of February 14, 2018.This information is current as

Infiltration into Antigen-Challenged SkinPerforin Directs Effector CD8 T Cell Neutrophil Expression of Fas Ligand and

Parameswaran, Neetu Gupta and Robert L. FairchildDanielle D. Kish, Anton V. Gorbachev, Neetha

ol.1102729http://www.jimmunol.org/content/early/2012/07/18/jimmun

published online 18 July 2012J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Neutrophil Expression of Fas Ligand and Perforin DirectsEffector CD8 T Cell Infiltration into Antigen-Challenged Skin

Danielle D. Kish,* Anton V. Gorbachev,* Neetha Parameswaran,* Neetu Gupta,*,† and

Robert L. Fairchild*,†

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by

hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior

CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing

hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and per-

forin expression. Although DNFB sensitization of gld/perforin2/2 mice induced hapten-specific CD8 T cells producing IFN-g and

IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when

transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS

when transferred to naive gld/perforin2/2 recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when

transferred to sensitized gld/perforin2/2 mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and

CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1,

CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-

primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune

responses in the skin. The Journal of Immunology, 2012, 189: 000–000.

Effector CD8 T cells are critical components of immuneresponses against intracellular pathogens and tumors.Following Ag priming in peripheral lymphoid organs, the

effector CD8 T cells must traffic to the tissue site of the inflam-matory insult to elicit the response. Current paradigms proposethat integrins and chemokines function synergistically to directAg-primed CD8 T cell arrest in the vasculature of inflammatorysites and into parenchymal tissues during the elicitation of immuneresponses (1). Additional factors required to induce the chemo-attractants directing the infiltration of CD8 T cells into paren-chymal tissues during elicitation of immune responses remainincompletely identified.The most frequently observed dermatosis in industrialized

countries is allergic contact dermatitis, or contact hypersensitivity(CHS), a T cell-mediated immune response to epidermal sensiti-zation and subsequent challenge with the sensitizing hapten (2–5).Hapten-specific CD8 T cell populations producing IFN-g and IL-17are primed during skin sensitization and are the primary effectorT cells mediating CHS responses to 2,4-dinitro-1-fluorobenzene(DNFB), oxazolone, and urushiol, the reactive hapten of poisonivy (6–11). Hapten challenge of sensitized individuals induces the

recruitment of these CD8 T cell populations into the skin challengesite and their activation to mediate the characteristic edema of theresponse. The factors directing the CD8 T cells through the vascularendothelial barrier and into the skin parenchymal tissue of thechallenge site remain poorly defined. Our previous studies haveindicated that skin challenge of hapten-sensitized mice is quicklyfollowed by the recruitment of the hapten-primed CD8 T cells to thevasculature of the challenge sitewhere they are activated to produceIL-17 and IFN-g by endothelial cells presenting the challengehapten (10, 12). These cytokines stimulate the endothelial cells toproduce the neutrophil chemoattractants CXCL1 and CXCL2,which direct the neutrophils into the skin parenchyma. The sub-sequent infiltration of the hapten-primed CD8 T cells into the skinparenchymal tissue of the hapten challenge site and the CHS re-sponse are inhibited when hapten-sensitized mice are given eitherCXCL1- and CXCL2-neutralizing Abs or neutrophil-depleting Absat the time of hapten challenge (13, 14), indicating that priorCXCL1/CXCL2-directed neutrophil infiltration and activation di-rect the subsequent infiltration of the hapten-primed CD8 T cellsinto the skin parenchymal tissue during elicitation of CHS. Theneutrophil functions directing this CD8 T cell infiltration during theelicitation of CHS have remained unknown.Because CD8 T cells are the major effector T cells in CHS

responses, there has been considerable interest in the possibilitythat these T cells express cytolytic functions to elicit the response.One group of investigators has reported the absence of CHSresponses following sensitization and challenge of gld/perforin2/2

mice, whereas mice with the single Fas ligand (FasL) or perforindeficiency had normal CHS responses (15). These observations ledto the proposal that CD8 T cells must express cytolytic activitythrough either the FasL or perforin/granzyme B pathway withinthe hapten challenge site to mediate CHS responses. Observationsof keratinocyte apoptosis during the elicitation of CHS wereconsistent with this proposal (16). However, examination ofhapten-primed CD8 T cells has failed to demonstrate expressionof FasL, and the primed CD8 T cells do not exhibit cytolytic

*Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland,OH 44195; and †Department of Pathology, Case Western Reserve University Schoolof Medicine, Cleveland, OH 44106

Received for publication September 21, 2011. Accepted for publication June 23,2012.

This work was supported by National Institutes of Allergy and Infectious DiseasesGrant R01 AI45888.

Address correspondence and reprint requests to Dr. Robert L. Fairchild, ClevelandClinic, Lerner Research Institute, 9500 Euclid Avenue, NB3-59, Cleveland, OH44195-0001. E-mail address: kishd@ccf.org

The online version of this article contains supplemental material.

Abbreviations used in this article: CHS, contact hypersensitivity; DNFB, 2,4-dinitro-1-fluorobenzene; FasL, Fas ligand; qRT-PCR, quantitative RT-PCR.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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functions when cocultured with hapten-labeled targets, raising thepossibility that the requirement for either FasL or perforin ismediated through expression by other cells participating in theelicitation of CHS responses (17–19). In this study, we investi-gated mechanisms underlying the absent CHS responses followingchallenge of hapten-sensitized gld/perforin2/2 mice. The resultsindicate a novel function for neutrophils in expressing both FasLand perforin, which induce T cell chemoattractants and promoteCD8 T cell infiltration into the skin to mediate CHS.

Materials and MethodsMice

C57BL/6 (H-2b) mice were obtained through Dr. Clarence Reeder (Na-tional Cancer Institute, Frederick, MD) and gld and perforin2/2 mice ona C57BL/6 background were obtained from The Jackson Laboratory (BarHarbor, ME). Perforin2/2 and gld mice were first crossed to produce gld+/2

/perforin+/2 mice and then these were intercrossed and the gld/perforin2/2

mice identified through PCR analysis of isolated tissue DNA. Female mice,8–10 wk age, were used throughout these studies.

Hapten sensitization and elicitation of CHS

Mice were sensitized to DNFB by painting the shaved abdomen with 25 ml0.25% DNFB (Sigma-Aldrich, St. Louis, MO) and 10 ml to each paw ondays 0 and +1 (10, 12). On day +5 hapten-sensitized and control, non-sensitized mice were challenged on each side of each ear with 10 mlDNFB. Ear thickness was measured using an engineer’s micrometer(Mitutoyo, Elk Grove Village, IL) and expressed in units of 1024 in. Theear swelling response is given as the mean increase in thickness to chal-lenge for each group of four individual animals 6 SEM.

Abs and cytokines

For flow cytometry the following Abs were used: anti-CD45 mAb, anti-mouse CD3 mAb, anti-mouse NK1.1 mAb, and F4/80 (BD Pharmingen,SanDiego,CA) and rat anti-mouseGr-1mAb(eBioScience,SanDiego,CA).

For in vivo depletion of CD4+ T cells, mice were injected with 100 mgeach anti-CD4 mAb, YTS 191 and GK1.5, i.p. on 3 consecutive daysbefore hapten sensitization on days 0 and +1 (14, 20). Treated sentinelmice were used to evaluate the efficiency of CD4+ T cell depletion by Abstaining and flow cytometry analysis of spleen and lymph node cells andwas always .95% when compared with cells from control, rat IgG-treatedmice. In vivo depletion of neutrophils was performed by injecting hapten-sensitized mice with 100 mg anti-Gr-1 mAb, RB6.8C5, on the day beforeand the day of hapten challenge (13, 14).

ELISPOT assays for enumeration of hapten-specific T cellsproducing IFN-g or IL-17

ELISPOT plates (Unifilter 350; Polyfiltronics Group, Rockland, MA) werecoated with IFN-g– or IL-17–specific mAbs, incubated overnight in thecold, and then blocked with 1% BSA in PBS. Responder lymph node cellsfrom nonsensitized or DNFB-sensitized mice were prepared on day +5postsensitization and enriched for CD8 T cells by removing CD4 T cellswith magnetic beads. Syngeneic spleen cells from naive mice were treatedwith 50 mg/ml mitomycin C and labeled with 100 mg/ml DNBS for use asstimulator cells. Stimulator cells were plated at 5 3 105 cells/well witheither 23 105, 53 105, or 13 106 responder cells/well in serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1 mM L-glutamine. Responder cells plated with unlabeled splenocytes were used asa negative (hapten specificity) control. After 24 h, cells were removed byextensive washing with PBS/0.05% Tween 20 and biotinylated anti–IFN-gmAb or biotinylated anti–IL-17 mAb was added. The plate was incubatedovernight at 4˚C, washed with PBS/0.05% Tween 20, and conjugatedstreptavidin-alkaline phosphatase was added to each well. After 2 h atroom temperature, the plate was washed with PBS/0.05% Tween 20 andNBT/5-bromo-4-cholor-30-indolyl substrate (Bio-Rad Laboratories, Her-cules, CA) added for detection of IFN-g or IL-17. Resulting spots werecounted with an ImmunoSpot series I analyzer (Cellular Technology,Cleveland, OH).

Histological analyses

Hapten-challenged skin was excised from sensitized wild-type and gld/per-forin2/2mice 24 h after challenge. For staining to detect leukocyte infiltrationinto the challenge site, the skin tissue was fixed in 10% formalin, embeddedin paraffin, and 8-mm sections were prepared and stained with H&E.

Quantitation of CXCL1 and CXCL2 in skin by immunoassay

CXCL1 and CXCL2 levels in skin were determined by ELISA. Mice weresensitized by application of 10 ml 0.25% DNFB to each side of each ear ondays 0 and +1. On day +5 the shaved trunk skin of both sensitized andnonsensitized mice was challenged with 25 ml DNFB and the challengedskin was excised 6 h later and homogenized in 500 ml proteinase inhibitormixture (Sigma-Aldrich) with gentle shaking for 30 min. Following cen-trifugation at 12,000 3 g for 10 min, supernatants were collected and totalprotein concentrations quantified using a Coomassie Plus protein assayreagent kit (Pierce, Rockford, IL). All samples were diluted to an equiv-alent total protein concentration and tested in CXCL1- and CXCL2-specific ELISA.

Analysis of tissue-infiltrating cells by flow cytometry

The shaved trunk skin of sensitized and nonsensitized wild-type and gld/perforin2/2 mice was challenged with DNFB and 6 or 18 h later the chal-lenged skin was removed and incubated in 0.5% dispase (Invitrogen LifeTechnologies, Carlsbad, CA) at 4˚C for 18 h. In some experiments groups ofsensitized gld/perforin2/2 mice and anti–Gr-1 mAb-treated wild-type micewere injected intradermally with 10 ng recombinant FasL (R&D Systems,Minneapolis, MN), either boiled for 15 min or not boiled, directly into four tofive sites in the skin challenge site 2 h after hapten challenge and thenthe challenged skin was removed 16 h after the injection. Following incubationof the excised skin, the epidermis was separated from the dermis and bothwere incubated in 0.5% trypsin (Sigma-Aldrich) at 37˚C/5% CO2 for 1 h. Bothtissues were pressed through a kidney dialysis bag and the harvested cellstreated with 0.2% DNase (Roche, Indianapolis, IN) for 10 min at roomtemperature. The cells were washed twice with staining buffer (Dulbecco’sPBS with 2% FCS/0.2% NaN3) and 106 cell aliquots were incubated onice in staining buffer with 1:100 Fc Block (BD Biosciences) for 30 min.The cells were washed and stained with fluorochrome-labeled anti-mousemAb to CD45, CD3, and Gr-1. After 30 min, the cells were washed,resuspended in staining buffer, and analyzed by two-color flow cytometryusing a FACSCalibur and CellQuest software (Becton Dickinson, SanJose, CA). The cells were gated to exclude residual tissue debris andnonviable cells, and sample data were collected on 2 3 104 CD45+ cells.

Cell transfer

For transfer of hapten-primed CD8 T cells, C57BL/6 wild-type and gld/perforin2/2 mice were depleted of CD4+ T cells prior to DNFB sensiti-zation. On day +4 lymph node cell suspensions were prepared and 10 3106 cell aliquots transferred to naive C57BL/6 or gld/perforin2/2 recipientsthat were immediately challenged either with 10 ml DNFB on each side ofeach ear to elicit the CHS response or with 25 ml DNFB on the shavedabdomen to assess cell infiltration into the challenge site. The challengedabdominal skin was excised 18 h after challenge and tissue homogenateswere prepared for flow cytometry analysis of infiltrating cells as detailedabove.

For isolation and transfer of neutrophils, C57BL/6 mice were sensitizedwith 0.25%DNFB on the shaved abdomen and paws and challenged on eachside of each ear with 0.2% DNFB on day +5. Two hours later, bone marrowwas extracted from the femurs and tibiae. Bone marrow cells were washedand incubated with PE-conjugated mAb to B220, CD3, F4/80, and NK1.1(BD Pharmingen) to label populations of B cells, T cells, macrophages andNK cells, respectively. Cells were subsequently incubated with anti-PE–coated magnetic beads, loaded onto a MACS column (Miltenyi Biotec,Auburn, CA), and placed in a magnetic field to eliminate the B, T, and NKcells. The resulting cell populations were .95% Gr-1+ cells and ,0.5%CD3+, NK1.1+, and F4/80+ cells and ,0.70% B220+ cells (SupplementalFig. 1). Aliquots of 2 3 107 RB6.8C5+ were transferred i.v. to sensitizedgld/perforin2/2 mice 2 h after challenge.

Analysis of gene expression by quantitative RT-PCR

Whole-cell RNA was prepared from excised skin, purified bone marrowneutrophils, and primed lymph node CD8+ T cells from either DNFB-sensitized or skin allograft recipient (A/J, H-2a → C57BL/6, H-2b) miceby dissolving the tissue or cells in TRIzol reagent (Invitrogen Life Tech-nologies) with subsequent chloroform extraction. cDNA was synthesizedfrom 2 mg mRNA using the TaqMan reverse transcription reagent kit(Applied Biosystems, Foster City, CA). PCR was performed using customprimers and FAM dye-labeled probes (Applied Biosystems) for mouseFasL, perforin, IL-17, IFN-g, CXCL9, CXCL10, CCL1, CCL2, CCL5,and Mrpl 32 (gene assay ID nos. Mm00438864_m1, Mm00812512_m1,Mm00439619_m1,Mm00801778_m1,Mm00434946_m1,Mm00445235_m1,Mm00441236_m1,Mm00441242_m1,Mm01302428_m1, andMm00777741_sH, respectively). The quality threshold of gene expression of one sample, the

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RNA isolated from the skin homogenate of naive, not challenged, wild-type mice, was arbitrarily set at 1.0 and used to determine the expressionlevels of the remaining samples.

Cell lysis and Western blot analysis

Neutrophils were enriched from the bone marrow of naive or sensitizedC57BL/6 wild-type or gld/perforin2/2 mice as detailed above. CD8+

T cells were enriched from the skin-draining lymph nodes of C57BL/6mice sensitized with DNFB or engrafted with complete MHC-mismatchedA/J skin allografts. All cells samples were lysed in buffer containing 20mM Tris-Cl (pH 8.3), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, andprotease and phosphatase inhibitors (5 mg/ml pepstatin A, 1 mM PMSF,0.5 mM iodoacetamide, 1 mM sodium metavanadate, and 10 mM sodiumfluoride) for 30 min on ice. Cell lysates were loaded onto precast gels priorto transfer to polyvinylidene difluoride membranes (Millipore, Hayward,CA). Membranes were probed with primary rabbit Abs to FasL or perforin(Abcam, Cambridge, MA) or to b-actin (Santa Cruz Biotechnology, SantaCruz, CA) followed by secondary HRP secondary anti-rabbit Ab (JacksonImmunoResearch Laboratories, West Grove, PA) prior to development byECL reagent (GE Healthcare Biosciences, Pittsburgh, PA). Resulting bandswere quantified using ImageJ software.

Statistical analysis

Statistical analysis to assess differences between experimental groups wasperformed using a Student t test. Differences were considered significantwhen p , 0.05.

ResultsAbsence of CHS responses following challenge ofhapten-sensitized gld/perforin2/2 mice

To first confirm the reported absence of CHS responses to sen-sitization and challenge of gld/perforin2/2 mice (15), groups ofwild-type C57BL/6 and gld/perforin2/2 mice were sensitizedwith DNFB and challenged on the ear with the hapten on day +5.The increase in ear thickness of sensitized wild-type mice afterchallenge was ∼5-fold the increase measured in naive micechallenged with the hapten (Fig. 1A). In contrast to sensitizedwild-type mice, the increase in ear thickness of sensitized gld/perforin2/2 mice was nearly equivalent to that of challengednaive mice (Fig. 1A). Histological analyses indicated that theabsent CHS response in gld/perforin2/2 mice was associated withdecreased cellular infiltration into the skin challenge site, andthe absence of the characteristic keratinocyte hyperplasia at 24 hpostchallenge and these features remained so for several daysafter challenge (Fig. 1B and data not shown). The absent CHSresponse in gld/perforin2/2 mice was further examined byexcising the skin challenge site 24 h after challenge, digestingthe tissue to prepare single-cell suspensions, and staining cellaliquots with Abs to assess T cell infiltration into the site.Consistent with the histological analyses, infiltrating CD3+

T cells were virtually absent in the skin challenge site of sen-sitized gld/perforin2/2 mice when compared with the challengesite of sensitized wild-type (Fig. 1C).

CD8 T cell priming is equivalent in sensitized wild-type andgld/perforin2/2 mice

The absence of CHS in sensitized gld/perforin2/2 mice raisedthe possibility of a defect in the priming of the IFN-g– and/orIL-17–producing CD8 T cell populations required for the re-sponse. The presence of these T cells was tested by ELISPOTanalyses of lymph node CD8 T cells from sensitized wild-typeversus gld/perforin2/2 mice. In contrast with the low numbersof T cells infiltrating the skin challenge site, numbers ofhapten-specific CD8 T cells producing either IFN-g or IL-17 inthe skin draining lymph nodes of sensitized gld/perforin2/2

mice were equivalent to those induced in sensitized wild-typemice (Fig. 2).

Hapten-primed CD8 T cells from sensitized gld/perforin2/2

mice function to mediate CHS in wild-type recipients

Because IL-17– and IFN-g–producing CD8 T cells appeared todevelop normally in response to hapten sensitization of gld/per-forin2/2 mice, the ability of the primed gld/perforin2/2 CD8 Tcells to mediate CHS responses following transfer to naive wild-type

FIGURE 1. Absence of contact hypersensitivity following challenge of

hapten-sensitized gld/perforin2/2mice. (A) Groups of wild-type C57BL/6

and gld/perforin2/2 mice were sensitized with 0.25% DNFB on days 0 and

+1 and these mice and groups of unsensitized mice were challenged on the

ears with 0.2% DNFB. The change in ear thickness was determined 24 h

later and is shown as the mean increase in ear thickness for each group of

four animals 6 SEM. *p , 0.02 when comparing increased ear thickness

of the sensitized wild-type group versus sensitized gld/perforin2/2 and

unsensitized groups. (B) DNFB-sensitized wild-type and gld/perforin2/2

mice were challenged on the shaved abdominal trunk skin with DNFB. The

challenged skin was excised 24 h after challenge, fixed in formalin, and

prepared paraffin-embedded sections were stained with H&E. Represen-

tative light microscopy images of skin from each group are shown.

Original magnification 3400. (C) On day +5, DNFB-sensitized wild-type

and gld/perforin2/2 mice were challenged on the shaved abdominal trunk

skin with DNFB. Challenged skin was excised 18 h later, digested to

prepare single-cell suspensions, and cell aliquots were stained with fluo-

rescent Abs to identify infiltrating (CD3+) T cells. A representative sample

from the challenged skin of one mouse per group is shown.

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recipients was tested. Aliquots of DNFB-primed CD8 T cellsuspensions from sensitized wild-type and gld/perforin2/2 micewere transferred to naive wild-type or gld/perforin-/- recipients thatwere then challenged with hapten. Primed CD8 T cells from bothsensitized wild-type and sensitized gld/perforin2/2 mice inducedequivalent CHS responses following transfer to naive wild-typemice and recipient challenge (Fig. 3A). However, CHS responsesin naive gld/perforin2/2 recipients of primed wild-type CD8T cells were reduced to near background levels. When the infil-tration of the primed CD8 T cells from the wild-type or gld/per-forin2/2 donors into the recipient skin challenge site tissue wasassessed 24 h after T cell transfer and recipient challenge, therewas clear infiltration of transferred hapten-primed CD8 T cellsfrom sensitized wild-type or sensitized gld/perforin2/2 donorsinto the challenged skin of naive wild-type recipients but not whenthe primed wild-type CD8 T cells were transferred to naive gld/perforin2/2 recipients (Fig. 3B).The activity of the hapten-specific CD8 T cell populations

producing IFN-g and IL-17 induces the initial production of

CXCL1 and CXCL2 by endothelial cells in the challenge site andit is these chemokines that direct the initial infiltration of neu-trophils during elicitation of CHS responses to DNFB and tooxazolone (10, 12). To further test the function of the hapten-primed CD8 T cells within the challenge site of sensitized gld/perforin2/2 mice, the induction of these neutrophil chemo-attractants and the infiltration of neutrophils into the challenge site6 h after hapten challenge were investigated. Initial experimentsclearly indicated high levels of IL-17 and IFN-g mRNA expres-sion in the hapten challenge sites of sensitized wild-type andsensitized gld/perforin2/2 mice but not following hapten chal-lenge of naive mice (data not shown). Consistent with these resultsequivalent levels of CXCL1 and CXCL2 protein were induced 6 hafter challenge of sensitized wild-type and gld/perforin2/2 mice(Fig. 4A). Furthermore, equivalent levels of neutrophil infiltrationwere observed in the challenge site of both sensitized wild-typeand gld/perforin2/2 mice 6 h after challenge (Fig. 4B). In con-junction with the absent CD8 T cell infiltration into the haptenchallenge site of sensitized gld/perforin2/2 mice 18–24 h afterchallenge, neutrophil infiltration into the site was also absent atthis time point.Overall, these results indicated that the absent CHS responses

observed following challenge of sensitized gld/perforin2/2 miceare not due to a defect in the priming or function of the hapten-specific CD8 T cells but rather to the environment within whichelicitation of the immune response occurs.

Upregulated expression of FasL and perforin in the skinchallenge site 6 h after hapten challenge of sensitized wild-typeanimals

Because the early neutrophil infiltration occurring prior to hapten-primed CD8 T cell infiltration into the challenge site was observedin both sensitized wild-type and gld/perforin2/2 mice, the mRNAexpression levels of FasL and perforin in the skin challenge sitewere tested. Hapten-challenged skin of sensitized wild-type andgld/perforin2/2 mice was excised 6 h after challenge, whole-cellRNAwas isolated from the tissue, and quantitative RT/PCR (qRT-PCR) was performed to compare expression levels of FasL andperforin. When compared with skin excised from the haptenchallenge site of naive wild-type mice, hapten challenge of sen-sitized wild-type mice upregulated expression of both FasL andperforin by .20-fold (Fig. 5A). Background levels of both FasLand perforin expression in the hapten challenge sites of sensitizedgld/perforin2/2 mice were observed and were $4-fold lower thanthe expression induced in the challenge site of sensitized wild-type mice.Neutrophil expression of FasL and perforin was directly tested

by qRT-PCR analysis of RNA isolated from bone marrow neu-trophils and lymph node CD8 T cells from hapten-sensitized mice.As a positive control, RNAwas prepared from CD8 T cells isolatedfrom the draining lymph nodes of MHC-mismatched skin allograftrecipients and as expected expressed high levels of both FasL andperforin (Fig. 5B). In contrast, RNA prepared from CD8 T cellsisolated from lymph nodes of hapten-sensitized mice expressedlow levels of both mediators. Neutrophils obtained from the bonemarrow of sensitized and challenged wild-type mice clearlyexpressed FasL and perforin mRNA, whereas neutrophils from thebone marrow of naive wild-type mice expressed slightly lowerlevels of perforin mRNA but low/background levels of FasLmRNA.These studies were extended by performing immunoblot anal-

yses of cell lysates prepared from hapten-primed CD8 T cells andneutrophils from the bone marrow of DNFB-sensitized and-challenged wild-type and gld/perforin2/2 mice. As a positive

FIGURE 2. Hapten-specific CD8 T cell priming in DNFB-sensitized

wild-type and gld/perforin2/2 mice. Wild-type C57BL/6 and gld/perforin2/2

mice were sensitized with DNFB and on day +5 lymph node cell suspen-

sions were prepared from naive wild-type and the sensitized mice. CD4

T cells were removed from the cells using anti-CD4 Ab-coated magnetic

beads. Aliquots of 5 3 105 of the enriched CD8 T cells were cultured with

5 3 105 DNBS-labeled or unlabeled syngeneic splenocytes in triplicate

cultures on IFN-g– or IL-17–coated ELISPOT plates. After 24 h, cells were

removed and the ELISPOTassay was developed to detect numbers of IFN-g–

or IL-17–producing cells. The mean number of hapten-specific CD8 T cells

producing IFN-g or IL-17 per 53 105 cells6 SEM for groups of three mice

is shown. The number of spots from control wells containing the enriched

CD8 T cells with unlabeled stimulator cells was always less than five spots

per well. Results are representative of two individual experiments. Numbers

of cytokine-producing CD8 T cells between sensitized wild-type and gld/

perforin2/2 mice are not significantly different.

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control, lysates of CD8 T cells enriched from the lymph nodes ofwild-type C57BL/6 recipients of complete MHC-mismatched A/Jskin allografts expressed both the 70-kDa perforin protein and the31-kDa FasL protein (Fig. 6A). Consistent with the mRNA anal-yses, lysates of DNFB-primed CD8 T cells enriched from thelymph nodes of DNFB-sensitized wild-type mice did not expresseither of these proteins, but neutrophils enriched from the bonemarrow of DNFB-sensitized and -challenged wild-type but notgld/perforin2/2 mice did express both perforin and FasL proteins.Neutrophils enriched from the bone marrow of unsensitized/naivewild-type mice did not express either of the proteins in theimmunoblot analyses. Densitometric analyses indicated similarlevels of perforin and FasL protein in the lysates of the controlskin allograft-primed CD8 T cells and the neutrophils from thebone marrow of hapten-sensitized and -challenged wild-type mice(Fig. 6B).

Reconstitution of CHS responses in gld/perforin2/2 mice byneutrophils from hapten-sensitized and -challenged wild-typemice

Because the results indicated the expression of FasL and perforinby wild-type neutrophils and not by hapten-primed CD8 T cells, theability of neutrophils from wild-type mice to reverse the defectiveCHS responses in sensitized gld/perforin2/2 mice was tested.Aliquots of bone marrow neutrophils or immune CD8 T cells fromhapten-sensitized wild-type mice were transferred to sensitizedgld/perforin2/2 mice 2 h after hapten challenge and the increasein ear thickness was determined 20 h later. As previously shown,delivery of hapten-primed CD8+ T cells from sensitized wild-type

mice did not induce CHS responses to challenge of gld/perforin2/2

recipients (Fig. 7A). However, delivery of bone marrow neu-trophils from sensitized and challenged wild-type mice inducedCHS responses similar to the responses observed in sensitized andchallenged wild-type mice. Consistent with the CHS responseobserved upon delivery of wild-type neutrophils to challenged gld/perforin2/2 mice, the wild-type neutrophil transfer also inducedinfiltration of both Gr-1+ cells and CD3+ T cells into the haptenchallenge site of sensitized gld/perforin2/2 mice 18 h after chal-lenge that was absent without the neutrophil transfer (Fig. 7B).FasL and perforin were expressed at low levels in isolated

neutrophils from the bone marrow of naive/unsensitized wild-typemice (Figs. 5, 6). Therefore, we compared the restoration ofCHS in sensitized gld/perforin2/2 mice that received aliquotsof neutrophils from the bone marrow of unsensitized/naive versusDNFB-sensitized and challenged wild-type donors (Fig. 7C). Incontrast to the ability of bone marrow neutrophils from sensitizedand challenged wild-type donors to reverse the defective CHSresponse following challenge of sensitized gld/perforin2/2 mice,transfer of bone marrow neutrophills from naive/unsensitizedwild-type donors had only a modest effect on the CHS response inthe sensitized gld/perforin2/2 recipients.

Delivery of soluble FasL into the hapten challenge sitepromotes the infiltration of hapten-primed CD8 T cells into thesite

Because transfer of wild-type neutrophils expressing FasL andperforin could correct the defective trafficking of hapten-primedCD8 T cells into the skin challenge site of sensitized gld/per-

FIGURE 3. Hapten-primed CD8 T cells from sensitized gld/perforin2/2 mice elicit CHS following transfer to naive wild-type recipients. C57BL/6 and

gld/perforin2/2 (dKO) mice were depleted of CD4+ T cells by treatment with anti-CD4 mAb and then sensitized with DNFB on days 0 and +1. On day +4,

lymph node cell suspensions were prepared and aliquots of 10 3 106 CD8+ T cells were transferred i.v. into groups of naive gld/perforin2/2 or wild-type

C57BL/6 mice. (A) Recipients of hapten-primed CD8 T cells from sensitized wild-type or gld/perforin2/2 mice and a group of naive/nonrecipient wild-type

mice were challenged on the ears with DNFB. The change in ear thickness was determined 24 h later and is shown as the mean increase in ear thickness for

each group of four animals6 SEM. *p , 0.02 when comparing increased ear thickness of wild-type recipients of CD8 T cells from sensitized wild-type or

gld/perforin2/2 donors versus gld/perforin2/2 recipients of primed wild-type CD8 T cells and unsensitized wild-type groups. (B) Recipients of hapten-

primed CD8 T cells from sensitized wild-type and from gld/perforin2/2 mice and a group of naive/nonrecipient wild-type mice were challenged on the

abdomen with DNFB. The challenged skin was excised 18 h later, digested to prepare single-cell suspensions, and cell aliquots were stained with fluo-

rescent Abs to identify skin-infiltrating (CD3+) T cells. Representative samples from one mouse per group for groups of four mice for two individual

experiments are shown.

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forin2/2 mice, the ability of soluble FasL to direct this traffickingwas investigated. As a proof of principle, the ability of the solubleFasL to induce hapten-primed CD8 T cell trafficking into thehapten-challenged skin of sensitized wild-type mice depleted ofneutrophils at the time of hapten challenge was first tested (Fig. 8).As previously reported (10, 14), depletion of neutrophils by treatinghapten-sensitized wild-type mice with anti–Gr-1 mAb at the timeof challenge led to an absence of T cell infiltration into the haptenchallenge site. Despite the absence of the depleted neutrophils inthe hapten challenge site, hapten-primed T cell infiltration intothe challenge site was restored when recombinant soluble FasLwas injected directly into the hapten-challenged skin of thesesensitized wild-type mice. The ability of the soluble FasL todirect this infiltration was destroyed by boiling the soluble FasLfor 15 min. Similarly, the defect in hapten-primed T cell infil-tration into hapten-challenged skin of sensitized gld/perforin2/2

mice was corrected when recombinant soluble FasL was injecteddirectly into the hapten-challenged skin of these sensitized miceand this activity was destroyed by boiling the soluble FasL for 15min (Fig. 9).

Expression of chemokines secreted by activated T cells isdownregulated in the skin challenge site of sensitized gld/perforin2/2 mice

To begin to investigate consequences of neutrophil FasL andperforin expression in the skin 6 h after challenge, the hapten-challenged skin from sensitized wild-type and gld/perforin2/2

mice was excised 6 h after challenge and whole-cell RNA wasisolated and tested for expression of genes that would be expectedto mediate the recruitment of the CD8 T cells and other leukocytesinto the challenge site (Fig. 10A). The expression of the T cellchemoattractants CXCL9 and CXCL10 were observed at high

FIGURE 4. Early neutrophil infil-

tration into the hapten challenge site of

sensitized gld/perforin2/2 mice. Wild-

type C57BL/6 and gld/perforin2/2

mice were sensitized with DNFB. On

day +5 the sensitized mice and a group

of naive wild-type mice were chal-

lenged on the shaved abdominal trunk

skin with DNFB. Challenged skin was

excised either 6 or 18 h later and

weighed. (A) Challenged skin homo-

genates were prepared and CXCL1 and

CXCL2 concentrations determined by

ELISA. The mean chemokine concen-

tration in picograms per milligram

excised skin tissue 6 SEM for four

individual mice per group is shown. (B)

Challenged skin was digested to pre-

pare single cells and cell aliquots were

stained with fluorescent Abs to identify

skin-infiltrating Gr-1+ cells. Percen-

tages shown in upper left quadrant in-

dicate percentage of infiltrating CD45+

cells expressing Gr-1. Representative

samples from one mouse per group for

groups of four mice are shown for two

individual experiments.

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levels in the skin challenge sites of both sensitized gld/perforin2/2

and wild-type mice although the expression of CXCL9 was sig-nificantly higher in the site of the sensitized gld/perforin2/2 mice.In contrast, expression levels of CCL1, CCL2, and CCL5 in thechallenged skin of sensitized gld/perforin2/2 mice were equiva-lent to levels of expression following challenge of the skin ofnaive mice.Finally, the ability of wild-type neutrophil transfer to restore

expression of these chemokines in challenged skin of sensitized

gld/perforin2/2 mice was tested. Bone marrow neutrophils wereisolated from hapten-sensitized and -challenged wild-type donorsand transferred to sensitized gld/perforin2/2 recipients (Fig. 10B).Consistent with the absent CHS responses observed by depletingsensitized wild-type mice of neutrophils at the time of haptenchallenge, neutrophil depletion of sensitized wild-type mice at thetime of challenge reduced the expression of CCL1, CCL2, andCCL5 to the levels observed following challenge of naive wild-type or sensitized gld/perforin2/2 mice. Transfer of the wild-typeneutrophils increased the expression of the chemoattractants in theskin challenge site of the sensitized gld/perforin2/2 mice severalfold above those observed in the wild-type mice.

DiscussionThe directed infiltration of leukocytes through the vasculature andinto parenchymal tissues under inflammatory duress is a highlyregulated process that functions to resolve the inflammatory insultand obviate the occurrence of inflammation at other tissue sites (1).Neutrophils are typically the first leukocytes to infiltrate sites oftissue inflammation directed by chemoattractants produced by thevascular endothelium (21, 22). The binding of chemoattractantsto receptors on the surface of neutrophils also activates the releaseof granules containing cytokines, chemokines, and extracellularmatrix-degrading enzymes that directly mediate tissue injury aswell as promote the infiltration of other leukocytes, including Ag-

FIGURE 5. Expression of FasL and perforin by neutrophils in the

hapten challenge site of sensitized wild-type mice. (A) Groups of DNFB-

sensitized wild-type C57BL/6 and gld/perforin2/2 mice and a group of

unsensitized wild-type mice were challenged with DNFB on the abdominal

skin on day +5. The challenged skin was excised 6 h later, and whole-cell

RNA was prepared from homogenates and tested by qRT-PCR for ex-

pression levels of FasL and perforin. The mean expression level for each of

four samples per group 6 SEM is shown. *p # 0.05 when comparing

mRNA expression levels of FasL and perforin from challenged skin of

sensitized wild-type group versus sensitized gld/perforin2/2 and unsensi-

tized wild-type groups. All results are representative of two individual

experiments. (B) CD8 T cells were enriched from the draining lymph

nodes of C57BL/6 (H-2b) recipients of A/J (H-2a) skin allografts and

DNFB-sensitized wild-type C57BL/6 mice. Bone marrow cells were

flushed from the femurs of naive and DNFB-sensitized and -challenged

C57BL/6 mice and T (CD3+) cells, B (B220+) cells, NK (NK1.1+) cells,

and macrophages (F4/80+) were removed by Ab staining and magnetic

bead negative selection. For each test cell population, mRNAwas prepared

and tested by qRT-PCR for expression levels of FasL and perforin. The

expression level for a single sample for each group is shown. All results are

representative of two individual experiments with similar results observed

each time.

FIGURE 6. FasL and perforin proteins are expressed in bone marrow

neutrophils but not CD8 T cells from hapten-sensitized wild-type mice.

Cell lysates were prepared from CD8 T cells enriched from the skin-

draining lymph nodes of C57BL/6 mice 1) engrafted with complete

MHC-mismatched A/J skin allografts or 2) sensitized with DNFB; from

neutrophils enriched from the bone marrow of DNFB-sensitized and

-challenged 3) wild-type or 4) gld/perforin2/2 mice; or 5) from neutrophils

enriched from the bone marrow of naive wild-type mice. (A) Equal

amounts of lysate sample protein (2–3 mg) were resolved on a 7% gel for

Western blotting and probed with Abs to perforin, FasL, or b-actin. Images

of immunoblots are representative of two individual experiments. (B)

Ratios of perforin or FasL signal to b-actin signal for each cell lysate

sample were calculated from densitometric analysis of the immunoblot.

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primed T cells, into the inflammation site (16, 21). Subcutaneousinjection of recombinant IL-8 promotes the initial infiltration ofhuman neutrophils into the skin that is quickly followed by theinfiltration of T cells in a neutrophil-dependent manner (20, 23).Depletion of neutrophils or inhibition of neutrophil trafficking hasbeen shown to delay or attenuate T cell infiltration during allograftrejection, delayed-type hypersensitivity, antiviral responses, andresponses to autoantigens in the CNS (24–28). Although neutrophilsregulate the progression of inflammatory events during many im-mune responses, the mechanisms expressed by neutrophils to in-duce the key chemoattractants directing T cell infiltration throughthe vasculature of inflammatory sites to mediate responses inextravascular tissues remain poorly understood.The present studies were prompted by the reported absence of

CHS responses in mice deficient in both FasL and perforin ex-pression (15). The authors had detected the presence of IFN-gmRNA in the challenge site 6 but not 24 h after challenge ofhapten-sensitized gld/perforin2/2 mice, leading them to conclude

that the hapten-primed CD8 T cells had infiltrated the skin chal-lenge site and that cytotoxic activity through either FasL- orperforin/granzyme B-mediated pathways were required for theelicitation of CHS responses. As confirmed in this study, DNFB-sensitized gld/perforin2/2 mice do not exhibit CHS responses, butthe current results question the role of CD8 T cell-mediated cy-tolysis as an effector mechanism in CHS. We had been unable todetect the expression of FasL on the surface of hapten-primedCD8 T cells (18), and the present studies confirm the absence ofthis expression as well as the absent expression of perforin inmRNA isolated from purified CD8 T cells from the lymph nodesof sensitized wild-type mice. Studies by several investigators havealso indicated the absence of hapten-primed CD8 T cell cytolysisof hapten-labeled target cells (17, 19, 29). TUNEL analyses ofsections prepared from the hapten challenge sites of sensitizedwild-type and gld/perforin2/2 mice indicated low but similarnumbers of TUNEL+ cells throughout the challenged skin, indi-cating that FasL- or perforin/granzyme B-mediated apoptosis

FIGURE 7. Neutrophils from the bone marrow of sensitized and challenged wild-type mice restore the absent CHS responses following challenge of

sensitized gld/perforin2/2 mice. Groups of wild-type C57BL/6 and gld/perforin2/2 mice were sensitized with DNFB. Aliquots of 10 3 106 CD8+ T cells

prepared from skin-draining lymph nodes of sensitized wild-type C57BL/6 mice or 2 3 107 neutrophils isolated from the bone marrow of either non-

sensitized/nonchallenged or DNFB-sensitized and -challenged wild-type C57BL/6 mice were transferred to groups of DNFB-sensitized gld/perforin2/2

mice at the time of hapten challenge as indicated. (A) The sensitized mice and a group of nonsensitized wild-type mice were challenged on the ear with

DNFB. The change in ear thickness was determined 24 h later and is shown as the mean increase in ear thickness for each group of four animals 6 SEM.

*p, 0.05 when comparing increased ear thickness of the sensitized and challenged wild-type group or the sensitized and challenged gld/perforin2/2 group

that had received neutrophils from sensitized and challenged wild-type mice versus sensitized and challenged gld/perforin2/2 mice and versus gld/per-

forin2/2 mice that received hapten-primed wild-type CD8+ T cells and versus unsensitized wild-type groups. (B) The sensitized mice and a group of

nonsensitized wild-type mice were challenged on the abdomen with DNFB. The challenged skin was excised 18 h later, digested to prepare single-cell

suspensions, and cell aliquots were stained with fluorescent Abs to identify skin-infiltrating (Gr-1+) neutrophils and (CD3+) T cells. Representative samples

from one mouse per group for four mice per group are shown for two individual experiments. (C) The groups of sensitized mice and a group of non-

sensitized wild-type mice were challenged on the ear with DNFB. The change in ear thickness was determined 24 h later and is shown as the mean increase

in ear thickness for each group of four animals6 SEM. *p, 0.01 when comparing increased ear thickness of the sensitized and challenged gld/perforin2/2

group that had received neutrophils from sensitized and challenged wild-type mice.

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of cells in the site is not required for the elicitation of CHS(A. Gorbachev, data not shown). Finally, CHS responses wereabsent in response to hapten challenge of naive gld/perforin2/2

recipients of hapten-primed CD8 T cells from sensitized wild-typedonors. These results suggested an entirely different mechanismthan hapten-specific CD8 T cell-mediated cytolysis underlyingthe absence of CHS responses to challenge of hapten-sensitizedgld/perforin2/2 mice.CHS responses are inhibited when sensitized wild-type mice are

treated with anti–Gr-1 mAb or anti-CXCL1 Ab at the time ofhapten challenge, and the intensity of neutrophil infiltration intothe hapten challenge site influences the number of hapten-specificCD8 T cells infiltrating the site (13, 14). These results led us topropose a role for neutrophils in directing the infiltration of primedCD8 T cells into the site. In addition to neutrophils, however,several other leukocyte populations express Gr-1, including plas-macytoid dendritic cells and monocyte/macrophage populations

and could have been affected by the anti-Gr-1 mAb treatment(30–33). To directly test the ability of neutrophils to promotehapten-primed CD8 T cell infiltration into hapten-challenged skin,transfer of highly purified neutrophils from the bone marrow ofwild-type mice to sensitized gld/perforin2/2 recipients correctedthe defective hapten-primed CD8 T cell infiltration into thechallenge site and CHS responses. Neutrophil transfer has beenshown to restore T cell-mediated responses to autoantigens andinfectious pathogens in murine models (24, 34). The neutrophilsisolated from the bone marrow express mRNA encoding bothFasL and perforin, accounting for the ability to promote CHSresponses following transfer to sensitized gld/perforin2/2 mice.The surprising expression of perforin was confirmed by staining ofcytospin preparations of the purified bone marrow neutrophils (D.Kish, data not shown). Neutrophils from both humans and rodentshave been shown to express FasL, and human peripheral bloodneutrophils have been reported to express perforin and granzymesA and B, although the function of these molecules in neutrophil-

FIGURE 8. Delivery of recombinant FasL to the skin challenge site of

neutrophil-depleted sensitized wild-type mice restores hapten-primed T

cell infiltration into the site. C57BL/6 mice were sensitized with DNFB on

days 0 and +1 then depleted of neutrophils by treatment with anti–Gr-1

mAb on day +4 and day +5. Untreated and neutrophil-depleted DNFB-

sensitized mice and a group of naive mice were challenged on the abdomen

with DNFB and 2 h later aliquots of 10 ng boiled or nonboiled recombi-

nant FasL was injected directly into four to five sites within the challenged

skin of sensitized mice depleted of neutrophils. The challenged skin was

excised 16 h later, digested to prepare single-cell suspensions, and cell

aliquots were stained with anti-CD45 mAb and mAb to identify skin-

infiltrating Gr-1+ or CD3+ cells. Representative samples from one mouse per

group for groups of four mice are shown from two individual experiments.

FIGURE 9. Delivery of recombinant FasL to the skin challenge site of

hapten-sensitized gld/perforin2/2 mice restores T cell infiltration into the

site. Groups of C57BL/6 and gld/perforin2/2 mice were sensitized with

DNFB on days 0 and +1. Sensitized wild-type and gld/perforin2/2 mice

and a group of naive wild-type mice were challenged on the abdomen with

DNFB and 2 h later aliquots of 10 ng boiled or nonboiled recombinant

FasL was injected directly into four to five sites within the challenged skin

of sensitized gld/perforin2/2 mice. The challenged skin was excised 16 h

later, digested to prepare single-cell suspensions, and cell aliquots were

stained with anti-CD45 mAb and mAb to identify skin infiltrating Gr-1+ or

CD3+ cells. Representative samples from one mouse per group for groups

of four mice are shown from two individual experiments.

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mediated inflammation has not been investigated (35–39). Im-portantly, note that in the present studies that neutrophils isolatedfrom the bone marrow of naive mice expressed very little FasLmRNA and lower amounts of perforin mRNA than did neutrophils

isolated from the bone marrow of DNFB-sensitized and -chal-lenged mice. Hapten application is likely to induce systemic in-flammatory mediators such as G-CSF that control the generationof neutrophils (40–42). Recent studies have distinguished distinct

FIGURE 10. FasL and perforin induced expression of leukocyte chemoattractant chemokines in the skin challenge site during elicitation of CHS. (A)

Groups of wild-type C57BL/6 and gld/perforin2/2 mice were sensitized with DNFB, challenged with DNFB on day +5, and the challenged skin was

excised 6 h later. Skin was also excised from groups of naive wild-type mice that were or were not (NNC WT) challenged with the hapten. Whole-cell RNA

was prepared from skin homgenates and tested by qRT-PCR for expression levels of CXCL9, CXCL10, CCL1, CCL2, and CCL5. The mean expression

level for each of four samples per group 6 SEM is shown. *p , 0.03 when comparing increased CXCL9 expression in sensitized and challenged gld/

perforin2/2 versus sensitized and challenged wild-type group; **p , 0.02 and ***p , 0.002 when comparing increased gene expression of the sensitized

wild-type group versus sensitized gld/perforin2/2 and unsensitized wild-type groups. (B) Groups of wild-type C57BL/6 and gld/perforin2/2 mice were

sensitized with DNFB and challenged with DNFB on day +5. The groups of sensitized wild-type mice were treated with 100 mg anti–Gr-1 mAb or rat IgG

i.p. on the day before and the day of DNFB challenge. Two hours after challenge, one of the groups of sensitized gld/perforin2/2 mice received aliquots of

2 3 107 neutrophils purified from the bone marrow of DNFB-sensitized and -challenged wild-type C57BL/6 mice. The challenged skin of all sensitized

mice and a group of naive mice was excised 6 h later, and whole-cell RNAwas prepared and tested by qRT-PCR for expression levels of CCL1, CCL2, and

CCL5. The mean expression level for each of four samples per group6 SEM is shown. *p, 0.025, **p, 0.05 when comparing increased gene expression

of the sensitized gld/perforin2/2 group that had received neutrophils from sensitized and challenged wild-type mice versus sensitized gld/perforin2/2 mice,

sensitized gld/perforin2/2 mice that received CD8+ T cells from sensitized wild-type mice, and unsensitized wild-type groups.

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populations of neutrophils (43), and FasL and/or perforin may beexpressed by specific neutrophil populations generated by haptenapplication to the skin.The FasL and perforin expressed by infiltrating neutrophils early

after hapten skin challenge of sensitizedmice results in the inductionof many chemokines that are chemoattractants for T cells. Weconsidered two possible sources of these chemokines: the neu-trophils themselves or other cells in the parenchymal tissue ofthe hapten challenge site induced to produce the chemokines byneutrophil-derived FasL and perforin. Certainly neutrophils area critical source of many chemokines during inflammatory pro-cesses, but their activities, particularly the production of acute phasecytokines and mediators such as oxygen radicals, also stimulateother cells to produce chemokines (44–47).We interrogated mRNAprepared from the neutrophils used in the transfer experiments andobserved low CCL2 and CCL5 and absent CCL1 expression (D.Kish, data not shown). This suggests that neutrophils are a source ofat least CCL2 and CCL5 during the elicitation of CHS but thatneutrophil expression of FasL and perforin induces other cells inthe challenge site to produce CCL1, CCL2, and CCL5. It is alsopossible that neutrophil functions are transcriptionally upregulatedupon infiltration into inflammatory sites, as has been observedduring lung injury (48). However, it is difficult to reconcile pre-formed or newly transcribed chemokine mRNA in FasL- andperforin-deficient neutrophils with the low levels of the chemokinesobserved in the challenge site of the gld/perforin2/2 mice.Following hapten challenge of sensitized gld/perforin2/2 mice,

higher IFN-g and IL-17 and equivalent CXCL1 and CXCL2 andneutrophil infiltration were observed when compared with wild-type mice. Higher levels of the IFN-g induced chemokinesCXCL9 accompanied the higher expression of IL-17 and IFN-g inthe challenge site of sensitized gld/perforin2/2 mice when com-pared with sensitized wild-type mice. These results suggest thatFasL- and/or perforin-mediated activities of neutrophils have a di-rect and/or indirect role in regulating the intensity of the initial andongoing inflammatory response in the vasculature of the haptenchallenge site. We have previously observed higher levels ofCXCL1 and CXCL2 when neutrophils are depleted at the time ofhapten challenge. We interpreted this finding as further indicationthat neutrophil activity is not required for the initial localization ofthe hapten-primed CD8 T cell populations to the hapten-presentingendothelial cells in the challenge as well as the digestion of theCXCL1 and CXCL2 by the infiltrating neutrophils as has beenobserved in other inflammatory models (49).Overall, the results of this study indicate a novel function for

neutrophils in expressing FasL and perforin during early stages of animmune response to induce the chemoattractants directing Ag-primed CD8 T cells through the vascular barrier into parenchy-mal tissues to mediate an immune response. These results suggestthe possible strategy of using isolated neutrophil transfer to promoteAg-primed T cell infiltration into sites of peripheral tissue inflam-mation to potentiate effective immune responses to tumors andviruses. Differences in the effector functions of Ag-primed CD8T cell populations, however, may dictate the requirements for priorneutrophil infiltration and activation to mediate effective immuneresponses. CD8 T cells may develop to express various effectorfunctions, including cytolytic function and/or production of dif-ferent cytokines when Ag priming occurs under different cytokineand costimulatory environments. Hapten-primed CD8 T cells ex-press little to no FasL and perforin and require the FasL andperforin provided by the infiltrating neutrophils whereas CD8T cells primed to viruses and allografts express high levels of FasLand perforin. In the latter case, these mediators may function todirect primed CD8 T cell infiltration into parenchymal tissues in the

absence of neutrophils expressing FasL and perforin. The mecha-nismsdirectingAg-primedCD8Tcells expressingvarious functionsinto extravascular tissues warrants further investigation to attainoptimal efficiency in immune responses as well as to regulate theintensity and duration of these immune responses.

AcknowledgmentsWe thank Drs. Anna Valujskikh, Booki Min, andWink Baldwin for valuable

discussions and advice during the course of this work and the staff of the

Cleveland Clinic Biological Resources Unit for excellent care of the animals

used in the study.

DisclosuresThe authors have no financial conflicts of interest.

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