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Regulation of HMGB-1 Cytokine Activity-Induced Dendritic Cell Maturation through

MycoplasmaSurfactant Protein-A Inhibits

Kristi L. Williams and Jo Rae WrightThomas, Katherine Evans, Derek W. Cain, Monica Kraft, Julie G. Ledford, Bernice Lo, Michele M. Kislan, Joseph M.

http://www.jimmunol.org/content/185/7/3884doi: 10.4049/jimmunol.1000387September 2010;

2010; 185:3884-3894; Prepublished online 1J Immunol 

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The Journal of Immunology

Surfactant Protein-A Inhibits Mycoplasma-Induced DendriticCell Maturation through Regulation of HMGB-1Cytokine Activity

Julie G. Ledford,* Bernice Lo,* Michele M. Kislan,* Joseph M. Thomas,*

Katherine Evans,* Derek W. Cain,† Monica Kraft,‡ Kristi L. Williams,*,†

and Jo Rae Wright*

During pulmonary infections, a careful balance between activation of protective host defense mechanisms and potentially injurious

inflammatory processes must be maintained. Surfactant protein A (SP-A) is an immune modulator that increases pathogen uptake

and clearance by phagocytes while minimizing lung inflammation by limiting dendritic cell (DC) and T cell activation. Recent

publications have shown that SP-A binds to and is bacteriostatic forMycoplasma pneumoniae in vitro. In vivo, SP-A aids in mainte-

nance of airway homeostasis during M. pneumoniae pulmonary infection by preventing an overzealous proinflammatory response

mediated byTNF-a. Although SP-Awas shown to inhibitmaturation of DCs invitro, the consequence of DC/SP-A interactions invivo

has notbeen elucidated. In this article,we show that the absence of SP-AduringM.pneumoniae infection leads to increasednumbers of

mature DCs in the lung and draining lymph nodes during the acute phase of infection and, consequently, increased numbers of

activated TandB cells during the course of infection. The findings that glycyrrhizin, a specific inhibitor of extracellular high-mobility

group box-1 (HMGB-1) abrogated this effect and that SP-A inhibits HMGB-1 release from immune cells suggest that SP-A inhibits

M. pneumoniae-induced DCmaturation by regulatingHMGB-1 cytokine activity. The Journal of Immunology, 2010, 185: 3884–3894.

Mycoplasma pneumoniae is recognized as one of the mostcommon causes of community-acquired pneumonia and.50% of chronic stable asthmatics have evidence of

airway infection with M. pneumoniae (1, 2). M. pneumoniae areatypical bacteria that form strong attachments to ciliated airwayepithelial cells where they release oxidative products that can causeairway tissue damage and contribute to exacerbations in chronicasthmatics (3). Infections with M. pneumoniae may persist, withmild symptoms, for several weeks with manifestations in the upperand lower respiratory tract.Because M. pneumoniae is primarily an extracellular pathogen

that invades and resides in the respiratory tract, it has the potentialto encounter pulmonary surfactant proteins that are produced byalveolar type II cells, Clara cells, and submucosal glands of therespiratory tract. Indeed, studies have demonstrated that surfactantprotein A (SP-A) binds M. pneumoniae through disaturated phos-phatidylglycerols and through a specific surface-binding protein,MPN372 (4, 5), and limits the growth of M. pneumoniae in vitro(5). SP-A also helps to maintain airway homeostasis and reduce

hyperresponsiveness by curtailing an overly ambitious proinflam-

matory immune response during M. pneumoniae infection in mice

in vivo (6).Several immune functions have been ascribed to SP-A, including

inhibition of T cell proliferation, augmentation of pathogen phago-

cytosis by acting as an opsonin, and modulation of chemotaxis and

cytokine production (reviewed inRef. 7). An additional role for SP-A

was established in mediating adaptive immune responses through

interactions with dendritic cells (DCs). For example, SP-A binds to

DCs and negatively regulates their maturation in vitro, thereby re-

ducing their T cell allostimulatory ability (8). The consequences of

this interaction during an infection in vivo, as well as the mechanism

by which SP-A modulates DC functional maturation, have not been

defined. Therefore, using mice deficient in SP-A, we tested the hy-

pothesis that SP-A regulates recruitment, activation, and maturation

of adaptive immune cells in response to M. pneumoniae by regu-

lating expression of the endogenous stress factor high-mobility

group box-1 (HMGB-1), which, if released in the context of infec-

tion, can activate DCs and lead to their maturation (9).We report in this article that M. pneumoniae infection leads to

increased numbers of exudative macrophages and DCs in the lung

parenchyma, a response that is augmented by the absence of SP-A.

Likewise, the total number and activation state of DCs that have

migrated to the mediastinal draining lymph nodes during the acute

phase (3 d) of infection are also increased in the absence of

SP-A. Additionally, elevated numbers of activated T and B cells

in the lungs and mediastinal lymph nodes (MLNs), as well as

M. pneumoniae-specific IgG in the serum, are observed in mice

lacking SP-A 9 d after M. pneumoniae infection. Treatment with

glycyrrhizin, a specific extracellular inhibitor of the potent pro-

inflammatory cytokine HMGB-1, protects the DCs in the MLNs

of mice lacking SP-A from M. pneumoniae-induced maturation,

suggesting that SP-A inhibits M. pneumoniae-induced DC matu-

ration by regulating HMGB-1 cytokine activity.

*Department of Cell Biology, †Department of Immunology, and ‡Department ofPulmonary, Allergy and Critical Care Medicine, Duke University Medical Center,Durham, NC 27710

Received for publication February 23, 2010. Accepted for publication July 26, 2010.

This work was supported by Grants F32HL091642, HL084917, and AI81672 fromthe National Institutes of Health.

Address correspondence and reprint requests to Dr. Jo Rae Wright, Department ofCell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710.E-mail address: j.wright@cellbio.duke.edu

The online version of this article contains supplemental material.

Abbreviations used in this paper: 0, saline only; BAL, bronchoalveolar lavage; BMDC,bone marrow-derived dendritic cell; C, control; DC, dendritic cell; HMGB-1, high-mobility group box-1; MIG, monokine induced by IFN-g; MLN, mediastinal lymphnode; NHBE, normal human bronchial epithelial cell; RAGE, receptor for advancedglycation end products; SP-A, surfactant protein-A; U, uninfected; WT, wild-type.

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

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Materials and MethodsM. pneumoniae culture

M. pneumoniae (catalog no. 15531) from American Type Culture Collection(Manassas, VA) was grown in SP4 broth (REMEL, Lenexa, KS) at 35˚C untiladherent. M. pneumoniae concentration was determined by plating serialdilutions of M. pneumoniae on pleuropneumonia-like organism agar plates(REMEL). Colonies were counted under 310 magnification on plates afterincubation for 14 d. For in vivo infection, adherent M. pneumoniae werewashed by centrifuging at 6000 rpm for 5 min and resuspended in sterilesaline for infection at a concentration of 1 3 108 M. pneumoniae/50 ml in-oculum. M. pneumoniae burden was assessed, as previously described,by plating bronchoalveolar lavage (BAL) or by RT-PCR using primersagainstM. pneumoniae-specific P1-adhesin gene relative to the housekeepercyclophilin (6).

Mice

An inbred strain of SP-A–deficient mice was generated by disrupting themurine gene encoding SP-A by homologous recombination, as previouslydescribed (10). SP-A–null mice were backcrossed for 12 generations withwild-type (WT) C57BL/6 background mice from Charles River Labora-tories (Wilmington, MA). WT C57BL/6 mice used as controls were pur-chased from Charles River Laboratories and bred in-house to account forany possible effects of environmental conditions. All mice used in ex-periments were age (8–12 wk) and sex (males) matched. Protocols wereapproved by the Institutional Animal Care and Use Committee at DukeUniversity.

Mice 8–12 wk of age were anesthetized via i.p. injections of a 12% ket-amine (100 mg/ml) and 5% xylazine (20 mg/ml) mix (10 ml/g body weight).Mice were infected with 50 ml sterile saline or 50 ml ∼1 3 108 M. pneu-moniae units in sterile saline by intranasal instillation. Some groups of micereceived i.p injections of glycyrrhizin (10 mg/kg body weight) 2 h prior toM. pneumoniae infection and 24 h postinfection, to neutralize HMGB-1cytokine activity, as previously described (11).

BAL collection, lung digestion, and analysis of pulmonary DCs

The lungs of mice were perfused with 10 ml PBS and then lavaged with PBScontaining 0.1 mM EDTA (warmed to 37˚C). Analysis of cytokines andchemokines present in the cell-free BAL of infected and uninfected micewas carried out by multiplex cytokine analysis (Luminex technology;Invitrogen, Carlsbad, CA). Lungs were removed, minced with a razor blade,and resuspended in 5 ml HBSS (containing calcium and magnesium) with1.0 mg/ml collagenase A and 0.2 mg/ml DNase I. The cell suspensions wereincubated for 1 h with shaking (200 rpm) at 37˚C for enzymatic digestionto occur. Lung digests were then filtered through 40-mm strainers.Remaining RBCs in the digests or BAL were lysed with Gey’s lysis solution(0.83% NH4Cl, 0.1% KHCO3). The suspended cells were layered on top ofa 4.0% solution of iodixanol (Optiprep; Axis-Shield, Norton, MA), placedabove a 14.5% iodixanol solution, and centrifuged at 6003 g (no brake) for20 min at room temperature. Low-density cells were isolated from the 4–14.5% interface and used for FACS analysis. Cells were tristained withallophycocyanin-labeled anti-CD11c, FITC- (or PE-) labeled anti-MHCclass II, and PE-anti–CD80 or PE-anti–CD86.

MLN digests and DC analysis

MLNs collected from each mouse were placed in individual wells of a six-well plate containing 5 ml PBS with 5% FCS, 1.0 mg/ml collagenase A,and 0.2 mg/ml DNase I. Lymph nodes were minced using a scalpel and in-cubated at 37˚C for∼35 min. Digestion was stopped by adding 1 ml 120mMEDTA in PBS and incubating for an additional 5 min at room temperature.Lymph node digests were pushed through a 40-mm strainer to obtain single-cell suspensions. Some lymph node digests were centrifuged through adensity gradient (Nycodenz; Axis-Shield) to enrich for DCs. RBCs werelysed with Gey’s solution (0.83% NH4Cl, 0.1% KHCO3). Cells were thenresuspended in HBSS with 5% FCS, 2 mM EDTA, and 100 U/ml penicillin-streptomycin and stained for FACS analysis. Cells were tristained with allo-phycocyanin-labeled anti-CD11c, FITC- labeled anti-MHC class II, PE-labeled anti-CD86, and PE-Texas Red–labeled anti-CD80.

Flow-cytometry analysis

Flow cytometry was performed in the Duke Human Vaccine Institute FlowCytometry Core Facility, which is supported by the National Institutes ofHealth Award AI-51445. Initially, cells were examined by forward scatterversusside scatter to separate thosesmallernongranular lymphoidpopulationsfrom the larger granular myeloid populations. The percentage of cells within

each of these gates was used to calculate the total number of lymphoid cellsand myeloid cells examined from the total number of cells obtained from thelung digests, as counted on a hemacytometer. Based on similar gating strat-egies published by Pecora et al. (12) and Lin et al. (13), alveolar macrophageswere defined as CD11bneg-low/CD11cmid/MHCIImid, DCs were defined asCD11bhigh/CD11chigh/MHCIIhigh, and inflammatory lung macrophages (alsoknown as exudate macrophages) were defined as CD11bhigh/CD11cneg-mid/MHCIImid.

Generation of bone marrow-derived DCs

Bone marrow-derived DCs (BMDCs) were generated as described previ-ously by Inaba et al. (14) and as modified by Brinker et al. (15). Briefly,marrow from the tibia, femur, and humerus of mice was harvested andcultured in RPMI 1640 supplemented with 5% FCS, antibiotics, and 50 mM2-ME plus 5% GM-CSF–conditioned media for 6 d. Loosely attached cellswere harvested and negatively selected with biotinylated Gr-1 Abs (BDPharmingen, San Diego, CA) and streptavidin paramagnetic microbeads(Miltenyi Biotec, Auburn, CA). DCs were matured for 24 h in 24-well platesat 53 105 cells/ml in the presence of 100 ng/ml LPS (serotype 055:B5), aspreviously described (8).

Cell culture-stimulation experiments

Undifferentiated THP-1 cells (American Type Culture Collection) weremaintained in RPMI 1640 with supplements (10% FCS, 2 mM L-glutamine,1 mMsodiumpyruvate, 0.1 mMnonessential amino acids, and streptomycin-penicillin) at 37˚C at 5% CO2. Clonetics normal human bronchial epithelialcells (NHBEs)were purchased fromLonza (Basel, Switzerland) and grown to∼80% confluence in bronchial epithelial growth media (Clonetics) withsupplements and growth factors (bovine pituitary extract, hydrocortisone,human epidermal growth factor, epinephrine, insulin, triiodothyronine, trans-ferrin, gentamicin/amphotericin-B, and retinoic acid), at which point cellswere placed in bronchial epithelial basal medium (no supplements) forstimulation experiments. THP-1 cells were resuspended in RPMI 1640 (nosupplements). DCs, NHBEs, and THP-1 cells were resuspended in the re-spective medium in the presence or absence of purified human SP-A (50mg/ml),whichwas purified as described (16), and∼53105 cellswere seeded perwell into 12-well plates. After 2 h, M. pneumoniae was added at a concen-tration of 10M. pneumoniaeCFU/cell (53 106/well) to somewells, whereasothers received saline as a control buffer for the M. pneumoniae. Somesample wells received neutralizing anti-TLR2 Ab (No. 16-9024; eBio-science, San Diego, CA) or isotype control prior to stimulation. After M.pneumoniae stimulation for ∼16 h, the cell-free supernatant was collected,and pelleted cells were lysed for Western blot analysis.

Western blot analysis

Cell-free lavages from uninfected and M. pneumoniae-infected WT and SP-A2/2 mice; stimulated DC, THP-1, or NHBE-free supernatants; and DC,THP-1, or NHBE lysates were denatured by heating at 95˚C for 5 minin buffer containing DTT prior to gel electrophoresis on 12.5% poly-acrylamide gels (Protogel, National Diagnostics, Atlanta, GA). For cell-freeBAL analysis, 25 ml from 1-ml lavages was loaded for each sample. For theDC, THP-1, and NHBE-free supernatants, ∼25 ml from a total well volumeof 250 ml was loaded for each sample, and 25 ml was loaded from 250 ml ofthe cell lysates. Gels were transferred onto nitrocellulose membrane, blockedwith 5% block (milk in TBS), and labeled with an HMGB-1 Ab (ab12029 orab65003; Abcam, Cambridge, MA) overnight at 1:1500. The secondary Ab,HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling Tech-nology, Beverly, MA), was used at 1:10,000 for 1 h. The SP-A Ab used wasrabbit anti-sheep SP-A (cross-reacts with mouse SP-A), which was used withan anti-rabbit HRP secondary Ab. GAPDH Ab (MAB374) was purchasedfromMillipore (Bedford, MA). SuperSignalWest Femto (Thermo Scientific,Rockford, IL) was used to visualize protein expression.

Cytokine/chemokine and IgG analysis

Cytokines and chemokines were analyzed by Luminex technologies usinga mouse 20-plex kit (Invitrogen); GM-CSF was analyzed by ELISA(eBioscience), according to the manufacturer’s instructions. M. pneumo-niae-specific IgG was analyzed by ELISA, as described previously (17).Briefly, sonicated heat-killed M. pneumoniae dissolved in coating buffer(0.05 M carbonate-bicarbonate buffer [pH 10]) was used at a concentrationof 10 mg/ml and was incubated overnight at 4˚C. Wells were then blockedfor 1 h with 2% BSA/PBS/0.05% Tween-20 mix, after which the serumsamples were diluted 1:10,000 in 1% BSA/PBS buffer and added fromuninfected and M. pneumoniae-infected mice. A biotin-goat anti-mouseIgG Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) andstreptavidin-HRP (BD Pharmingen) were used for the detection. Samples

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were analyzed at 450 nm absorbance on the FLUOstar Optima plate reader(BMG Labtech, Offenburg, Germany).

Microscope image acquisition

Images of the lung were taken on a Nikon Eclipse 50i light microscope at310 (aperture 0.3) or 340 (aperture 0.75) at room temperature by digitalphotography of bright-field images using a Nikon Infinity 2 camera(Nikon, Melville, NY). Infinity Capture software was used for image ac-quisition, and Adobe Photoshop (Adobe Systems, San Jose, CA) was usedfor color-contrast settings and figure presentation.

Statistical analysis

All data measurements were analyzed with Prism software (GraphPad, SanDiego, CA), first to determine whether data were distributed normally,followed by the t test to determine significance. Data sets with significantvariance between comparison groups were analyzed by the t test using theWelch correction, as assessed in Prism.

ResultsIncreased numbers of APCs in lungs of SP-A–null mice duringM. pneumoniae infection

To assess the role of SP-A in regulating immune cell responsesin vivo, the cells present in the lungs due to M. pneumoniae infec-tion and the resulting inflammationwere analyzed inWTand SP-A–null mice. Although the total numbers of myeloid cells present inthe lung digests were not significantly different in the infected SP-A–null mice compared with infected WT mice, the cellular com-position, which was analyzed by flow cytometry, showed differ-ences in certain cell populations. Inflammatory monocytes andPMNs increased in number in response to M. pneumoniae chal-lenge, although levels were equivalent in the lungs of WT and SP-A–null mice (data not shown). Among the other cell populations,DCs were examined from the lung digests of infected and un-infected mice. Lung DCs were defined based on their pattern of sidescatter versus forward scatter and high levels of expression ofCD11c and MHC class II cell surface markers (12, 18). Very fewDCs (,1% of total cells) were present in the lung digests of thesaline-treated mice (Fig. 1A). After M. pneumoniae infection, thenumber of DCs observed in WT mice was only slightly elevatedover those observed in the saline-treated mice. However, the num-ber of cells observed within the DC gate was significantly increasedin the SP-A–null mice (Fig. 1A).The numbers of macrophages (MHCIIintCD11c+) isolated from

lungs of saline-treated mice were not significantly different be-tween the WT control and the SP-A–null mice. However, 72 h afterM. pneumoniae infection, there were significantly more macro-phages (including resident and inflammatory) in both groups ofinfected mice compared with saline-treated animals in each group(Fig. 1B). Although the number of macrophages increased in theinfectedWTmice by 2-fold, the number ofmacrophages in the lungsof the infected SP-A–null mice increased almost 4-fold over theirsaline controls and was significantly greater in comparison with theinfected WT mice (p, 0.01).Expression of the cell-surfacemarker CD11bwas also assessed on

macrophage populations in the M. pneumoniae-infected mice todifferentiate inflammatory newly recruited macrophages (CD11b+),also known as exudate macrophages, which had migrated into thelung during pulmonary infection from the resident alveolar macro-phages (CD11b2) (13, 19). Similar to the alveolar macrophagesanalyzed in the BAL fluid of saline-treated mice, very few of thetissue macrophages expressed CD11b in the saline-treated mice,suggesting that thosewere primarily residentmacrophages (Fig. 1B).However, CD11b expression was significantly elevated on macro-phages in the WT and SP-A–null mice after M. pneumoniae in-fection, suggesting that thesewere predominantly newly recruited ordifferentiated inflammatory macrophages. Additionally, there were

significantlymore CD11b+macrophages inM. pneumoniae-infectedmice deficient in SP-A comparedwith infectedWTmice, suggestingthat SP-A is important in inhibiting the influx of these inflammatorymacrophages into the lung duringM. pneumoniae infection.

Increased chemoattractant signals in BAL duringM. pneumoniae infection in the absence of SP-A

The function of the pulmonary DC network is altered dramaticallyduring inflammatory conditions as the result of signals produced bypulmonary epithelium and myofibroblasts of the airways that attractimmature DCs from the bloodstream into areas of pulmonary chal-lenge (20). To determine whether any such chemokine or cytokinemediators were differentially expressed in response to M. pneumo-niae infection in the absence of SP-A, BAL fluid was harvested after3 d of infection and analyzed. As shown in Table I, neither MCP-1nor MIP-1a, factors known to be chemotactic for immature DCs(21, 22), was detectable in BAL fluid of saline-treated mice. How-ever, in BAL from M. pneumoniae-infected animals lacking SP-A,MCP-1 was detected at 15.6 pg/ml (not detectable in infected WTmice), and MIP-1a was detected at 21.9 pg/ml (compared with 9.7pg/ml in infected WT mice). The amount of GM-CSF, a factorknown to enhance the differentiation of monocytes into immuno-stimulatory DCs in the lung vasculature, was increased in BAL from

FIGURE 1. Analysis of APCs in the lungs. Mice were instilled with 13108 M. pneumoniae and samples were collected 72 h postinfection. Lungs

were perfused, and cells were harvested from lung tissue after enzymatic

digest and density-gradient centrifugation. Cells were labeled with fluo-

rescent Abs against cell surface markers and analyzed by flow cytometry.

DCs were identified as MHCIIhiCD11c+ cells (A) and macrophages were

identified as MHCIIintCD11c+ cells (B) by flow cytometry. Total numbers

of DCs, macrophages, and CD11b+ macrophages were determined by

multiplying the percentage of each respective cell population by hema-

cytometer counts per sample. Results shown are the mean 6 SEM from

three independent experiments (n = 12/group). ppp , 0.01.

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WT and SP-A–null M. pneumoniae-infected mice compared withthe untreated control sample.

Increased T lymphocytes inM. pneumoniae-infected SP-A–nullmice

Analysis of cell-surface markers from the lymphoid panel revealeda dramatic influx of CD3+ lymphocytes (mean, 1.6 3 106 per totallung cells) into the lung tissue of SP-A2/2 mice after 3 d ofM. pneumoniae infection compared with their respective salinecontrols (p , 0.001) or infected WT mice (p , 0.001) (Fig. 2A).Interestingly, the number of CD3+ lymphocytes present in theM. pneumoniae-infected WT mice at day three (mean = 0.7 3 106)was not significantly elevated over noninfected mice (mean = 0.73106). T lymphocytes were also analyzed 9 d postinfection to de-termine whether the inflammatory response was diminished, main-tained, or augmented over levels detected after 3 d of infection.Despite no significant T cell infiltration in WT mice 3 d post-infection, they did present with significantly enhanced numbers ofCD3+ T lymphocytes in the lungs 9 d postinfection compared withnoninfected controls (Fig. 2A). Additionally, CD3+ T cells detectedin the lungs of SP-A–null mice after 9 d of infection were signifi-cantly increased over the numbers present at 3 d of infection in micelacking SP-A (p, 0.001), as well as over the numbers present in theWT mice after 9 d of infection (p, 0.001). Interestingly, the T cellchemoattractant monokine induce by IFN-g was also significantlyincreased inM. pneumoniae-infected mice lacking SP-A comparedwith infected WT mice (Table I).Those cells expressing CD3 were further analyzed by flow

cytometry for their surface expression of CD4 and CD8. M.pneumoniae-infected SP-A2/2 mice had significantly elevatednumbers of CD3+CD4+ and CD3+CD8+ lymphocytes comparedwithM. pneumoniae-infected WT mice (Fig. 2B). The cell-surfaceexpression of CD25, which plays dual roles in lymphocyte differ-entiation and activation/proliferation, on CD4+ T cells was alsosignificantly greater in the SP-A2/2 M. pneumoniae-infected micecompared with infected control mice (data not shown).To determine the activation status of those T lymphocytes pres-

ent in the lung after 9 d of infection, we analyzed CD69 expression,a very early activation Ag, on CD3+CD4+ and CD3+CD8+ T cells byflow cytometry. CD69 was not upregulated in WT mice 9 d post-infection on CD4+ or CD8+ populations. However, there was sig-nificant expression of CD69 on CD4+ and CD8+ cells in the SP-A–null mice compared with infectedWTmice and saline controls (Fig.2C). In support of these findings, expression of IL-12, which isinvolved in the differentiation of T cells and is associated with T cellactivation, was also significantly increased in the BAL of infectedSP-A mice compared with infected WT mice (Table I).T cells present in the MLNs after 9 d ofM. pneumoniae infection

were also examined. There were no appreciable increases in the

total number of CD4+ or CD8+ T cells recovered in MLNs fromthe infected WT mice. However, in M. pneumoniae-infected SP-A–deficient mice, there were significantly more CD4+ and CD8+

T cells compared with infected WT mice (Fig. 2E). The state ofT cell activation was determined by the presence of CD25 andCD69 on the cell surface. The numbers of CD8+ activated cellswere quite low in all groups of mice examined (Fig. 2F). However,as shown in Fig. 2F, the numbers of CD4+ T cells expressing theseactivation markers were significantly increased in the MLNs ofinfected SP-A–deficient mice compared with infected WT miceand saline control mice.

Increased M. pneumoniae burden does not correlate withincreased T lymphocytes

Mycoplasma burden in SP-A–deficient mice was previously de-scribed in our studies after 3 d of infection, which showed thatalthough there was no difference in M. pneumoniae burden fromBAL fluid at day 3 of infection, significantly more M. pneumoniaewere colonizing the airway by binding to epithelial cells (6). Al-though WT and SP-A–deficient mice were clearing M. pneumo-niae by day 9, interestingly, only 20% of WT mice tested positivefor M. pneumoniae in the BAL compared with .80% of the SP-A–deficient mice (Fig. 3A). Likewise, M. pneumoniae burden inassociation with lung tissue, as determined by RT-PCR, was alsosignificantly increased in SP-A–deficient mice at day 9 of in-fection (Fig. 3B). Although some mycoplasmas are believed to bemitogenic for T cells, we did not find a correlation between M.pneumoniae burden and T cell number in our system (Fig. 3C).

Heightened B cell responses in M. pneumoniae-infectedSP-A–null mice

Cells that expressed the cell surface marker B220 were also sig-nificantly increased in mice lacking SP-A, including the saline-treated and infected mice, at 3 and 9 d postinfection (Fig. 4A).The number of B cells did not increase significantly in WT miceduring the infection. However, B cells in the lungs of SP-A–nullmice were significantly increased over their saline-treated controlsand at each time point examined compared with infected WT con-trols (Fig. 4A). Although there were significantly more B220+ cellsin saline-treated SP-A–null mice, the total number of B220+ cellsconsidered to be activated, as determined by coexpression of IgMand CD69, was equivalent in the saline-treated groups (Fig. 4B).There also was no increase in the total number of activated cells(B220+IgM+CD69+) isolated from M. pneumoniae-infected WTmice after 9 d of infection compared with their saline controls. Instriking contrast, M. pneumoniae-infected SP-A–null mice hadsignificantly greater numbers of activated cells isolated from thelungs after 9 d of infection compared with their saline controls andinfected WT mice at the same day of infection (Fig. 4B).

Table I. Cytokine and chemokine values (pg/ml) detected in BAL fluid 3 d post-M. pneumoniae infection

Cytokine or Chemokine(pg/ml) WT Saline SP-A2/2 Saline WT M. pneumoniae SP-A2/2 M. pneumoniae

IL-5 ND ND ND 221.4 6 29*IL-12 ND ND 43.9 6 7.6 166.2 6 33.4**MCP-1 ND ND ND 15.6 6 6.4*MIG ND ND 1.4 6 0.7 15 6 2.4**MIP-1a ND ND 9.7 6 1.6 21.9 6 2.0**GM-CSF 20 6 4.4 34 6 1.2 120 6 25.4*** 164 6 39.2***

The various cytokines and chemokines were measured in BAL by Luminex multiplex technology or ELISA (n = 8–12/group). Data are combined from three separate experiments.

pp , 0.05, SP-A2/2 M. pneumoniae group versus the lowest detectable value on the curve for WT M. pneumoniae samplesbecause IL-5 and MCP-1 were not detectable; ppp , 0.01, SP-A2/2 M. pneumoniae group versus WT M. pneumoniae group;pppp , 0.05, M. pneumoniae-infected groups versus saline-treated groups.

MIG, monokine induced by IFN-g; ND, not detectable.

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FIGURE 2. Increases in T lymphocytes in the lungs and MLNs ofM. pneumoniae-challenged mice. WTand SP-A2/2mice were instilled with 13 108 M.

pneumoniae, and samples were collected 3 or 9 d postinfection. Lungs were perfused, and cells were harvested from lung tissue or MLNs after enzymatic

digest and density-gradient centrifugation. Cells were labeled with fluorescent Abs against cell surface markers and analyzed by flow cytometry. CD3+ T cells

(A) and CD3+CD4+ and CD3+CD8+ cells (B) detected by flow cytometry from the total lung cell digest at the indicated times. ppp , 0.01 comparison of

infected SP-A deficient mice at both days 3 and 9 versus SP-A deficient saline controls and both day 3 and day 9 infected WT mice; #p, 0.01 comparison of

infected SP-A deficient mice at day 9 versus infected SP-A deficient mice at day 3; ^p, 0.05 coomparison of infected WT mice at day 9 versus infected WT

mice at day 3. C, The activation status of CD3+CD4+ and CD3+CD8+ T cells from the total lung cell digest at day 9, as determined by the presence of CD25

and CD69 by flow cytometry.D, Histological representation taken at310 and340magnification of lymphocytic foci in infected lungs fromWTand SP-A2/2

mice. Circles represent areas of mononuclear inflammation. E, The total number of CD3+CD4+ and CD3+CD8+ T cells harvested from the MLNs after 9 d of

infection. F, The activation status of CD3+CD4+ and CD3+CD8+ T cells from MLN digest, as determined by the presence of CD25 and CD69 by flow

cytometry. Results shown are the mean 6 SEM from three independent experiments (n = 12/group). pp , 0.05; ppp , 0.01.

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B cells were also analyzed from the MLNs of infected mice after9 d of infection. The total numbers of B220+ cells harvested fromthe MLNs were unchanged in infected WT mice compared with

saline-treated WT mice. However, significantly more B220+ cellswere collected from infected SP-A–deficient mice compared withsaline-treated control mice and infected WT mice (Fig. 4C). Ad-ditionally, the number of B220+ activated cells was significantlyincreased in infected WT and SP-A–deficient mice (Fig. 4D).However, the total number of activated B cells was significantlyenhanced in infected SP-A–deficient mice compared with infectedWT mice.Because the M. pneumoniae-infected SP-A–deficient mice dis-

played a strikingly enhanced B cell response compared with in-fected WT mice, we sought to determine whether M. pneumoniae-specific IgG Abs were produced by day 9 of the pulmonary in-fection. Serum was collected from WT and SP-A–null mice thatwere infected with M. pneumoniae, and M. pneumoniae-specificIgG was determined by ELISA. The amount of M. pneumoniae-specific IgG is expressed as the fold increase over the baselinelevels measured from the serum of noninfected mice. As shown inFig. 4E, M. pneumoniae-specific IgG levels were significantlyelevated in the serum of infected SP-A–null mice compared withinfected WT mice.

More mature DCs in MLNs in M. pneumoniae-infected micelacking SP-A

Given that increased numbers of activated CD4+ T cells werediscovered in the MLNs of infected SP-A–deficient mice at thelater time point (day 9), we examined the activation state of DCs inthe MLNs at an earlier time point (day 3), because their early acti-vation state could directly influence the conversion of naive T cellsinto activated T cells at the later time point. WTand SP-A–null micewere instilled with M. pneumoniae or saline, and the MLNs wereharvested after 3 d. Lymph nodes were enzymatically digested to aidin the release of DCs, a single-cell suspension was obtained, andthe cells were stained and analyzed by flow cytometry. There are atleast two populations of CD11c+ cells that can be differentiated in thelymph nodes. The more mature population expressing higher lev-els of MHC class II and CD86 are predicted to be the DCs thathave migrated from the lungs (23, 24).M. pneumoniae-infected SP-A–null mice had a greater percentage ofMHCIIhiCD11c+ cells in theMLNs than infected WT mice (data not shown). The percentageof cells that wereMHCIIhiCD11c+, which was determined by gatingthe flow-cytometry measurements, was used to calculate the totalnumber of MHCIIhiCD11c+ cells based on hemacytometer countsfrom individual lymph node preparations. As shown in Fig. 5A, thetotal number of MHCIIhi CD11c+ cells was also significantly greaterin infected mice lacking SP-A, suggesting that, in the absence ofSP-A, more DCs in the infected pulmonary environment migrateinto the draining lymph nodes. The number of DCs in the MLNsand lungs at day 9 were back to levels observed in saline-treatedanimals; likewise, the lymphocytic inflammation had subsided byday 15 in WT and SP-A2/2 mice (data not shown). Expres-sion of the cell-surface markers associated with DC maturation,CD80 and CD86, were also analyzed. Because the total number ofMHCIIhiCD11c+ cells was higher in the infected SP-A–deficientmice, CD80 and CD86 levels are shown as the fold increase in ex-pression within the DC populations. CD80 and CD86 cell-surfaceexpression was significantly greater on the MHCIIhi CD11c+ cells ofthe SP-A–null mice compared with cells harvested from the infectedWT mice (Fig. 5B).

Inhibition of M. pneumoniae-induced DC maturation bySP-A in vitro

Previous studies from our laboratory demonstrated a role for SP-A ininhibiting LPS-induced DC maturation in vitro (8). Our in vivostudies examining DC maturation in M. pneumoniae–infected SP-

FIGURE 3. M. pneumoniae burden in WT and SP-A2/2 infected mice.

WT and SP-A2/2 mice were infected with 1 3 108 M. pneumoniae in-

tranasally, and theM. pneumoniae burden was assessed after 9 d from BAL

plated on pleuropneumonia-like organism agar (A) or lung tissue by RT-

PCR for the M. pneumoniae specific P1-adhesin gene relative to the

housekeeper gene, cyclophilin (B). Data are from two experiments com-

bined, with n = 8 per group. pp , 0.05; ppp , 0.01. C, WT and SP-A2/2

mice were infected with 13 108 M. pneumoniae intranasally; after 3 d, the

M. pneumoniae burden was determined in the lung tissue by RT-PCR of

the M. pneumoniae specific P1-adhesin gene relative to the housekeeper

gene, cyclophilin, and the number of CD3+ T cells was determined by flow

cytometry using specific cell-surface markers. Data are from two experi-

ments combined and were assessed statistically using Prism software.

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A–null mice compared with control WT mice also supported a rolefor SP-A in limiting DC maturation in response to M. pneumoniae.

To determine whether SP-A directly carries out this protective role

in response to a clinically relevant pulmonary pathogen, DC mat-

uration in response to M. pneumoniae was examined in the pres-

ence or absence of SP-A. BMDCs were plated, and some samples

were preincubated with SP-A, after which liveM. pneumoniaewere

added to the DCs, and M. pneumoniae-induced maturation in the

presence or absence of SP-A was determined after 24 h of culture

by flow cytometry. MHCIIhiCD11c+ cells were analyzed for ex-

pression of the maturation marker CD86.As described previously, addition of SP-A to nonstimulated DCs

leads to decreased expression of maturation markers compared

with media alone. These results were repeated as controls and are

in agreement with published reports (8) (Fig. 5C). Addition of

M. pneumoniae to DCs resulted in increased expression of CD86

compared with those DCs receiving media alone or those pre-

incubated with SP-A alone (no M. pneumoniae stimulation). In-

terestingly, those samples that had been preincubated with SP-A

prior to stimulation with M. pneumoniae were significantly pro-

tected from the upregulation of the maturation marker CD86

compared with the M. pneumoniae-stimulated DCs that were

treated with vehicle only (Fig. 5C).

Elevated HMGB-1 expression in M. pneumoniae-infectedSP-A–deficient mice

HMGB-1, although most commonly associated with necrotic cellsand used as a marker of tissue damage, has more recently beenidentified as a potent proinflammatory cytokine released by activatedmacrophages and monocytes in the lung during acute inflammation(9, 25, 26). Because HMGB-1 cytokine activity was shown to beintegral for DC maturation and is necessary for Ag presentationleading to the activation of T cells (27), we sought to determinewhether levels of HMGB-1were elevated inM. pneumoniae-infectedSP-A–null mice. BAL fluid from uninfected and M. pneumoniae-infected mice was collected, and the presence of HMGB-1 was ex-amined by Western blot analysis. Viability of the cells recovered inBAL was assessed using trypan blue and microscopy; no signifi-cant differences in cell death were noted between groups. Asshown in Fig. 6A, levels of HMGB-1 in BAL of WTand SP-A–nullmice were similar in the uninfected mice. However, levels ofHMGB-1, albeit increased in infected WT mice over uninfectedcontrols, were dramatically increased in infected mice lackingSP-A (Fig. 6A).To determine whether the heightened HMGB-1 secretion and its

potential cytokine activity observed in the absence of SP-A couldbe influencing the maturation of DCs in the M. pneumoniae-

FIGURE 4. Increased B lymphocytes in lungs and MLNs of M. pneumoniae-infected mice. WT and SP-A2/2 mice were instilled with 1 3 108

M. pneumoniae, and samples were collected 3 or 9 d postinfection. Lungs were perfused, and cells were harvested from lung tissue or MLNs after en-

zymatic digest and density-gradient centrifugation. Cells were labeled with fluorescent Abs against cell surface markers and analyzed by flow cytometry.

B220+ cells (A) and B220+IgM+CD69+ cells (B) from the digested lung tissue and B220+ cells (C) and B220+CD69+ cells (D) from the digested MLNs, as

determined by flow cytometry. E, M. pneumoniae-specific IgG, as determined by ELISA from serum of infected mice, shown as the fold increase over the

serum level of noninfected mice. Results shown are the mean 6 SEM from three independent experiments (n = 12/group). pp , 0.05; ppp , 0.01.

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infected mice, we used glycyrrhizin, a direct inhibitor of HMGB-1cytokine activity (28). Cells from the MLNs were collected andanalyzed from mice that were infected with M. pneumoniae andtreated with vehicle (saline) and were compared with those thatwere infected with M. pneumoniae and treated with glycyrrhizin.The total number of cells collected from the MLNs, which wereenriched for DCs by gradient centrifugation, of uninfected WTand SP-A–null mice were not significantly different. However, thetotal number of cells collected from the MLNs of the WT and SP-A–deficientM. pneumoniae-infected mice were elevated over theiruninfected controls (data not shown). When the DCs (MHCIIhi

CD11c+) isolated from the MLNs were analyzed for the presenceof the maturation marker CD86, again we detected significantlymore cells expressing CD86 in the infected mice lacking SP-Athat were treated with vehicle compared with WT mice (Fig. 6B).In contrast, SP-A–null mice that were treated with the HMGB-1inhibitor, glycyrrhizin, had significantly fewer DCs expressingCD86 compared with those receiving vehicle (Fig. 6B), indicatingthat SP-A can inhibit M. pneumoniae-induced DC maturation bymodulating HMGB-1 cytokine activities.

SP-A regulates HMGB-1 expression from M. pneumoniae-stimulated human cells

To determine whether exogenously added SP-A inhibits the releaseof HMGB-1 from M. pneumoniae-activated cells into the culturesupernatant, additional experiments were carried out in vitro usinga human acute monocytic cell line (THP-1) as well as NHBEs.The levels of HMGB-1 in the supernatant from THP-1 cells treatedwith saline or SP-A alone were below the level of detection byWestern blot analysis. However, when cells were infected with 10M. pneumoniae CFU per cell, secreted HMGB-1 was readily de-tected in the supernatants after 16 h of stimulation (Fig. 7A). In-cubation of the cells prior to infection with exogenous human SP-A(50 mg/ml) inhibited secretion of HMGB-1 into the culture su-pernatant. Importantly, cellular viability (.85%) was not signifi-cantly altered in stimulated conditions, as determined by trypanblue exclusion and by lactate dehydrogenase assays (SupplementalFig. 1).Although monocytes are the most likely source of the secreted

HMGB-1 present in the inflamed pulmonary environment, humanepithelial cells might also produce HMGB-1 upon stimulation.

FIGURE 5. DCs from MLNs of M. pneumoniae-infected mice 3 d post-

infection. A, Total number of MHCIIhiCD11c+ DCs present in the MLNs of

uninfected and M. pneumoniae-infected mice on day 3 of infection (n = 8/

group). Data are representative of three experiments.B, The fold expression,

relative to M. pneumoniae-infected WT mice, of the maturation markers

CD80 and CD86 on the MHCIIhiCD11c+ DCs (n = 15–17/group). Data are

combined from four experiments. C, BMDCs were cultured for 6 d in the

presence of GM-CSF, purified, and stimulated withM. pneumoniae for 16 h

in the presence or absence of SP-A (40 mg/ml). The percentage of DCs un-

dergoing maturation was assessed by flow cytometry for expression of

MHC II, CD11c, and CD86 (n = 3 experiments). pp, 0.05; ppp, 0.01.

A

B

FIGURE 6. Inhibition of HMGB-1 cytokine activity limits DC matura-

tion in response to M. pneumoniae. A, Western blot analysis of cell-free

BAL harvested 3 d afterM. pneumoniae infection of WTand SP-A2/2 mice

for HMGB-1 expression and compared with HMGB-1 levels measured in

saline-treated mice. The same total volume was loaded for each sample.

Control (C) is brain lysate. Representative of three separate experiments.

B, WT and SP-A2/2 mice were treated with glycyrrhizin (10 mg/kg body

weight) or vehicle (saline) prior to and during infection withM. pneumoniae.

Threedayspostinfection,MLNswereharvested, and cellswere collected after

enzymatic digestion and density-gradient centrifugation to enrich forDCs and

labeled with fluorescent Abs for flow-cytometry analysis. The number of

MHCIIhi CD11c+ DCs from the MLNs that were CD86+ are shown. WTand

SP-A2/2 uninfected (U) mice were included as baseline controls (n = 10–12/

group). Data are from three combined experiments. pp, 0.05; ppp, 0.01.

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Therefore,we also examined the ability ofSP-A to regulateHMGB-1release from M. pneumoniae-stimulated NHBEs. Similar to obser-vations with the THP-1 cells, levels of HMGB-1 were also un-detectable in samples with the addition of saline or SP-A alone.However, stimulation of cells with 10M. pneumoniae CFU per cellalso induced HMGB-1 secretion from NHBEs, whereas the additionof exogenous SP-A attenuated this response in M. pneumoniae-stimulated samples (Fig. 7B).

SP-A binding toM. pneumoniae limits HMGB-1 secretion fromDCs

Although SP-A could be acting directly on DCs to inhibit M.pneumoniae-induced maturation, it could also be functioning bybinding to M. pneumoniae, which is known to occur through abinding protein (MPN372) and surface lipids (4, 5), and, thereby,protecting the cells from interactions with M. pneumoniae. SP-Ainhibited DC maturation when immature (Fig. 8A) or mature (Fig.8B) DCs were preincubated with human SP-A. To determinewhich aspect of these DC–SP-A–M. pneumoniae interactions waskey to limiting DC maturation, we preincubated DCs with SP-Aand washed away unbound SP-A prior to the addition of M.pneumoniae. In parallel, we precoated M. pneumoniae with SP-Aand washed away unbound SP-A prior to adding it to DCs. In-terestingly, preincubation of DCs with SP-A prior to stimulationdid not lead to reduced levels of HMGB-1 secretion, in fact,HMGB-1 levels were still quite high (Fig. 8C). However, in theparallel experiment, M. pneumoniae that had been precoatedwith SP-A was not able to elicit HMGB-1 secretion from DCsin contrast with M. pneumoniae that was not coated with SP-A(Fig. 8C).Because M. pneumoniae is known to work almost exclusively

through TLR2, additional experiments were conducted to de-terminewhether this receptor was a key receptor inM. pneumoniae-induced HMGB-1 secretion from these cells. ATLR2-neutralizingAb or an isotype control was incubated with DCs prior to stim-ulation with M. pneumoniae. Interestingly, inhibition of TLR2 re-

sulted in no detectable HMGB-1 secretion upon M. pneumoniaestimulation (Fig. 8D).

DiscussionOur findings describe a novel role for SP-A in limiting M. pneu-moniae -induced DC maturation via inhibition of HMGB-1 cyto-kine activity. The absence of SP-A in mice during M. pneumoniaeinfection leads to increased numbers, as well as the activation state,of APCs in the lung and draining lymph nodes during the acutephase of infection and, consequently, increased numbers of acti-vated T and B cells later during the course of infection. Thesefindings are consistent with reports that describe SP-A as a vitalcomponent of the pulmonary innate immune system that limits in-flammation and inhibits LPS-induced DC maturation in vitro. Incontrast, studies have only recently begun to focus on the extra-cellular cytokine activity of HMGB-1 as an important mediator ofinflammation that is actively secreted from stimulated myeloidcells and induces DC activation. Although no association between

FIGURE 7. SP-A inhibition of HMGB-1 secretion from human cells.

THP-1 cells (A) or NHBEs (B) were incubated with 50 mg/ml purified

human SP-A (+) or an equivalent volume of sterile Tris buffer (2) for 2 h

prior to stimulation with M. pneumoniae 10 CFU/cell or with saline only

(0). Cell-free supernatants and lysates were harvested 16 h poststimulation,

and HMGB-1 and GAPDH expression was assessed by Western blot

analysis. Figure is representative of two experiments.

FIGURE 8. SP-A interaction with M. pneumoniae-stimulated DCs.

BMDCs were cultured for 6 d in the presence of GM-CSF, purified, and

stimulated with M. pneumoniae (10 CFU/cell) for 16 h in the presence or

absence of SP-A (50 mg/ml). Supernatants and lysates were harvested for

analysis by SDS-PAGE andWestern immunoblotting forHMGB-1, SP-A, or

GAPDH. Immature DCs were used immediately after purification on day 6

(A) ormatureDCswere stimulated for 24 h in the presence of LPS (B) prior to

studies withM. pneumoniae.C, DCs were treated for 16 hwith nothing (lane

1); preincubatedwith SP-A,washed, and then stimulatedwithmedia (lane 2)

orM. pneumoniae (lane 3); or stimulated with SP-A–coatedM. pneumoniae

(lane 4) or noncoated M. pneumoniae (lane 5). D, DCs were preincubated

with a TLR-2–neutralizing Ab or isotype-control Ab prior to stimulation

with M. pneumoniae for 16 h. Blots are representative of experiments con-

ducted with three independent BMDC cultures.

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HMGB-1 and SP-A has been described, several phenotypicparameters measured in M. pneumoniae-infected SP-A–null micesuggested that SP-A may play a role in regulating HMGB-1 extra-cellular cytokine activity. Using a specific inhibitor of HMGB-1cytokine activity (glycyrrhizin), our research describes a role forSP-A in regulating HMGB-1 activity during M. pneumoniae pul-monary infection.M. pneumoniae infection of mice lacking SP-A induced a dra-

matic and significant increase in the total number of CD3+ T cellspresent in the lung and MLNs at the early time point (3 d) and thelater time point (9 d) compared with infected WT mice. M. pneu-moniae likely is not acting as a mitogen in this response because thelevels ofM. pneumoniae detected at 9 d of infection is significantlydecreased in the lungs of WT and SP-A–null mice compared withthe burden at 3 d, whereas the numbers of T cells are continuing toincrease (data not shown). Cell-surface markers on T cells indica-tive of activation were also significantly increased in the lung andMLNs of infected mice lacking SP-A. Additionally, compared withinfected WT mice, SP-A–null M. pneumoniae–infected mice had∼4-foldmoreBAL IL-12,which is involved in T cell differentiation.Although one of the many known roles of SP-A is to inhibit

T cell proliferation and attenuate the initial Ca2+ spike (29), anadditional likely explanation for the increase in activated T cells ininfected SP-A–null mice can be ascribed to the role of SP-A in DCmaturation. Previous work from our laboratory determined thatSP-A inhibits basal and LPS-induced DC maturation in vitro.Therefore, it was important to determine whether this role would becarried out in vivo in response to a respiratory pathogen, such asM.pneumoniae. More DCs were found in the lung of M. pneumoniae-infected mice that lacked SP-A, and more functionally mature DCswere detected in the MLNs, where they would act as potent T cellstimulators. Additionally, MCP and MIP-1, factors known to bechemotactic for immature DCs (21, 22), were present in BAL fluidof infected mice at much higher levels in mice lacking SP-A,suggesting that more DCs migrated into the lung from the blood-stream of M. pneumoniae-infected mice in the absence of SP-A asthe result of greater chemokine production. The amount of GM-CSF, a factor vital to enhancing the differentiation of monocytesinto immunostimulatory DCs in the lung vasculature, was signifi-cantly increased in BAL from WT and SP-A–null M. pneumoniae-infected mice. Collectively, these findings suggest that, in the ab-sence of SP-A, more DCs infiltrate the airways as the result ofincreases in chemoattractant signals; however, because GM-CSF isincreased comparably in infected WT and SP-A–null mice, it is notlikely that the increase in GM-CSF accounts for the difference inDC maturation observed.Because differential increases in GM-CSF in SP-A–null mice

were not observed, we investigated the possibility that other medi-ators may be responsible for the SP-A–mediated inhibition of DCmaturation. Recent studies suggested that endogenous stress factors,such asHMGB-1, released during infection can aid inmaturingDCsthat will then further contribute to the initiation and maintenance ofan immune response against an invading pathogen (9). We thoughtthat HMGB-1 was a good candidate for mediating the SP-A–dependent modulation of DCs and T cells because HMGB-1 can beactively secreted from activated monocytes and macrophages, isactively involved in regulating the maturation of DCs (30), and isthought to be necessary for the proliferation and polarization ofnaiveCD4+ T cells (27). Indeed, we found that the level of HMGB-1was dramatically increased in the BAL ofM. pneumoniae-infectedmice when SP-Awas absent. There was no indication of increasedcell death in thesemice, as determined by cell staining from theBALand lung tissue, suggesting that increased HMGB-1 was releasedfrom activated, but not necrotic, airwaymonocytes ormacrophages.

To determine whether the increased HMGB-1 present in thelungs after M. pneumoniae infection was responsible for the in-creased DCmaturation observed in the absence of SP-A, we used aninhibitor of HMGB-1 cytokine activity, glycyrrhizin. Glycyrrhizin,a product produced by the licorice plant, has been shown to bindHMGB-1 directly and block its extracellular functions (28, 31).Taking advantage of the ability of glycyrrhizin to functionally inhibitHMGB-1 cytokine activity, we were able to examine the maturationstate of those DCs that had migrated to the MLNs followingM. pneumoniae infection and determinewhether HMGB-1was a keymediator in this process. In mice lacking SP-A, more mature DCswere again observed in the M. pneumoniae-infected vehicle-treatedmice compared with infected vehicle-treated infected WT mice.However, inmice lacking SP-A treatedwith glycyrrhizin, the level ofDC maturation of migrated cells was significantly reduced to thoselevelsmeasured in the infectedWTmice. These findings suggest thatSP-A inhibits DC maturation in vivo in response toM. pneumoniaeinfection, at least in part by limiting HMGB-1 extracellular cytokineactivity, which can directly influence initiation of an adaptive im-mune response.Although we used cell-surface markers most commonly used to

distinguish macrophages from DCs, we acknowledge that pheno-typic distinction of these two populations of APCs is increasinglynebulous. Although the total numbers of phenotypically activatedDCs were significantly increased in M. pneumoniae-infected SP-A–nullmice, the percentage of activatedT lymphocyteswas similar.This profile suggests that the increase in APCs may be due toincreases in the pool of activated macrophages rather than differ-entiated DCs, the latter of which should induce activation of T cells.Therefore, more detailed studies were conducted using a purifiedpopulation of BMDCs in which we found that exogenous SP-Aadded at physiologic levels could inhibit DC maturation inducedby M. pneumoniae stimulation. However, functional assays exam-ining Ag-specific lymphocyte proliferation should be conductedto strengthen our understanding of the inhibitory role of SP-A inDCdifferentiation, independent of any macrophage participation.Additional experiments were carried out in vitro using a human

monocyte cell line (THP-1) and human bronchial epithelial cells todetermine whether exogenously added SP-A inhibits the release ofHMGB-1 from M. pneumoniae-activated cells into the culture su-pernatant. The levels of HMBG-1 in the supernatant from THP-1cells treated with saline or SP-A alone were below the level of de-tection byWestern blot analysis. However, when cells were infectedwith 10 M. pneumoniae CFU per cell, secreted HMGB-1 was de-tected in the supernatants, as was described for human monocytes(32). Incubation of the cells prior to infection with exogenous hu-manSP-A inhibited secretion ofHMGB-1 into the supernatant in thehuman monocyte cells and bronchial epithelial cells. Importantly,cellular viability and membrane permeability were not significantlyaltered in stimulated conditions, indicating that the increasedHMGB-1 was not from increased cell death or membrane leakagewithin those samples.The amount of HMGB-1 detected in BAL from infected SP-A–

null mice is increased compared with infected WT mice, whichsuggests that SP-A may regulate the amount of HMGB-1 secretedfrom the activated cells. Although it is possible that SP-A directlybinds extracellular HMGB-1, coimmunoprecipitation experimentswith BAL fluid from infected WT mice showed no detectable bind-ing, whereas HMGB-1 and SP-A were readily observed. Furtherstudies determined that the interaction and binding of SP-A toM. pneumoniae were vital components in limiting M. pneumoniae-induced HMGB-1 secretion from cultured DCs. Additionally, whena neutralizing Ab was used to inhibit TLR2, M. pneumoniae wasunable to elicit HMGB-1 secretion from these cells. Taken together,

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these findings suggest that the binding of SP-A toM. pneumoniae iscritical in curtailing HMGB-1 secretion from DCs by restricting theinteraction ofM. pneumoniae with its primary receptor, TLR2.M. pneumoniae is known to colonize the respiratory tract where

it initiates a cascade of immune-response amplification, includingproliferation of lymphocytes, proinflammatory cytokine release, andproduction of Igs. Several studies reported increased IgG serumlevels inM. pneumoniae-infected individuals (33, 34). Our findingsshowed that, in the absence of SP-A, more M. pneumoniae-specificIgG was present in the serum of infected mice compared withinfected WT mice. The production of IgG Abs is predominantlyassociated with the secondary immune response. This finding furthersupports an indirect role for SP-A in limiting the advancement of anadaptive immune response by regulating the initiation of the Abresponse toM. pneumoniae that could be a direct result of increasedDC maturation and migration in the innate phase of the response.In summary, our findings support an inhibitory role for SP-A

inM. pneumoniae-inducedDCmaturation, which is a key step in theinitiation of an immune response. Additionally, our studies showedthat one mechanism by which SP-A inhibits M. pneumoniae-induced DC maturation is by regulating HMGB-1 secretion in vivoand in vitro in human cells. Previous studies showed that the re-ceptor for advanced glycation end products (RAGE) and HMGB-1are required for maturation of human DCs (30). Because SP-Ainhibits DC maturation in the presence and absence of stimulation,further experiments testing whether SP-A interferes with HMGB-1binding via RAGE and limiting DC maturation through this in-teraction may be of interest. These findings might be of value indesigning therapies, because HMGB1, as well as other proin-flammatory ligands, and RAGE are present in myriad acute andchronic inflammatory diseases, such as sepsis, diabetes, athero-sclerosis, and renal failure (35). Additionally, chronic inflammationand inflammatory diseases in the lung often result in epithelialdamage and airway remodeling, therefore, blocking HMGB-1 bypharmacological interventions or potentially with surfactant treat-ment may alleviate lung damage.

AcknowledgmentsWe thank Charles Giamberardino for technical assistance, Pamela Hesker

for HMGB-1 Ab advice, and Sambuddho Mukherjee and Amy Pastva for

helpful discussions.

DisclosuresThe authors have no financial conflicts of interest.

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