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2008;180;6317-6324 J. Immunol.

Zhou, John R. Ledford and Marsha Wills-KarpKristen Page, Kristin M. Lierl, Valerie S. Hughes, Ping

Cockroach FrassNeutrophils in Response to GermanTLR2-Mediated Activation of

http://www.jimmunol.org/cgi/content/full/180/9/6317

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TLR2-Mediated Activation of Neutrophils in Response toGerman Cockroach Frass 1

Kristen Page,2

*

Kristin M. Lierl,* Valerie S. Hughes,* Ping Zhou,* John R. Ledford,*and Marsha Wills-Karp

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthmapathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether Germancockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intra-tracheal inhalation of GC frass. Proinammatory cytokines were signicantly increased in the bronchoalveolar lavage uid at 3 hand were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as earlyas 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2or TLR4. Newly inltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Usingneutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airwaysfollowing GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production viaTLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-decient mice. Activation of cytokine expression occurred via GC frass-induced NF- B translocation and DNA binding. These data show that GC frasscontains a TLR2 agonist and, to our knowledge, this is the rst report of an allergen directly activating cells of the innate immunesystem via TLR2 and suggests an important link between innate and adaptive immunity. The Journal of Immunology, 2008, 180:63176324.

T he incidence and severity of asthma has risen dramaticallyover the past two decades. There is growing evidence thatthe indoor environment may play an important role in thepathogenesis of childhood asthma. Cockroach, house dust mite,and cat exposure are the most commonly associated with asthmaexacerbations (1). Cockroach allergens are important sensitizingagents, particularly in individuals who live in substandard, multi-family dwellings located in highly populated areas (2). A few stud-ies have suggested that sensitization to cockroach might be moreimportant than other allergens. Children who were both allergic tocockroach allergen and exposed to high levels of this allergen hadgreater hospitalization rates, unscheduled medical visits, and moredays of wheezing than children allergic to and exposed to dustmites or cat dander (3). A prospective birth cohort study of chil-dren of asthmatic/allergic parents showed that the level of cock-roach allergen, but not that of dust mite or cat allergen, was asignicant predictor of recurrent wheeze (4). Fecal remnants(frass) are a likely source of allergen exposure, because desiccatedfrass is thought to integrate into dust particles and become depos-ited into the airways (5). These studies highlight the importance of

cockroach in the asthma phenotype and substantiate the use of German cockroach (GC) 3 frass in the current study.

Although the specic cytokines that induce Th1 vs Th2 re-sponses are well described, the early innate response to allergen isnot fully understood. An early and transient inltration of neutro-phils into the airways was shown following allergen challenge inhumans and following OVA challenge in mice (6). In a model of OVA-sensitized mice, allergen challenge resulted in increased air-way neutrophilia at 8 h with resolution at 48 h. The initial neu-trophilia was followed later by an inux of eosinophils and lym-phocytes and much later by airway hyperresponsiveness (7). Thefactor(s) regulating transient neutrophil inux into the airways arenot well dened. Using the neutrophil as an early marker of innateimmune responses, we asked whether GC frass may have a directeffect on innate immunity.

The innate immune response has evolved to recognize patho-gen-associated molecular patterns (PAMPs), which are common tomany classes of pathogens. PAMPs are recognized by pathogenrecognition receptors, which include TLRs. TLRs recognize con-served microbial molecular signatures. For example, TLR2 recog-nizes peptidoglycan and lipoteichoic acid from Gram-positive bac-

teria, TLR4 recognizes Gram-negative bacterial LPS, TLR5recognizes agella, and TLR9 recognizes unmethylated DNA.TLRs are expressed on most cell types; however, their expressiondoes not guarantee recognition of the pathogen because corecep-tors and adaptor molecules may also be required. It has recentlybeen shown that polymorphisms in TLR2 were a major determi-nant of allergy and asthma susceptibility in children of European

*Division of Critical Care Medicine and Division of Immunobiology, CincinnatiChildrens Hospital Medical Center and Cincinnati Childrens Research Foundationand Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45229

Received for publication October 17, 2007. Accepted for publication February26, 2008.

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 the National Institutes of Health Grant HL075568 (toK.P.) and HL67736 (to M.W.-K.).2 Address correspondence and reprint requests to Dr. Kristen Page, CincinnatiChildrens Hospital Medical Center, 3333 Burnet Avenue, ML 7006, Cincinnati,OH 45229. E-mail address: [email protected]

3 Abbreviations used in this paper: GC, German cockroach; BAL, bronchoalveolarlavage; MPO, myeloperoxidase; Pam 3 CSK 4 , (S)-[2,3-bis(palmitoyloxy)-(2- RS)-pro-pyl]- N -palmitoyl-( R)-Cys-( S)-Ser-( S)-Lys 4 -OH, 3HCl; PAMP, pathogen-associatedmolecular pattern.

Copyright 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

The Journal of Immunology

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farmers (8). A few studies have examined the role of TLR2 onexperimentally induced murine models of asthma by sensitizingmice with a TLR2 agonist (911). The results of these studies werecontradictory. In this study we investigated whether GC frasscould directly induce early innate immune responses via TLR-dependent activation in early responding immune cells. To ourknowledge, an allergen has never been shown to activate TLRsignaling.

Materials and MethodsCockroach frass

The frass from one cage of GCs was transferred to a sterile container andstored at 4 o C. Frass was resuspended in endotoxin-free, double-distilledwater (2 h at 4C while rocking) and frozen in aliquots for use throughoutthe entire study. Extracts were centrifuged to remove debris (13,000 gfor 5 min at 4C), supernatants were harvested, and total protein was mea-sured using the Bio-Rad protein assay dye (Bio-Rad). Endotoxin levelswere determined by Charles River Laboratories using the Limulus amebo-cyte lysate assay.

Animals

Six-week-old female BALB/c, C57BL/6, C3H/HeOuJ (control), and C3H/ HeJ (spontaneous mutation in TLR4) mice were obtained from The Jack-

son Laboratory and housed in a laminar hood in a virus-free animal facility.TLR2-decient mice were obtained from Dr. S. Akira (12). In some ex-periments, mice were injected i.p. with the anti-granulocyte mAb RB5-8C5(also referred to as Ly6g; BD Pharmingen) at a concentration of 100 g/ mouse (13) 24 h before inhalation. In all experiments, mice were anesthe-tized with ketamine (45 mg/kg)/xylazine (8 mg/kg) before PBS or GC frass(40 g/40 l) exposure by a single inhalation as previously described (14).Mice were given a lethal dose of sodium pentobarbital 324 h later (in moststudies after 18 h). Animal care was provided in accordance with NationalInstitutes of Health guidelines. These studies were approved by the Cin-cinnati Childrens Hospital Medical Center Institutional Animal Care andUse Committee (Cincinnati, OH).

Assessment of airway inammation

Lungs were lavaged thoroughly with 1 ml of HBSS without calcium ormagnesium. The lavage uid was centrifuged (1,800 rpm for 10 min) and

the supernatant was removed for cytokine analysis and immediately storedat 80C. Total cell numbers were counted on a hemocytometer. Smearsof bronchoalveolar lavage (BAL) cells prepared with a Cytospin II centri-fuge (Shandon Thermo) were stained with Diff-Quick (Thermo Electron)solution for differential cell counting.

Myeloperoxidase (MPO) assay

Whole lungs were snap frozen in liquid nitrogen and homogenized in asolution containing 0.5% hexadecyltrimethylammonium bromide dis-solved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30min at 20000 g at 4C. An aliquot of the supernatant was allowed toreact with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H 2 O2 .The rate of change in absorbance was measured by spectrophotometry at650 nm. MPO activity was dened as the quantity of enzyme degrading 1

mol of hydrogen peroxide per minute at 37C and expressed in units per100-mg weight of tissue (15).

Cell culture

HL-60 promyelocytic leukemia cells (American Type Culture Collection)were cultured in RPMI 1640 medium supplemented with 10% FBS, 50

g/ml streptomycin, 2 U/ml penicillin, and 2 mM L-glutamine. For differ-entiation, cells (1 106 /ml were incubated in the presence of 1% DMSOfor 4 days. Cells were centrifuged, washed, and deprived of serum for 6 h.In some cases cells were pretreated with isohelenin (30 M for 1 h; EMDBiosciences) or neutralizing Abs against TLR2, TLR4, or isotype control(10 g/ml; eBioscience) before the addition of GC frass (100 ng/ml) for16 h. Selected cells were also treated with LPS from Escherichia coli(O111:B4; Sigma-Aldrich) that had been puried by ion exchange chro-matography or with 1 g/ml (S)-[2,3-bis(palmitoyloxy)-(2- RS)-propyl]- N -palmitoyl-( R)-Cys-( S)-Ser-( S)-Lys 4 -OH, 3HCl (Pam 3 CSK 4 ; Calbiochem).

Primary human neutrophil isolation

Polymorphonuclear leukocytes were isolated from blood collected fromhealthy volunteers. Blood was drawn into heparin and then subjected to

dextran T500 sedimentation, Ficoll-Histopaque density gradient centrifu-gation, and hypotonic erythrocyte lysis (16). All steps during the neutro-phil isolation were maintained constant and all assays were performedimmediately after isolation was complete. Polymorphonuclear cellswere resuspended in serum-deprived medium before treatment with GCfrass (10100 ng/ml) for 4 h (for RNA isolation and real-time PCRexperiments) or 18 h (for cytokine ELISA experiments) with continualrocking.

Primary mouse bone marrow-derived neutrophils

Femurs and tibias were removed from C57BL/6 or TLR2-decient mice.Bone marrow was isolated and rinsed and RBCs were lysed. Resuspendedcells were layered onto a three-step Percoll gradient (52, 64, and 72%) andcentrifuged (1,000 rpm for 30 min at room temperature). Neutrophils con-tained in the bottom layer (6472%) were collected, counted, and plated.

Isolation of neutrophils from mouse BAL uid

Naive mice were administered a single intratracheal inhalation of GCfrass, and BAL uid was harvested 18 h later. Neutrophils were mag-netically labeled with an anti-Ly-6G microbead kit (Miltenyi Biotech).Neutrophils were isolated by placing the magnetically labeled sampleon a column placed in the magnetic eld of a MACS separator. Mag-netically labeled Ly-6G cells were retained on the column while non-labeled cells were run through. After removal of the column from themagnetic eld, the neutrophils were counted and either stained for owcytometry or placed in culture for determination of cytokine expression.Over 90% of the cells were neutrophils as determined by differentialcell counting using Diff-Quick solution (Thermo Electron).

Flow cytometry

Neutrophils (1 106 ) isolated from the BAL uid of mice were washedand incubated with PE-labeled isotype control or PE-labeled anti-mouseTLR2 Abs (eBioscience) for 1 h on ice. Cells were washed to removeunbound Ab and xed with 1% paraformaldehyde (EM Sciences). Sampleswere analyzed on the FACSCalibur ow cytometer using the CellQuest Prosoftware for analysis.

ELISA

Cell supernatants were collected and claried (13,000 rpm for 10 min at4C) before being analyzed for IL-8, IL-6, or TNF- by ELISA accordingto the manufacturers specications (Amersham Biosciences).

Immunoblot analysis

Differentiated HL-60 cells were cultured in 6-well plates and serum-starvedfor 24 h before treatment. Selected wells were treated with frass, and celllysates were harvested and resolved electrophoresis on a 10% SDS- poly-acrylamide gel as previously described (17). After incubation with an anti-I B (Santa Cruz Biotechnology), signals were amplied and visualizedusing ECL.

EMSA

Differentiated HL-60 cells were treated with GC frass (100 ng/ml) for 1 h.Cells were harvested and nuclear proteins were isolated as previously de-

scribed (18). All nuclear extraction procedures were performed on ice withice-cold reagents. Protein concentrations were determined by Bradford as-say (Bio-Rad) and stored at 70C until use.

The probe was labeled with [ -32 P]ATP using T4 polynucleotide kinase(Invitrogen) and puried in MicroBiospin chromatography column. Thegel was run using 10 g of nuclear protein as previously described (18). Insome instances, Abs against p65 (Rel A) or p50 (NF- B1; Santa CruzBiotechnology) were added (10 min at room temperature). An oligonucle-otide probe encoding the consensus sequence of NF- B was purchasedfrom Santa Cruz Biotechnology. Cold-specic or nonspecic probes wereadded at 5 the concentration of the radiolabeled probe. Gels were trans-ferred to Whatman 3M paper, dried under a vacuum at 80C for 1 h, andexposed using a PhosphorImager (GE Healthcare).

Statistical analysis

When applicable, statistical signicance was assessed by one-wayANOVA. Differences identied by ANOVA were pinpointed by the Stu-dent-Newman-Keuls multiple range test.

6318 TLR2-DEPENDENT ACTIVATION OF NEUTROPHILS


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Results A single intratracheal inhalation of GC frass increased cytokineexpression and neutrophilia in BALB/c mice

Naive BALB/c mice were given a single intratracheal inhalation of PBS or GC frass (40 g) and killed 3, 6, 18, or 24 h later. Within3 h there was a statistically signicant increase in the level of KCin the BAL uid of GC frass-treated mice compared with PBS-treated mice (Fig. 1 A). The levels of KC began to decrease at 6 hbut were still signicantly higher at 18 h compared with levels inthe PBS control. TNF- levels were maximal between 3 and 6 h,after which time the levels began to decrease but were still signif-icantly higher than in the PBS controls (Fig. 1 B). IL-6 was alsosignicantly increased at 3, 6, and 18 h following inhalation of GCfrass compared with PBS (data not shown). The increase in cyto-kines preceded the increase in neutrophilia, which was small butstatistically signicant at 3 h postinhalation of GC frass, reachedmaximal levels around 6 h, and remained stable for 24 h (Fig. 1 C ).At the 18-h time point MPO levels in whole lung were signi-

cantly increased in the GC frass-treated mice compared with PBS-treated mice (12.9 1.9 U per 100 mg of tissue for PBS-treatedmice compared with 119.6 4.6 U per 100 mg of tissue for frass-treated mice).

The initial increase in cytokine production is not mediated byTLR2 or TLR4

Because innate immune responses are thought to be regulated byTLRs, we asked whether TLR2 or TLR4 played a role in the initialcytokine release into the airways 3 h postinhalation. Although thetrend toward a decrease was detected, there was no signicantdifference between neutrophil inltration (Fig. 2 A) or the levels of KC and IL-6 in the airways of wild-type or TLR2- or TLR4-de-cient mice (Fig. 2, B and C ). Because GC frass contains endo-toxin (923 ng/mg frass; a 40- g inhalation of GC frass contained

3 hr 6 hr 18 hr 24 hr

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37 ng of endotoxin), these data also suggest that the endotoxin

alone in GC frass is not responsible for the activation of cytokines.In addition, these data suggest that GC frass-mediated early releaseof cytokines occurred independently of TLR2 or TLR4.

Maintenance of cytokine levels in the airways is dependent onneutrophil recruitment

We hypothesized that neutrophils played a role in maintaining thecytokine levels in the BAL uid of mice. To investigate this, wepretreated mice with R6B-8C5 (an Ab that depletes circulatingneutrophils) 24 h before a single intratracheal challenge. We mea-sured cytokine levels 18 h following PBS or GC frass treatment,because this time point correlated with high levels of BAL uidneutrophilia. Pretreatment with the R6B-8C5 Ab totally abolishedneutrophil recruitment into the BAL uid as expected (Fig. 3 A).Importantly, the increase in KC and TNF- expression followingGC frass inhalation was completely abolished in the RB68C5-

pretreated mice compared with mice pretreated with isotype con-trol Ab (Fig. 3, B and C ). Of note, we also measured cytokineproduction 3 h following PBS or GC frass treatment and found thatalthough R6B-8C5 abolished neutrophil inltration, cytokine ex-pression in the BAL uid was only slightly reduced (data notshown). These data suggest that there is a population of cells thatsecrete cytokines for the immediate attraction of neutrophils and,once neutrophils inltrate into the airways, they play a signicantrole in regulating cytokine expression.

TLR2, but not TLR4, regulated GC frass-induced cytokinemaintenance

We next asked whether TLR2 or TLR4 played a role in GC frass-induced cytokine release into the airways 18 h postinhalation. Tothis end, we treated naive wild-type (C3H/HeOuJ or C57BL/6),

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FIGURE 3. Neutrophil recruitment directly affected cytokine produc-tion. BALB/c mice were given a single injection of control Ab or RB5-8C5(100 g/mouse) 24 h before a single intratracheal inhalation of PBS (40 l)or GC frass (40 g/40 l). Eighteen hours following the challenge withPBS or GC frass, mice were given a lethal dose of sodium pentobarbitaland BAL uid was harvested and claried and neutrophils were quantiedfollowing differential staining. In all cases, means SEM (n 57 miceper group) were reported. Statistical signicance of the frass-treated micecompared with PBS-treated mice was determined by ANOVA (nd, nonedetected). A, Neutrophil inux into BAL uid ( , p 0.001). B, KCELISA of BAL uid ( , p 0.001). C , TNF- ELISA of BAL uid ( , p0.001).

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TLR4-decient (C3H/HeJ), or TLR2-decient (TLR2 / ) micewith a single inhalation of PBS or GC frass and measured cytokineproduction and cellular inltration into the airways 18 h later. Neu-trophil inltration into the airways was unaffected following GCfrass treatment in TLR4- and TLR2-decient mice compared withtheir wild-type controls (Fig. 4 A). Interestingly, cytokine expres-sion was signicantly attenuated in the TLR2-decient mice com-pared with the TLR4-decient mice (Fig. 4, B and C ). These datasuggest that although TLR2 is not required for neutrophil recruit-ment into the airways, it is necessary for neutrophil-derived cyto-kine expression. This led us to hypothesize that GC frass directlyaffected neutrophil function via TLR2.

Mouse neutrophils express TLR2 and secrete cytokines

Naive BALB/c mice were administered a single intratracheal in-halation of GC frass and BAL uid was recovered 18 h later.Neutrophils were magnetically labeled with anti-Ly-6G mi-crobeads and isolated using a magnetic column. Ninety-ve per-cent of the newly inltrated neutrophils express TLR2 on their cellsurface as determined by ow cytometry (Fig. 5) and secreteTNF- (300.6 36 pg/ml 106 cells) and KC (43 2 pg/ml106 cells). These data demonstrate that neutrophils recruited intothe airways following GC frass inhalation express TLR2 and aresecreting cytokines.

GC frass increased IL-8 and TNF- expression in primaryhuman neutrophils and differentiated HL-60 cells

To address whether GC frass would directly affect neutrophil func-tion, we used primary human neutrophils from normal healthy do-nors. We conrmed that 98% of the cells isolated were neutro-phils. Incubation of isolated neutrophils with GC frass resulted ina dose-dependent increase in IL-8 and TNF- protein expression(Fig. 6, A and B). GC frass treatment increased IL-8 and TNF-mRNA levels in primary human neutrophils (4.3- and 11.6-fold,respectively when cells were treated with 100 ng/ml GC frass for4 h), suggesting transcriptional up-regulation. Incubation of cellswith 100 ng/ml frass resulted in the addition of 92 pg/ml endo-toxin. However, treatment of the cells with 100 pg/ml column-puried endotoxin did not increase IL-8 expression, nor did poly-myxin B have an effect on GC frass-induced IL-8 proteinexpression (Fig. 6 C ). We acknowledge that the addition of puried

endotoxin from E. coli to cells should be interpreted with caution,as this may not represent the same source of endotoxin or thecomplexity of components in GC frass (i.e., TLR4 adaptor mole-cules or coreceptors). However, combined with the polymyxin Bexperiments and the in vivo data in TLR4 mutant mice, togetherthese data suggest that GC frass can mediate cytokine expressionand release from neutrophils independently of endotoxin. In addi-tion, treatment of cells with boiled frass (boiled for 1 h before use)attenuated GC frass-induced IL-8 production from primary humanneutrophils, suggesting that the TLR2 agonist activity is heat sen-sitive (Fig. 6 C ). Lastly, treatment of cells with the TLR2 agonistPam 3 CSK 4 increased IL-8 expression in neutrophils, conrmingthe activation of TLR2 on primary human neutrophils (Fig. 6 C ).

FIGURE 5. TLR2 is expressed on the cell surface of neutrophils re-cruited into the airways. BALB/c mice were given a single intratrachealinhalation of GC frass (40 g/40 l) and BAL uid was harvested 18 hlater. Neutrophils were magnetically labeled and isolated before stainingwith either PE-conjugated isotype control (line) or PE-conjugated TLR2(lled area) Abs. This experiment was repeated twice.

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FIGURE 6. GC frass increased IL-8 and TNF- protein abundance inprimary human neutrophils. Primary human neutrophils were isolated andtreated with increasing concentrations of GC frass (10100 ng/ml) for 18 h.Supernatant was harvested and claried and run on an ELISA for cytokineexpression. A, IL-8 ELISA. Data are expressed as means SEM for fourexperiments ( , p 0.003; , p 0.001; ANOVA). B, TNF- ELISA.Data are expressed as means SEM for four experiments ( , p 0.008;

, p 0.001; ANOVA). C , Primary human neutrophils were pretreatedwith polymyxin B (50 mg/ml for 1 h) before treatment with GC frass (100ng/ml) for 18 h. Some cells were treated with LPS (100 pg/ml), boiled frass(100 ng/ml, boiled for 1 h), or Pam-3-Cys (1 g/ml). IL-8 ELISA wasperformed. Data are expressed as means SEM for four experiments(compared with control (cont); , p 0.009; , p 0.011; , p0.007; ANOVA).

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We also tested the effects of GC frass on DMSO-differentiatedHL-60 cells. We found a similar level of IL-8 activation (100ng/ml GC frass increased IL-8 levels from a baseline of 6.0 0.6ng/ml to 35.2 5.3 ng/ml, n 7; p 0.001). Because we wereobtaining a similar cytokine regulation using HL-60 cells com-pared with primary human neutrophils, we performed the remain-ing mechanistic studies using HL-60 cells.

GC frass regulates cytokine production via TLR

To assess the roles of TLR2 and TLR4 in GC frass-mediated cy-tokine expression, we pretreated differentiated HL-60 cells with aneutralizing Ab against TLR2 or TLR4 before treatment with GCfrass. GC frass-mediated IL-8 expression was inhibited by theTLR2-neutralizing Ab, but not the TLR4-neutralizing Ab (Fig.7 A). To further test this, we isolated bone marrow derived-neutro-phils from TLR2-knockout and wild-type mice and treated them exvivo with GC frass. Neutrophils from wild-type mice were respon-sive to GC frass as depicted by the signicant increase of TNF-(Fig. 7 B). In contrast, neutrophils from TLR2-knockout mice didnot respond to GC frass, suggesting the importance of TLR2 forGC frass-mediated activation of neutrophils. Importantly, thesedata suggest that GC frass contains a TLR2 agonist.

GC frass increased NF- B translocation and transactivation inhuman neutrophils

We initially investigated the role of NF- B in GC frass-inducedcytokine expression by using a chemical inhibitor of the NF- Bsignaling pathway. We found that pretreatment of DMSO-differ-entiated HL-60 cells with isohelenin completely abolished GCfrass-induced IL-8 release (Fig. 8 A). To determine the role of NF- B in GC frass-mediated cytokine expression in HL-60 cells,we asked whether GC frass regulated I B degradation. As wouldbe expected with NF- B activation, frass induced degradation of I B in a time-dependent fashion (Fig. 8, B and C ). Next, wetreated HL-60 cells with GC frass for 1 h and nuclear extracts wereharvested for EMSA. GC frass increased the binding of nuclear

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800

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T N F p g / m l

1000

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wild typeTLR2 mutant *

control frass

FIGURE 7. TLR2 is required for GC frass-induced regulation of cy-tokine production from neutrophils. A, Differentiated HL-60 cells werepretreated with neutralizing Abs against TLR2, TLR4, or isotype con-trol Abs for 1 h before stimulation with GC frass (100 ng/ml). Cellmedium was harvested at 18 h and analyzed by ELISA for IL-8 pro-duction. Data are expressed as means SEM for four experiments. GCfrass increased IL-8 production in the presence of the isotype controland TLR4 Abs, but not the TLR2 Ab ( , p 0.001; ANOVA). B,Primary bone marrow-derived mouse neutrophils were treated with GCfrass. Cell medium was harvested at 18 h and analyzed by ELISA forTNF- production. Data are expressed as means SEM for three ex-periments. GC frass increased TNF- production compared with controltreatment ( , p 0.001; ANOVA).

FIGURE 8. GC frass regulated NF- B activa-tion. A, HL-60 cells were pretreated with isohelenin(30 M) for 1 h before treatment with GC frass for18 h. Supernatants were harvested, claried, and runon IL-8 ELISA. Data are expressed as meansSEM for four experiments ( , p 0.001; ANOVA). B, HL-60 cells were treated with frass (100 ng/ml)

for the times as indicated (0120 min). Cell lysatewas harvested, run on a Western blot, and probed forI B . These data are representative of four separateexperiments. C , Quantication of the I B Westernblots. Data are expressed as percentage of I B re-maining compared with the untreated sample(means SEM, n 4; , p 0.046; , p 0.023;ANOVA). D, HL-60 cells were treated with GCfrass for 1 h and nuclear extracts were harvested forEMSA. Coincubation of nuclear extracts from frass-treated cells with Abs against p65 Rel A and p50NF- B1 were also performed. These data are rep-resentative of three separate experiments.



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proteins to an oligonucleotide encoding the consensus sequenceNF- B binding site. Coincubation of nuclear extracts with Absagainst p65 RelA and p50 NF- B1 each induced a supershift of theDNA binding complex, demonstrating the presence of theseNF- B family transcription factors (Fig. 8 D). Taken together,these data suggest that GC frass increased neutrophil-derived cy-tokine expression in a NF- B-dependent manner.

DiscussionIn this study we found that a single intratracheal inhalation of GCfrass in naive mice signicantly increased neutrophil recruitmentinto the airways in a TLR2- and TLR4-independent manner. Anovel nding in this study is that GC frass contains a TLR2 agonistand that an allergen directly regulated neutrophil function viaTLR2. Direct proof of this came from the following ndings: 1)neutralizing Abs against TLR2, but not TLR4, inhibited GC frass-mediated cytokine production in differentiated HL-60 cells; and 2)bone marrow-derived neutrophils from TLR2-decient micetreated ex vivo with GC frass failed to increase cytokine produc-tion compared with neutrophils from wild-type mice. In addition,GC frass-induced cytokine expression in neutrophils was shown tobe dependent on NF- B activation. One limitation of this study isthat the NF- B studies were performed in the differentiated HL-60cell line and not in primary human neutrophils because the isola-tion of neutrophils resulted in autoactivation, as evidenced by in-creased I B activation (K. Page, unpublished observations). Nev-ertheless, these data present strong evidence that GC frass containsa TLR2 agonist that directly activated neutrophils via TLR2 andNF- B and suggests a link between innate and adaptive immunityto GC frass.

Neutrophils are important regulators of airway cytokine expres-sion, as inhibition of neutrophil inltration abrogated BAL cyto-kine expression following GC frass inhalation. The inux of neu-trophils into the airways occurred independently of TLR2 agonistsas evidenced by the increase in BAL neutrophils in GC frass-

treated TLR2-knockout mice. Although neutrophil recruitmentinto the airways appeared to be unaffected by TLR2, maintenanceof airway cytokine levels is dependent on TLR2. Increased levelsof these cytokines may be important to maintain neutrophil re-cruitment, prolong the life of the neutrophil, or attract other in-ammatory regulatory cells. A recent study showed that neutro-phils treated with the TLR2-specic agonist Pam 3 CSK 4 hadincreased IL-8 production and phagocytosis (19), suggesting animportant role for TLR2 in regulating innate immune functions. Inanother study, stimulation of TLR2 with Pam 3 CSK 4 increasedneutrophil function as determined by increased respiratory burst,enhanced IL-8 generation, and prolonged life span in highly pu-ried neutrophils (20). Our data conrm the role of TLR2 agonists

in regulating neutrophil cytokine production. Although we did notstudy all of the parameters of neutrophil function (i.e., phagocy-tosis, chemotaxis, and respiratory burst), our data suggest an im-portant role for GC frass in mediating neutrophil function in theairways.

The rst cells to respond to GC frass inhalation and secretecytokines to mediate immune and inammatory reactions wouldmost likely be airway epithelial cells or resident alveolar macro-phages, both of which express TLR2 (2123). Our preliminarydata suggest that although mouse tracheal epithelial cells respondto GC frass by secreting cytokines, the cytokine levels in TLR2-decient mice are not signicantly altered (K. Page, unpublishedobservation). Although it has been shown that both alveolar epi-thelial cells and tracheobronchial epithelial cells express TLR2, thepresence of TLR2 does not guarantee participation in respondingto a pathogen. Consider that airway epithelial cells express TLR4

but are not responsive to endotoxin due to low levels of an essen-tial adaptor molecule, MD-2 (24). It is not completely clear whatadditional adaptor proteins or coreceptors are involved in ampli-fying TLR2 signaling. It is known that TLR2 forms a heterodimerwith either TLR1 or TLR6. In the current study, we did not in-vestigate the effect of GC frass on either TLR1- or TLR6-decientmice. It is possible that the presence of adaptor proteins or theprole of TLR2 heterodimers in airway epithelium compared with

neutrophils may be important factors in GC frass-inducedsignaling.The role of TLR2 in asthma is controversial. Previous studies

have suggested that TLR2 agonists administered during sensitiza-tion enhanced allergic inammation (10, 11), while other studieshave suggested these agonists inhibit allergic airway inammation(9, 25). It was shown that priming with the TLR2 agonistPam 3 CSK 4 increased OVA-induced experimental asthma in mice(10). Two studies have recently shown that Pam 3 CSK 4 induced aTh2 bias in murine bone marrow-derived dendritic cells (10) anduncommitted, immature human monocyte-derived dendritic cells(26) as determined by increased Th2-associated effector moleculesand up-regulation of costimulatory molecules. This would impli-

cate an important role for the TLR2 agonist in GC frass, as acti-vation of TLR2 could mediate allergic asthma. These data, alongwith our recent study, suggest that the TLR2 agonist in the allergenGC frass could be the signal that skews the immune response to-ward Th2 and the allergic phenotype.

Cockroach feces (frass) are likely sources of allergen exposure(27), as desiccated fecal remnants may easily incorporate into dustparticles. It has been shown that after disturbance, dust particlescontaining cockroach proteins 10 m in diameter may be de-posited in the airway (5). GC frass is a complex mixture of avariety of biologically active molecules, including the allergensBla g 1 and Bla g 2 (27) and active serine proteases and endotoxin(28). Interestingly, our data do not show a role for endotoxin or

TLR4 in either neutrophil recruitment or activation. The fact thatGC frass contains a TLR2 agonist suggests a possible mechanismby which cockroach may be a potent allergen, i.e., it contains notonly the Ag but a TLR agonist (a PAMP) that may help induce theasthma phenotype. Interestingly it has been shown that Ag expo-sure in the absence of PAMPs failed to induce lung DC maturation(29). It is currently thought that lipopeptides and lipoproteins frommicroorganisms and also peptidoglycans derived from Gram-pos-itive bacteria are TLR2 agonists (30). Although we have not yetidentied the TLR2 agonist in GC frass, it is possible that frasscontains peptidoglycans or lipoproteins from digestive processesor from microbial life in the cockroach gut.

During status asthmaticus, neutrophils are the dominant leuko-cyte subpopulation within the airways (31). In fact, the importanceof neutrophilia in the asthma phenotype is suggested by the ndingof a closer correlation of neutrophils to airway obstruction andseverity of asthma than eosinophils in some studies (31, 32). Wehave found that once inhaled GC frass maintains its activity in theairways for at least 3 h (K. Page, unpublished observation), sug-gesting that GC frass would have an opportunity to interact withneutrophils in the airways. Through this investigation we havefound that GC frass contains a TLR2 agonist, that has a directeffect on neutrophils to regulate cytokine expression in a NF- B-dependent manner. This suggests not only a link between innateand adaptive immune responses but also suggests that in a severeasthma state in which neutrophils are present in the airways, in-halation of GC frass could directly activate inltrated neutrophilsand exacerbate airway inammation.

6323The Journal of Immunology


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AcknowledgmentsWe thank Dr. Shizuo Akira for providing the TLR2 / mice and Dr. Le-sley Doughty and Dan Marmer for outstanding assistance with owcytometry.

DisclosuresThe authors have no nancial conict of interest.

References1. Gelber, L. E., L. H. Seltzer, J. K. Bouzoukis, S. M. Pollart, M. D. Chapman, and

T. A. Platts-Mills. 1993. Sensitization and exposure to indoor allergens as risk factors for asthma patients presenting to hospital. Am. Rev. Resp. Dis. 147:573587.

2. Leaderer, B. P., K. Belanger, E. Triche, T. Holford, D. R. Gold, Y. Kim,T. Jankun, P. Ren, J. E. McSherry, T. A. E. Platts-Mills, et al. 2002. Dust mite,cockroach, cat, and dog allergen concentrations in homes of asthmatic children inthe Northeastern United States: impact of socioeconomic factors and populationdensity. Environ. Health Perspect. 110: 419425.

3. Rosenstreich, D. L., P. Eggleston, M. Kattan, D. Baker, R. G. Slavin, P. Gergen,H. Mitchell, K. McNiff-Mortimer, H. Lynn, D. Ownby, and F. Malveaux. 1997.The role of cockroach allergy and exposure to cockroach allergen is causingmorbidity among inner city children with asthma. N. Eng. J. Med. 336:13561363.

4. Gold, D. R., H. A. Burge, V. Carey, D. K. Milton, T. A. Platts-Mills, andS. T. Weiss. 1999. Predictors of repeated wheeze in the rst year of life: therelative roles of cockroach, birth weight, acute lower respiratory illness and ma-ternal smoking. Am. J. Resp. Crit. Care Med. 160: 227236.

5. De Lucca, S. D., D. J. Taylor, T. J. OMeara, A. S. Jones, and E. R. Tovey. 1999.Measurement and characterization of cockroach allergens detected during normaldomestic activity. J. Allergy Clin. Immunol. 104: 672680.

6. Lommatzsch, M., P. Julius, M. Kuepper, H. Garn, K. Bratke, S. Irmsher,W. Luttmann, H. Renz, A. Braun, and J. C. Virchow. 2006. The course of aller-gen-induced leukocyte inltration in human and experimental asthma. J. Allergy Clin. Immunol. 118: 9197.

7. Taube, C., A. Dakhama, Y. H. Rha, K. Takeda, A. Joetham, J. W. Park,A. Balhorn, T. Takai, K. R. Poch, J. A. Nick, and E. W. Gelfand. 2003. Transientneutrophil inltration after allergen challenge is dependent on specic antibodiesand Fc III receptors. J. Immunol. 170: 43014309.

8. Eder, W., W. Klimecki, L. Yu, E. von Mutius, J. Riedler, C. Braun-Fahrlander,D. Nowak, F. D. Martinez, and A. S. Team. 2004. Toll-like receptor 2 as a majorgene for asthma in children of European farmers. J. Allergy Clin. Immunol. 113:482488.

9. Akdis , C., F. Kussebi , B. Pulendran, M. Akdis , R. P. Lauener,C. B. Schmidt-Weber, S. Klunker, G. Isitmangil, N. Hansjee, T. A. Wynn, et al.2003. Inhibition of T helper 2-type responses, IgE production, and eosinophilia

by synthetic lipopeptides. Eur. J. Immunol. 33: 27172726.10. Redecke, V., H. Hacker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, and

E. Raz. 2004. Activation of Toll-like receptor 2 induces a Th2 immune responseand promotes experimental asthma. J. Immunol. 172: 27392743.

11. Chisholm, D., L. Libet, T. Hayashi, and A. A. Horner. 2004. Airway peptidogly-can and immunostimulatory DNA exposures have divergent effects on the de-velopment of airway allergen hypersensitivities. J. Allergy Clin. Immunol. 113:448454.

12. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda,and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443451.

13. Czuprynski, C. J., J. F. Brown, N. Maroushek, R. D. Wagner, and H. Steinberg.1994. Administration of anti-granulocyte mAb RB6 8C5 impairs the resistanceof mice to Listeria monocytogenes infection. J. Immunol. 152: 18361846.

14. Walters, D. M., P. N. Breysse, and M. Wills-Karp. 2001. Ambient urban Balti-more particulate-induced airway hyperresponsiveness and inammation in mice. Am. J. Respir. Crit. Care Med. 164: 14381443.

15. Aneja, R., P. W. Hake, T. J. Burroughs, A. G. Denenberg, H. R. Wong, andB. Zingarelli. 2004. Epigallocatechin, a green tea polyphenol, attenuates myo-cardial ischemia reperfusion injury in rats. Mol. Med. 10: 5562.

16. Tennenberg, S. D., F. P. Zemlan, and J. S. Solomkin. 1988. Characterization of N -formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. J. Im-munol. 141: 39373944.

17. Page, K., V. S. Strunk, and M. B. Hershenson. 2003. Cockroach proteases in-crease IL-8 expression in human bronchial epithelial cells via activation of pro-tease-activated receptor (PAR)-2 and ERK. J. Allergy Clin. Immunol. 112:11121118.

18. Allen, G. L., I. Y. Menendez, M. A. Ryan, R. L. Mazor, J. R. Wispe,M. A. Fielder, and H. R. Wong. 2000. Hyperoxia synergistically increases TNF-

-induced interleukin-8 gene expression in A549 cells. Am. J. Physiol. 278:L253L260.

19. Hayashi, F., T. K. Means, and A. D. Luster. 2003. Toll-like receptors stimulatehuman neutrophil function. Blood 102: 26602669.

20. Sabroe, I., L. R. Prince, E. C. Jones, M. J. Horsburgh, S. J. Foster, S. N. Vogel,S. K. Dower, and M. K. B. Whyte. 2003. Selective roles for toll-like receptor(TLR)2 and TLR4 in the regulation of neutrophil activation and life span. J. Im-munol. 170: 52685275.

21. Soong, G., B. Reddy, S. Sokol, R. Adamo, and A. Prince. 2004. TLR2 is mobi-lized into an apical lipid raft receptor complex to signal infection in airwayepithelial cells. J. Clin. Invest. 113: 14821489.

22. Armstrong, L., A. R. Medford, K. M. Uppington, J. Robertson, I. R. Witherden,T. D. Tetley, and A. B. Millar. 2004. Expression of functional toll-like receptor-2and -4 on alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 31: 241245.

23. Hertz, C. J., Q. Wu, E. M. Porter, Y. J. Zhang, K. H. Weismuller, P. J. Godowski,T. Ganz, S. H. Randell, and R. L. Modlin. 2003. Activation of Toll-like receptor2 on human tracheobronchial epithelial cells induces the antimicrobial peptidehuman defensin-2. J. Immunol. 171: 68206826.

24. Jia, H. P., J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, andP. B. J. McCray. 2004. Endotoxin responsiveness of human airway epithelia islimited by low expression of MD-2. Am. J. Physiol. 287: L428L437.

25. Patel, M., D. Xu, P. Kewin, B. Choo-Kang, C. McSharry, N. C. Thomson, andF. Y. Liew. 2005. TLR2 agonist ameliorates established allergic airway inam-mation by promoting Th1 response and not via regulatory T cells. J. Immunol.174: 75587563.

26. Agrawal, S., A. Agrawal, B. Doughty, A. Gerwitz, J. Blenis, T. Van Dyke, andB. Pulendran. 2003. Different Toll-like receptor agonists instruct dendritic cells toinduce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J. Immunol. 171:49844989.

27. Yun, Y. Y., S. H. Ko, J. W. Park, I. Y. Lee, H. I. Ree, and C. S. Hong. 2001.Comparison of allergic components between German cockroach whole body andfecal extracts. Ann. Allergy Asthma Immunol. 86: 551556.

28. Hughes, V. S., and K. Page. 2007. German cockroach frass proteases cleavepro-matrix metalloproteinase-9. Exp. Lung Res. 33: 135150.

29. Brimnes, M. K., L. Bonifaz, R. M. Steinman, and T. M. Moran. 2003. Inuenzavirus-induced dendritic cell maturation is associated with the induction of strongT cell immunity to a coadministered normally nonimmunogenic protein. J. Exp. Med. 198: 133144.

30. Kaisho, T., and S. Akira. 2001. Toll-like receptors and their signaling mechanismin innate immunity. Acta Odontol. Scand. 59: 124130.

31. Jatakanon, A., C. Uasuf, W. Maziak, S. Lim, K. F. Chung, and P. J. Barnes. 1999.Neutrophilic inammation in severe persistent asthma. Am. J. Respir. Crit. Care Med. 160: 15321539.

32. Ordonez, C. L., T. E. Shaughnessy, M. A. Matthay, and J. V. Fahy. 2000. In-creased neutrophil numbers and IL-8 levels in airway secretions in acute severeasthma: clinical and biological signicance. Am. J. Respir. Crit. Care Med. 161:11851190.


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