-Kb that carries a �-galactosidaseconstruct driven by NF-AT elements from the IL-2 promoter. T cell acti-vation was measured using a colorimetric assay for LacZ activity witho-nitrophenyl-�-D-galactopyranoside as a substrate. Background absor-bance values obtained for neutrophils cultured in the absence of OVA weresubtracted. �, p � 0.05 compared with neutrophils cultured with OVA atpH 7.3.

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as a danger signal able to stimulate both innate and adaptive im-mune responses. In the present study, we show that acidosis stim-ulates neutrophil function by activating PI3K and ERK pathways.Moreover, in agreement with our previous results in dendriticcells, we found that acidosis stimulates endocytosis, enabling neu-trophils to cross-present extracellular Ags through a MHC class-Irestricted pathway. Further studies are needed to evaluate in vivothe influence of extracellular acidosis on the function of both neu-trophils and dendritic cells.

AcknowledgmentsWe thank Selma Tolosa and Nelly Villagra for their technical assistanceand Maria Rita Furnkorn for her secretarial assistance.

DisclosuresThe authors have no financial conflict of interest.

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