Infiltration Into Antigen-Challenged Skin Perforin Directs Effector CD8 T

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

This information is current as Danielle D. Kish, Anton V. Gorbachev, Neetha of September 26, 2021. Parameswaran, Neetu Gupta and Robert L. Fairchild J Immunol 2012; 189:2191-2202; Prepublished online 18 July 2012; doi: 10.4049/jimmunol.1102729

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Neutrophil Expression of Fas Ligand and Perforin Directs Effector 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 perforin 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 Downloaded from 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: 2191–2202. http://www.jimmunol.org/

ffector CD8 T cells are critical components of immune recruitment of these CD8 T cell populations into the skin challenge responses against intracellular pathogens and tumors. site and their activation to mediate the characteristic edema of the E Following Ag priming in peripheral lymphoid organs, the response. The factors directing the CD8 T cells through the vascular effector CD8 T cells must traffic to the tissue site of the inflam- endothelial barrier and into the skin parenchymal tissue of the matory insult to elicit the response. Current paradigms propose challenge site remain poorly defined. Our previous studies have that integrins and chemokines function synergistically to direct indicated that skin challenge of hapten-sensitized mice is quickly Ag-primed CD8 T cell arrest in the vasculature of inflammatory followed by the recruitment of the hapten-primed CD8 T cells to the sites and into parenchymal tissues during the elicitation of immune vasculature of the challenge site where they are activated to produce by guest on September 26, 2021 responses (1). Additional factors required to induce the chemo- IL-17 and IFN-g by endothelial cells presenting the challenge attractants directing the infiltration of CD8 T cells into paren- hapten (10, 12). These cytokines stimulate the endothelial cells to chymal tissues during elicitation of immune responses remain produce the neutrophil chemoattractants CXCL1 and CXCL2, incompletely identified. which direct the neutrophils into the skin parenchyma. The sub- The most frequently observed dermatosis in industrialized sequent infiltration of the hapten-primed CD8 T cells into the skin countries is allergic contact dermatitis, or contact hypersensitivity parenchymal tissue of the hapten challenge site and the CHS re- (CHS), a T cell-mediated immune response to epidermal sensiti- sponse are inhibited when hapten-sensitized mice are given either zation and subsequent challenge with the sensitizing hapten (2–5). CXCL1- and CXCL2-neutralizing Abs or neutrophil-depleting Abs Hapten-specific CD8 T cell populations producing IFN-g and IL-17 at the time of hapten challenge (13, 14), indicating that prior are primed during skin sensitization and are the primary effector CXCL1/CXCL2-directed neutrophil infiltration and activation di- T cells mediating CHS responses to 2,4-dinitro-1-fluorobenzene rect the subsequent infiltration of the hapten-primed CD8 T cells (DNFB), oxazolone, and urushiol, the reactive hapten of poison into the skin parenchymal tissue during elicitation of CHS. The ivy (6–11). Hapten challenge of sensitized individuals induces the neutrophil functions directing this CD8 T cell infiltration during the elicitation of CHS have remained unknown. Because CD8 T cells are the major effector T cells in CHS *Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, responses, there has been considerable interest in the possibility † OH 44195; and Department of Pathology, Case Western Reserve University School that these T cells express cytolytic functions to elicit the response. of Medicine, Cleveland, OH 44106 One group of investigators has reported the absence of CHS Received for publication September 21, 2011. Accepted for publication June 23, 2/2 2012. responses following sensitization and challenge of gld/perforin This work was supported by National Institutes of Allergy and Infectious Diseases mice, whereas mice with the single Fas ligand (FasL) or perforin Grant R01 AI45888. deficiency had normal CHS responses (15). These observations led Address correspondence and reprint requests to Dr. Robert L. Fairchild, Cleveland to the proposal that CD8 T cells must express cytolytic activity Clinic, Lerner Research Institute, 9500 Euclid Avenue, NB3-59, Cleveland, OH through either the FasL or perforin/granzyme B pathway within 44195-0001. E-mail address: [email protected] the hapten challenge site to mediate CHS responses. Observations The online version of this article contains supplemental material. of keratinocyte apoptosis during the elicitation of CHS were Abbreviations used in this article: CHS, contact hypersensitivity; DNFB, 2,4-dinitro- consistent with this proposal (16). However, examination of 1-fluorobenzene; FasL, Fas ligand; qRT-PCR, quantitative RT-PCR. hapten-primed CD8 T cells has failed to demonstrate expression Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 of FasL, and the primed CD8 T cells do not exhibit cytolytic www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102729 2192 NEUTROPHIL MEDIATORS DIRECTING T CELLS INTO SKIN functions when cocultured with hapten-labeled targets, raising the Quantitation of CXCL1 and CXCL2 in skin by immunoassay possibility that the requirement for either FasL or perforin is CXCL1 and CXCL2 levels in skin were determined by ELISA. Mice were mediated through expression by other cells participating in the sensitized by application of 10 ml 0.25% DNFB to each side of each ear on elicitation of CHS responses (17–19). In this study, we investi- days 0 and +1. On day +5 the shaved trunk skin of both sensitized and gated mechanisms underlying the absent CHS responses following nonsensitized mice was challenged with 25 ml DNFB and the challenged challenge of hapten-sensitized gld/perforin2/2 mice. The results skin was excised 6 h later and homogenized in 500 ml proteinase inhibitor mixture (Sigma-Aldrich) with gentle shaking for 30 min. Following cen- indicate a novel function for neutrophils in expressing both FasL trifugation at 12,000 3 g for 10 min, supernatants were collected and total and perforin, which induce T cell chemoattractants and promote protein concentrations quantified using a Coomassie Plus protein assay CD8 T cell infiltration into the skin to mediate CHS. reagent kit (Pierce, Rockford, IL). All samples were diluted to an equivalent total protein concentration and tested in CXCL1- and CXCL2- Materials and Methods specific ELISA. Mice Analysis of tissue-infiltrating cells by flow cytometry C57BL/6 (H-2b) mice were obtained through Dr. Clarence Reeder (Na- The shaved trunk skin of sensitized and nonsensitized wild-type and gld/ tional Cancer Institute, Frederick, MD) and gld and perforin2/2 mice on perforin2/2 mice was challenged with DNFB and 6 or 18 h later the chal- a C57BL/6 background were obtained from The Jackson Laboratory (Bar lenged skin was removed and incubated in 0.5% dispase (Invitrogen Life Harbor, ME). Perforin2/2 and gld mice were first crossed to produce gld+/2/ Technologies, Carlsbad, CA) at 4˚C for 18 h. In some experiments groups of perforin+/2 mice and then these were intercrossed and the gld/perforin2/2 sensitized gld/perforin2/2 mice and anti–Gr-1 mAb-treated wild-type mice mice identified through PCR analysis of isolated tissue DNA. Female mice, were injected intradermally with 10 ng recombinant FasL (R&D Systems, 8–10 wk age, were used throughout these studies. Minneapolis, MN), either boiled for 15 min or not boiled, directly into four to five sites in the skin challenge site 2 h after hapten challenge and then Downloaded from Hapten sensitization and elicitation of CHS the challenged skin was removed 16 h after the injection. Following incubation Mice were sensitized to DNFB by painting the shaved abdomen with 25 ml of the excised skin, the epidermis was separated from the dermis and both were incubated in 0.5% trypsin (Sigma-Aldrich) at 37˚C/5% CO for 1 h. Both 0.25% DNFB (Sigma-Aldrich, St. Louis, MO) and 10 ml to each paw on 2 tissues were pressed through a kidney dialysis bag and the harvested cells days 0 and +1 (10, 12). On day +5 hapten-sensitized and control, nonsensitized mice were challenged on each side of each ear with 10 ml treated with 0.2% DNase (Roche, Indianapolis, IN) for 10 min at room temperature. The cells were washed twice with staining buffer (Dulbecco’s DNFB. Ear thickness was measured using an engineer’s micrometer 6 2 (Mitutoyo, Elk Grove Village, IL) and expressed in units of 10 4 in. The PBS with 2% FCS/0.2% NaN3) and 10 cell aliquots were incubated on http://www.jimmunol.org/ ear swelling response is given as the mean increase in thickness to chal- ice in staining buffer with 1:100 Fc Block (BD Biosciences) for 30 min. lenge for each group of four individual animals 6 SEM. The cells were washed and stained with fluorochrome-labeled anti-mouse mAb to CD45, CD3, and Gr-1. After 30 min, the cells were washed, Abs and cytokines resuspended in staining buffer, and analyzed by two-color flow cytometry using a FACSCalibur and CellQuest software (Becton Dickinson, San For flow cytometry the following Abs were used: anti-CD45 mAb, anti- Jose, CA). The cells were gated to exclude residual tissue debris and mouse CD3 mAb, anti-mouse NK1.1 mAb, and F4/80 (BD Pharmingen, nonviable cells, and sample data were collected on 2 3 104 CD45+ cells. San Diego, CA) and rat anti-mouse Gr-1 mAb (eBioScience, San Diego, CA). For in vivo depletion of CD4+ T cells, mice were injected with 100 mg Cell transfer each anti-CD4 mAb, YTS 191 and GK1.5, i.p. on 3 consecutive days For transfer of hapten-primed CD8 T cells, C57BL/6 wild-type and gld/ before hapten sensitization on days 0 and +1 (14, 20). Treated sentinel 2/2 + by guest on September 26, 2021 mice were used to evaluate the efficiency of CD4+ T cell depletion by Ab perforin mice were depleted of CD4 T cells prior to DNFB sensitization. On day +4 lymph node cell suspensions were prepared and 10 3 staining and flow cytometry analysis of spleen and lymph node cells and 6 2/2 was always .95% when compared with cells from control, rat IgG-treated 10 cell aliquots transferred to naive C57BL/6 or gld/perforin recipients mice. In vivo depletion of neutrophils was performed by injecting hapten- that were immediately challenged either with 10 ml DNFB on each side of sensitized mice with 100 mg anti-Gr-1 mAb, RB6.8C5, on the day before each ear to elicit the CHS response or with 25 ml DNFB on the shaved and the day of hapten challenge (13, 14). abdomen to assess cell infiltration into the challenge site. The challenged abdominal skin was excised 18 h after challenge and tissue homogenates ELISPOT assays for enumeration of hapten-specific T cells were prepared for flow cytometry analysis of infiltrating cells as detailed producing IFN-g or IL-17 above. For isolation and transfer of neutrophils, C57BL/6 mice were sensitized ELISPOT plates (Unifilter 350; Polyfiltronics Group, Rockland, MA) were with 0.25% DNFB on the shaved abdomen and paws and challenged on each coated with IFN-g– or IL-17–specific mAbs, incubated overnight in the side of each ear with 0.2% DNFB on day +5. Two hours later, bone marrow cold, and then blocked with 1% BSA in PBS. Responder lymph node cells was extracted from the femurs and tibiae. Bone marrow cells were washed from nonsensitized or DNFB-sensitized mice were prepared on day +5 and incubated with PE-conjugated mAb to B220, CD3, F4/80, and NK1.1 postsensitization and enriched for CD8 T cells by removing CD4 T cells (BD Pharmingen) to label populations of B cells, T cells, macrophages and with magnetic beads. Syngeneic spleen cells from naive mice were treated NK cells, respectively. Cells were subsequently incubated with anti-PE– with 50 mg/ml mitomycin C and labeled with 100 mg/ml DNBS for use as coated magnetic beads, loaded onto a MACS column (Miltenyi Biotec, stimulator cells. Stimulator cells were plated at 5 3 105 cells/well with Auburn, CA), and placed in a magnetic field to eliminate the B, T, and NK either 2 3 105,53 105,or13 106 responder cells/well in serum-free HL- cells. The resulting cell populations were .95% Gr-1+ cells and ,0.5% + + + + 1 medium (BioWhittaker, Walkersville, MD) supplemented with 1 mM L- CD3 , NK1.1 , and F4/80 cells and ,0.70% B220 cells (Supplemental glutamine. Responder cells plated with unlabeled splenocytes were used as Fig. 1). Aliquots of 2 3 107 RB6.8C5+ were transferred i.v. to sensitized a negative (hapten specificity) control. After 24 h, cells were removed by gld/perforin2/2 mice 2 h after challenge. extensive washing with PBS/0.05% Tween 20 and biotinylated anti–IFN-g mAb or biotinylated anti–IL-17 mAb was added. The plate was incubated Analysis of gene expression by quantitative RT-PCR overnight at 4˚C, washed with PBS/0.05% Tween 20, and conjugated Whole-cell RNA was prepared from excised skin, purified bone marrow streptavidin-alkaline phosphatase was added to each well. After 2 h at neutrophils, and primed lymph node CD8+ T cells from either DNFB- room temperature, the plate was washed with PBS/0.05% Tween 20 and sensitized or skin allograft recipient (A/J, H-2a → C57BL/6, H-2b) mice NBT/5-bromo-4-cholor-30-indolyl substrate (Bio-Rad Laboratories, Her- by dissolving the tissue or cells in TRIzol reagent (Invitrogen Life Tech- g cules, CA) added for detection of IFN- or IL-17. Resulting spots were nologies) with subsequent chloroform extraction. cDNA was synthesized counted with an ImmunoSpot series I analyzer (Cellular Technology, from 2 mg mRNA using the TaqMan reverse transcription reagent kit Cleveland, OH). (Applied Biosystems, Foster City, CA). PCR was performed using custom Histological analyses primers and FAM dye-labeled probes (Applied Biosystems) for mouse FasL, perforin, IL-17, IFN-g, CXCL9, CXCL10, CCL1, CCL2, CCL5, Hapten-challenged skin was excised from sensitized wild-type and gld/per- and Mrpl 32 (gene assay ID nos. Mm00438864_m1, Mm00812512_m1, forin2/2 mice 24 h after challenge. For staining to detect leukocyte infiltration Mm00439619_m1, Mm00801778_m1, Mm00434946_m1, Mm00445235_m1, into the challenge site, the skin tissue was fixed in 10% formalin, embedded Mm00441236_m1, Mm00441242_m1, Mm01302428_m1, and Mm00777741_ in paraffin, and 8-mm sections were prepared and stained with H&E. sH,respectively). The quality threshold of gene expression of one sample, the The Journal of Immunology 2193

RNA isolated from the skin homogenate of naive, not challenged, wild- type mice, was arbitrarily set at 1.0 andusedtodeterminetheexpression levels of the remaining samples. Cell lysis and Western blot analysis Neutrophils were enriched from the bone marrow of naive or sensitized C57BL/6 wild-type or gld/perforin2/2 mice as detailed above. CD8+ T cells were enriched from the skin-draining lymph nodes of C57BL/6 mice sensitized with DNFB or engrafted with complete MHC-mismatched A/J skin allografts. All cells samples were lysed in buffer containing 20 mM Tris-Cl (pH 8.3), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease and phosphatase inhibitors (5 mg/ml pepstatin A, 1 mM PMSF, 0.5 mM iodoacetamide, 1 mM sodium metavanadate, and 10 mM sodium fluoride) for 30 min on ice. Cell lysates were loaded onto precast gels prior to 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, Santa Cruz, CA) followed by secondary HRP secondary anti-rabbit Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) prior to development by ECL reagent (GE Healthcare Biosciences, Pittsburgh, PA). Resulting bands were quantified using ImageJ software.

Statistical analysis Downloaded from Statistical analysis to assess differences between experimental groups was performed using a Student t test. Differences were considered signiﬁcant when p , 0.05.

Results http://www.jimmunol.org/ Absence of CHS responses following challenge of hapten-sensitized gld/perforin2/2 mice To first confirm the reported absence of CHS responses to sensitization and challenge of gld/perforin2/2 mice (15), groups of wild-type C57BL/6 and gld/perforin2/2 mice were sensitized with DNFB and challenged on the ear with the hapten on day +5. The increase in ear thickness of sensitized wild-type mice after challenge was ∼5-fold the increase measured in naive mice challenged with the hapten (Fig. 1A). In contrast to sensitized by guest on September 26, 2021 wild-type mice, the increase in ear thickness of sensitized gld/ perforin2/2 mice was nearly equivalent to that of challenged naive mice (Fig. 1A). Histological analyses indicated that the absent CHS response in gld/perforin2/2 mice was associated with decreased cellular infiltration into the skin challenge site, and the absence of the characteristic keratinocyte hyperplasia at 24 h FIGURE 1. Absence of contact hypersensitivity following challenge of 2/2 A postchallenge and these features remained so for several days hapten-sensitized gld/perforin mice. ( ) Groups of wild-type C57BL/6 and gld/perforin2/2 mice were sensitized with 0.25% DNFB on days 0 and after challenge (Fig. 1B and data not shown). The absent CHS 2/2 +1 and these mice and groups of unsensitized mice were challenged on the response in gld/perforin mice was further examined by ears with 0.2% DNFB. The change in ear thickness was determined 24 h excising the skin challenge site 24 h after challenge, digesting later and is shown as the mean increase in ear thickness for each group of the tissue to prepare single-cell suspensions, and staining cell four animals 6 SEM. *p , 0.02 when comparing increased ear thickness aliquots with Abs to assess T cell infiltration into the site. of the sensitized wild-type group versus sensitized gld/perforin2/2 and Consistent with the histological analyses, infiltrating CD3+ unsensitized groups. (B) DNFB-sensitized wild-type and gld/perforin2/2 T cells were virtually absent in the skin challenge site of sen- mice were challenged on the shaved abdominal trunk skin with DNFB. The sitized gld/perforin2/2 mice when compared with the challenge challenged skin was excised 24 h after challenge, fixed in formalin, and site of sensitized wild-type (Fig. 1C). prepared paraffin-embedded sections were stained with H&E. Represen- tative light microscopy images of skin from each group are shown. CD8 T cell priming is equivalent in sensitized wild-type and Original magnification 3400. (C) On day +5, DNFB-sensitized wild-type 2/2 gld/perforin2/2 mice and gld/perforin mice were challenged on the shaved abdominal trunk 2/2 skin with DNFB. Challenged skin was excised 18 h later, digested to The absence of CHS in sensitized gld/perforin mice raised prepare single-cell suspensions, and cell aliquots were stained with fluo- the possibility of a defect in the priming of the IFN-g–and/or rescent Abs to identify infiltrating (CD3+) T cells. A representative sample IL-17–producing CD8 T cell populations required for the re- from the challenged skin of one mouse per group is shown. sponse. The presence of these T cells was tested by ELISPOT analyses of lymph node CD8 T cells from sensitized wild-type 2/2 versus gld/perforin2/2 mice. In contrast with the low numbers Hapten-primed CD8 T cells from sensitized gld/perforin of T cells infiltrating the skin challenge site, numbers of mice function to mediate CHS in wild-type recipients hapten-specific CD8 T cells producing either IFN-g or IL-17 in Because IL-17– and IFN-g–producing CD8 T cells appeared to the skin draining lymph nodes of sensitized gld/perforin2/2 develop normally in response to hapten sensitization of gld/per- 2 2 2 2 mice were equivalent to those induced in sensitized wild-type forin / mice, the ability of the primed gld/perforin / CD8 T mice (Fig. 2). cells to mediate CHS responses following transfer to naive wild-type 2194 NEUTROPHIL MEDIATORS DIRECTING T CELLS INTO SKIN

CXCL1 and CXCL2 by endothelial cells in the challenge site and it is these chemokines that direct the initial infiltration of neutrophils during elicitation of CHS responses to DNFB and to oxazolone (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 chemoattractants and the infiltration of neutrophils into the challenge site 6 h after hapten challenge were investigated. Initial experiments clearly indicated high levels of IL-17 and IFN-g mRNA expression in the hapten challenge sites of sensitized wild-type and sensitized gld/perforin2/2 mice but not following hapten challenge of naive mice (data not shown). Consistent with these results equivalent levels of CXCL1 and CXCL2 protein were induced 6 h after challenge of sensitized wild-type and gld/perforin2/2 mice (Fig. 4A). Furthermore, equivalent levels of neutrophil infiltration were observed in the challenge site of both sensitized wild-type and gld/perforin2/2 mice 6 h after challenge (Fig. 4B). In con- junction with the absent CD8 T cell infiltration into the hapten 2 2 challenge site of sensitized gld/perforin / mice 18–24 h after Downloaded from challenge, neutrophil infiltration into the site was also absent at this time point. Overall, these results indicated that the absent CHS responses observed following challenge of sensitized gld/perforin2/2 mice are not due to a defect in the priming or function of the hapten-

specific CD8 T cells but rather to the environment within which http://www.jimmunol.org/ elicitation of the immune response occurs. Upregulated expression of FasL and perforin in the skin challenge site 6 h after hapten challenge of sensitized wild-type FIGURE 2. Hapten-specific CD8 T cell priming in DNFB-sensitized animals 2 2 2 2 wild-type and gld/perforin / mice. Wild-type C57BL/6 and gld/perforin / Because the early neutrophil infiltration occurring prior to hapten- mice were sensitized with DNFB and on day +5 lymph node cell suspen- primed CD8 T cell infiltration into the challenge site was observed sions were prepared from naive wild-type and the sensitized mice. CD4 in both sensitized wild-type and gld/perforin2/2 mice, the mRNA T cells were removed from the cells using anti-CD4 Ab-coated magnetic expression levels of FasL and perforin in the skin challenge site by guest on September 26, 2021 beads. Aliquots of 5 3 105 of the enriched CD8 T cells were cultured with 3 5 were tested. Hapten-challenged skin of sensitized wild-type and 5 10 DNBS-labeled or unlabeled syngeneic splenocytes in triplicate 2/2 cultures on IFN-g– or IL-17–coated ELISPOT plates. After 24 h, cells were gld/perforin mice was excised 6 h after challenge, whole-cell removed and the ELISPOT assay was developed to detect numbers of IFN-g– RNA was isolated from the tissue, and quantitative RT/PCR (qRT- or IL-17–producing cells. The mean number of hapten-specific CD8 T cells PCR) was performed to compare expression levels of FasL and producing IFN-g or IL-17 per 5 3 105 cells 6 SEM for groups of three mice perforin. When compared with skin excised from the hapten is shown. The number of spots from control wells containing the enriched challenge site of naive wild-type mice, hapten challenge of sen- CD8 T cells with unlabeled stimulator cells was always less than five spots sitized wild-type mice upregulated expression of both FasL and per well. Results are representative of two individual experiments. Numbers perforin by .20-fold (Fig. 5A). Background levels of both FasL of cytokine-producing CD8 T cells between sensitized wild-type and gld/ 2/2 and perforin expression in the hapten challenge sites of sensitized perforin mice are not significantly different. gld/perforin2/2 mice were observed and were $4-fold lower than the expression induced in the challenge site of sensitized wild- recipients was tested. Aliquots of DNFB-primed CD8 T cell type mice. suspensions from sensitized wild-type and gld/perforin2/2 mice Neutrophil expression of FasL and perforin was directly tested were transferred to naive wild-type or gld/perforin-/- recipients that by qRT-PCR analysis of RNA isolated from bone marrow neu- were then challenged with hapten. Primed CD8 T cells from both trophils and lymph node CD8 T cells from hapten-sensitized mice. sensitized wild-type and sensitized gld/perforin2/2 mice induced As a positive control, RNAwas prepared from CD8 T cells isolated equivalent CHS responses following transfer to naive wild-type from the draining lymph nodes of MHC-mismatched skin allograft mice and recipient challenge (Fig. 3A). However, CHS responses recipients and as expected expressed high levels of both FasL and in naive gld/perforin2/2 recipients of primed wild-type CD8 perforin (Fig. 5B). In contrast, RNA prepared from CD8 T cells T cells were reduced to near background levels. When the infil- isolated from lymph nodes of hapten-sensitized mice expressed tration of the primed CD8 T cells from the wild-type or gld/per- low levels of both mediators. Neutrophils obtained from the bone forin2/2 donors into the recipient skin challenge site tissue was marrow of sensitized and challenged wild-type mice clearly assessed 24 h after T cell transfer and recipient challenge, there expressed FasL and perforin mRNA, whereas neutrophils from the was clear infiltration of transferred hapten-primed CD8 T cells bone marrow of naive wild-type mice expressed slightly lower from sensitized wild-type or sensitized gld/perforin2/2 donors levels of perforin mRNA but low/background levels of FasL into the challenged skin of naive wild-type recipients but not when mRNA. the primed wild-type CD8 T cells were transferred to naive gld/ These studies were extended by performing immunoblot anal- perforin2/2 recipients (Fig. 3B). yses of cell lysates prepared from hapten-primed CD8 T cells and The activity of the hapten-specific CD8 T cell populations neutrophils from the bone marrow of DNFB-sensitized and producing IFN-g and IL-17 induces the initial production of -challenged wild-type and gld/perforin2/2 mice. As a positive The Journal of Immunology 2195 Downloaded from

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FIGURE 3. Hapten-primed CD8 T cells from sensitized gld/perforin mice elicit CHS following transfer to naive wild-type recipients. C57BL/6 and http://www.jimmunol.org/ 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 animals 6 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 fluorescent 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. by guest on September 26, 2021 control, lysates of CD8 T cells enriched from the lymph nodes of mice did not induce CHS responses to challenge of gld/perforin2/2 wild-type C57BL/6 recipients of complete MHC-mismatched A/J recipients (Fig. 7A). However, delivery of bone marrow neu- skin allografts expressed both the 70-kDa perforin protein and the trophils from sensitized and challenged wild-type mice induced 31-kDa FasL protein (Fig. 6A). Consistent with the mRNA anal- CHS responses similar to the responses observed in sensitized and yses, lysates of DNFB-primed CD8 T cells enriched from the challenged wild-type mice. Consistent with the CHS response lymph nodes of DNFB-sensitized wild-type mice did not express observed upon delivery of wild-type neutrophils to challenged gld/ either of these proteins, but neutrophils enriched from the bone perforin2/2 mice, the wild-type neutrophil transfer also induced marrow of DNFB-sensitized and -challenged wild-type but not infiltration of both Gr-1+ cells and CD3+ T cells into the hapten gld/perforin2/2 mice did express both perforin and FasL proteins. challenge site of sensitized gld/perforin2/2 mice 18 h after chal- Neutrophils enriched from the bone marrow of unsensitized/naive lenge that was absent without the neutrophil transfer (Fig. 7B). wild-type mice did not express either of the proteins in the FasL and perforin were expressed at low levels in isolated immunoblot analyses. Densitometric analyses indicated similar neutrophils from the bone marrow of naive/unsensitized wild-type levels of perforin and FasL protein in the lysates of the control mice (Figs. 5, 6). Therefore, we compared the restoration of skin allograft-primed CD8 T cells and the neutrophils from the CHS in sensitized gld/perforin2/2 mice that received aliquots bone marrow of hapten-sensitized and -challenged wild-type mice of neutrophils from the bone marrow of unsensitized/naive versus (Fig. 6B). DNFB-sensitized and challenged wild-type donors (Fig. 7C). In 2 2 contrast to the ability of bone marrow neutrophils from sensitized Reconstitution of CHS responses in gld/perforin / mice by and challenged wild-type donors to reverse the defective CHS neutrophils from hapten-sensitized and -challenged wild-type 2 2 response following challenge of sensitized gld/perforin / mice, mice transfer of bone marrow neutrophills from naive/unsensitized Because the results indicated the expression of FasL and perforin wild-type donors had only a modest effect on the CHS response in by wild-type neutrophils and not by hapten-primed CD8 T cells, the the sensitized gld/perforin2/2 recipients. ability of neutrophils from wild-type mice to reverse the defective CHS responses in sensitized gld/perforin2/2 mice was tested. Delivery of soluble FasL into the hapten challenge site Aliquots of bone marrow neutrophils or immune CD8 T cells from promotes the infiltration of hapten-primed CD8 T cells into the hapten-sensitized wild-type mice were transferred to sensitized site gld/perforin2/2 mice 2 h after hapten challenge and the increase Because transfer of wild-type neutrophils expressing FasL and in ear thickness was determined 20 h later. As previously shown, perforin could correct the defective trafficking of hapten-primed delivery of hapten-primed CD8+ T cells from sensitized wild-type CD8 T cells into the skin challenge site of sensitized gld/per- 2196 NEUTROPHIL MEDIATORS DIRECTING T CELLS INTO SKIN

FIGURE 4. Early neutrophil infiltration 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 challenged on the shaved abdominal trunk skin with DNFB. Challenged skin was Downloaded from excised either 6 or 18 h later and weighed. (A) Challenged skin homogenates were prepared and CXCL1 and CXCL2 concentrations determined by ELISA. The mean chemokine concentration in picograms per milligram excisedskintissue6 SEM for four http://www.jimmunol.org/ individual mice per group is shown. (B) Challenged skin was digested to prepare single cells and cell aliquots were stained with fluorescent Abs to identify skin-infiltrating Gr-1+ cells. Percen- tages shown in upper left quadrant indicate percentage of infiltrating CD45+ cells expressing Gr-1. Representative

samples from one mouse per group for by guest on September 26, 2021 groups of four mice are shown for two individual experiments.

forin2/2 mice, the ability of soluble FasL to direct this trafficking mice was corrected when recombinant soluble FasL was injected was investigated. As a proof of principle, the ability of the soluble directly into the hapten-challenged skin of these sensitized mice FasL to induce hapten-primed CD8 T cell trafficking into the and this activity was destroyed by boiling the soluble FasL for 15 hapten-challenged skin of sensitized wild-type mice depleted of min (Fig. 9). neutrophils at the time of hapten challenge was first tested (Fig. 8). As previously reported (10, 14), depletion of neutrophils by treating Expression of chemokines secreted by activated T cells is downregulated in the skin challenge site of sensitized gld/ hapten-sensitized wild-type mice with anti–Gr-1 mAb at the time 2/2 of challenge led to an absence of T cell infiltration into the hapten perforin mice challenge site. Despite the absence of the depleted neutrophils in To begin to investigate consequences of neutrophil FasL and the hapten challenge site, hapten-primed T cell infiltration into perforin expression in the skin 6 h after challenge, the hapten- the challenge site was restored when recombinant soluble FasL challenged skin from sensitized wild-type and gld/perforin2/2 was injected directly into the hapten-challenged skin of these mice was excised 6 h after challenge and whole-cell RNA was sensitized wild-type mice. The ability of the soluble FasL to isolated and tested for expression of genes that would be expected direct this infiltration was destroyed by boiling the soluble FasL to mediate the recruitment of the CD8 T cells and other leukocytes for 15 min. Similarly, the defect in hapten-primed T cell infil- into the challenge site (Fig. 10A). The expression of the T cell tration into hapten-challenged skin of sensitized gld/perforin2/2 chemoattractants CXCL9 and CXCL10 were observed at high The Journal of Immunology 2197 Downloaded from http://www.jimmunol.org/

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 by guest on September 26, 2021 amounts of lysate sample protein (2–3 mg) were resolved on a 7% gel for FIGURE 5. Expression of FasL and perforin by neutrophils in the Western blotting and probed with Abs to perforin, FasL, or b-actin. Images hapten challenge site of sensitized wild-type mice. (A) Groups of DNFB- 2 2 of immunoblots are representative of two individual experiments. (B) sensitized wild-type C57BL/6 and gld/perforin / mice and a group of Ratios of perforin or FasL signal to b-actin signal for each cell lysate unsensitized wild-type mice were challenged with DNFB on the abdominal sample were calculated from densitometric analysis of the immunoblot. 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- 2/2 pression levels of FasL and perforin. The mean expression level for each of gld/perforin mice was tested. Bone marrow neutrophils were four samples per group 6 SEM is shown. *p # 0.05 when comparing isolated from hapten-sensitized and -challenged wild-type donors mRNA expression levels of FasL and perforin from challenged skin of and transferred to sensitized gld/perforin2/2 recipients (Fig. 10B). 2/2 sensitized wild-type group versus sensitized gld/perforin and unsensi- Consistent with the absent CHS responses observed by depleting tized wild-type groups. All results are representative of two individual sensitized wild-type mice of neutrophils at the time of hapten B experiments. ( ) CD8 T cells were enriched from the draining lymph challenge, neutrophil depletion of sensitized wild-type mice at the nodes of C57BL/6 (H-2b) recipients of A/J (H-2a) skin allografts and time of challenge reduced the expression of CCL1, CCL2, and DNFB-sensitized wild-type C57BL/6 mice. Bone marrow cells were CCL5 to the levels observed following challenge of naive wild- flushed from the femurs of naive and DNFB-sensitized and -challenged 2/2 C57BL/6 mice and T (CD3+) cells, B (B220+) cells, NK (NK1.1+) cells, type or sensitized gld/perforin mice. Transfer of the wild-type and macrophages (F4/80+) were removed by Ab staining and magnetic neutrophils increased the expression of the chemoattractants in the 2/2 bead negative selection. For each test cell population, mRNA was prepared skin challenge site of the sensitized gld/perforin mice several and tested by qRT-PCR for expression levels of FasL and perforin. The fold above those observed in the wild-type mice. expression level for a single sample for each group is shown. All results are representative of two individual experiments with similar results observed Discussion each time. The directed infiltration of leukocytes through the vasculature and into parenchymal tissues under inflammatory duress is a highly levels in the skin challenge sites of both sensitized gld/perforin2/2 regulated process that functions to resolve the inflammatory insult and wild-type mice although the expression of CXCL9 was sig- and obviate the occurrence of inflammation at other tissue sites (1). nificantly higher in the site of the sensitized gld/perforin2/2 mice. Neutrophils are typically the first leukocytes to infiltrate sites of In contrast, expression levels of CCL1, CCL2, and CCL5 in the tissue inflammation directed by chemoattractants produced by the challenged skin of sensitized gld/perforin2/2 mice were equiva- vascular endothelium (21, 22). The binding of chemoattractants lent to levels of expression following challenge of the skin of to receptors on the surface of neutrophils also activates the release naive mice. of granules containing cytokines, chemokines, and extracellular Finally, the ability of wild-type neutrophil transfer to restore matrix-degrading enzymes that directly mediate tissue injury as expression of these chemokines in challenged skin of sensitized well as promote the infiltration of other leukocytes, including Ag- 2198 NEUTROPHIL MEDIATORS DIRECTING T CELLS INTO SKIN Downloaded from http://www.jimmunol.org/

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 7 prepared from skin-draining lymph nodes of sensitized wild-type C57BL/6 mice or 2 3 10 neutrophils isolated from the bone marrow of either non- by guest on September 26, 2021 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 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 , 0.01 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. primed T cells, into the inflammation site (16, 21). Subcutaneous that the hapten-primed CD8 T cells had infiltrated the skin chal- injection of recombinant IL-8 promotes the initial infiltration of lenge site and that cytotoxic activity through either FasL- or human neutrophils into the skin that is quickly followed by the perforin/granzyme B-mediated pathways were required for the infiltration of T cells in a neutrophil-dependent manner (20, 23). elicitation of CHS responses. As confirmed in this study, DNFB- Depletion of neutrophils or inhibition of neutrophil trafficking has sensitized gld/perforin2/2 mice do not exhibit CHS responses, but been shown to delay or attenuate T cell infiltration during allograft the current results question the role of CD8 T cell-mediated cy- rejection, delayed-type hypersensitivity, antiviral responses, and tolysis as an effector mechanism in CHS. We had been unable to responses to autoantigens in the CNS (24–28). Although neutrophils detect the expression of FasL on the surface of hapten-primed regulate the progression of inflammatory events during many im- CD8 T cells (18), and the present studies confirm the absence of mune responses, the mechanisms expressed by neutrophils to in- this expression as well as the absent expression of perforin in duce the key chemoattractants directing T cell infiltration through mRNA isolated from purified CD8 T cells from the lymph nodes the vasculature of inflammatory sites to mediate responses in of sensitized wild-type mice. Studies by several investigators have extravascular tissues remain poorly understood. also indicated the absence of hapten-primed CD8 T cell cytolysis The present studies were prompted by the reported absence of of hapten-labeled target cells (17, 19, 29). TUNEL analyses of CHS responses in mice deficient in both FasL and perforin ex- sections prepared from the hapten challenge sites of sensitized pression (15). The authors had detected the presence of IFN-g wild-type and gld/perforin2/2 mice indicated low but similar mRNA in the challenge site 6 but not 24 h after challenge of numbers of TUNEL+ cells throughout the challenged skin, indi- hapten-sensitized gld/perforin2/2 mice, leading them to conclude cating that FasL- or perforin/granzyme B-mediated apoptosis The Journal of Immunology 2199 Downloaded from http://www.jimmunol.org/

FIGURE 8. Delivery of recombinant FasL to the skin challenge site of FIGURE 9. Delivery of recombinant FasL to the skin challenge site of by guest on September 26, 2021 2 2 neutrophil-depleted sensitized wild-type mice restores hapten-primed T hapten-sensitized gld/perforin / mice restores T cell infiltration into the 2 2 cell infiltration into the site. C57BL/6 mice were sensitized with DNFB on site. Groups of C57BL/6 and gld/perforin / mice were sensitized with 2 2 days 0 and +1 then depleted of neutrophils by treatment with anti–Gr-1 DNFB on days 0 and +1. Sensitized wild-type and gld/perforin / mice mAb on day +4 and day +5. Untreated and neutrophil-depleted DNFB- and a group of naive wild-type mice were challenged on the abdomen with sensitized mice and a group of naive mice were challenged on the abdomen DNFB and 2 h later aliquots of 10 ng boiled or nonboiled recombinant with DNFB and 2 h later aliquots of 10 ng boiled or nonboiled recombi- FasL was injected directly into four to five sites within the challenged skin 2 2 nant FasL was injected directly into four to five sites within the challenged of sensitized gld/perforin / mice. The challenged skin was excised 16 h skin of sensitized mice depleted of neutrophils. The challenged skin was later, digested to prepare single-cell suspensions, and cell aliquots were excised 16 h later, digested to prepare single-cell suspensions, and cell stained with anti-CD45 mAb and mAb to identify skin infiltrating Gr-1+ or aliquots were stained with anti-CD45 mAb and mAb to identify skin- CD3+ cells. Representative samples from one mouse per group for groups infiltrating Gr-1+ or CD3+ cells. Representative samples from one mouse per of four mice are shown from two individual experiments. group for groups of four mice are shown from two individual experiments. and could have been affected by the anti-Gr-1 mAb treatment of cells in the site is not required for the elicitation of CHS (30–33). To directly test the ability of neutrophils to promote (A. Gorbachev, data not shown). Finally, CHS responses were hapten-primed CD8 T cell infiltration into hapten-challenged skin, absent in response to hapten challenge of naive gld/perforin2/2 transfer of highly purified neutrophils from the bone marrow of 2 2 recipients of hapten-primed CD8 T cells from sensitized wild-type wild-type mice to sensitized gld/perforin / recipients corrected donors. These results suggested an entirely different mechanism the defective hapten-primed CD8 T cell infiltration into the than hapten-specific CD8 T cell-mediated cytolysis underlying challenge site and CHS responses. Neutrophil transfer has been the absence of CHS responses to challenge of hapten-sensitized shown to restore T cell-mediated responses to autoantigens and gld/perforin2/2 mice. infectious pathogens in murine models (24, 34). The neutrophils CHS responses are inhibited when sensitized wild-type mice are isolated from the bone marrow express mRNA encoding both treated with anti–Gr-1 mAb or anti-CXCL1 Ab at the time of FasL and perforin, accounting for the ability to promote CHS 2 2 hapten challenge, and the intensity of neutrophil infiltration into responses following transfer to sensitized gld/perforin / mice. the hapten challenge site influences the number of hapten-specific The surprising expression of perforin was confirmed by staining of CD8 T cells infiltrating the site (13, 14). These results led us to cytospin preparations of the purified bone marrow neutrophils (D. propose a role for neutrophils in directing the infiltration of primed Kish, data not shown). Neutrophils from both humans and rodents CD8 T cells into the site. In addition to neutrophils, however, have been shown to express FasL, and human peripheral blood several other leukocyte populations express Gr-1, including plas- neutrophils have been reported to express perforin and granzymes macytoid dendritic cells and monocyte/macrophage populations A and B, although the function of these molecules in neutrophil- 2200 NEUTROPHIL MEDIATORS DIRECTING T CELLS INTO SKIN Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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 RNA was prepared and tested by qRT-PCR for expression levels of CCL1, CCL2, and CCL5. The mean expression level for each of four samples per group 6 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. mediated inflammation has not been investigated (35–39). Im- isolated from the bone marrow of DNFB-sensitized and -chal- portantly, note that in the present studies that neutrophils isolated lenged mice. Hapten application is likely to induce systemic in- from the bone marrow of naive mice expressed very little FasL flammatory mediators such as G-CSF that control the generation mRNA and lower amounts of perforin mRNA than did neutrophils of neutrophils (40–42). Recent studies have distinguished distinct The Journal of Immunology 2201 populations of neutrophils (43), and FasL and/or perforin may be absence of neutrophils expressing FasL and perforin. The mecha- expressed by specific neutrophil populations generated by hapten nisms directing Ag-primed CD8 T cells expressingvarious functions application to the skin. into extravascular tissues warrants further investigation to attain The FasL and perforin expressed by infiltrating neutrophils early optimal efficiency in immune responses as well as to regulate the after hapten skin challenge of sensitized mice results in the induction intensity and duration of these immune responses. of many chemokines that are chemoattractants for T cells. We considered two possible sources of these chemokines: the neu- Acknowledgments trophils themselves or other cells in the parenchymal tissue of We thank Drs. Anna Valujskikh, Booki Min, and Wink Baldwin for valuable the hapten challenge site induced to produce the chemokines by discussions and advice during the course of this work and the staff of the neutrophil-derived FasL and perforin. Certainly neutrophils are Cleveland Clinic Biological Resources Unit for excellent care of the animals a critical source of many chemokines during inflammatory pro- used in the study. cesses, but their activities, particularly the production of acute phase cytokines and mediators such as oxygen radicals, also stimulate Disclosures other cells to produce chemokines (44–47). We interrogated mRNA The authors have no financial conflicts of interest. prepared from the neutrophils used in the transfer experiments and observed low CCL2 and CCL5 and absent CCL1 expression (D. References Kish, data not shown). This suggests that neutrophils are a source of 1. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the at least CCL2 and CCL5 during the elicitation of CHS but that site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu- neutrophil expression of FasL and perforin induces other cells in nol. 7: 678–689. Downloaded from the challenge site to produce CCL1, CCL2, and CCL5. It is also 2. Eisen, H. N., L. Orris, and S. Belman. 1952. Elicitation of delayed allergic skin reactions with haptens; the dependence of elicitation on hapten combination with possible that neutrophil functions are transcriptionally upregulated protein. J. Exp. Med. 95: 473–487. upon infiltration into inflammatory sites, as has been observed 3. Enk, A. H. 1997. Allergic contact dermatitis: understanding the immune response and potential for targeted therapy using cytokines. Mol. Med. Today 3: during lung injury (48). However, it is difficult to reconcile pre- 423–428. formed or newly transcribed chemokine mRNA in FasL- and 4. Flax, M. H., and J. B. Caulfield. 1963. Cellular and vascular components of allergic contact dermatitis. Am. J. Pathol. 43: 1031–1053. perforin-deficient neutrophils with the low levels of the chemokines http://www.jimmunol.org/ 2/2 5. Usatine, R. P., and M. Riojas. 2010. Diagnosis and management of contact observed in the challenge site of the gld/perforin mice. dermatitis. Am. Fam. Physician 82: 249–255. 2 2 Following hapten challenge of sensitized gld/perforin / mice, 6. Bour, H., E. Peyron, M. Gaucherand, J.-L. Garrigue, C. Desvignes, D. Kaiserlian, J.-P. Revillard, and J.-F. Nicolas. 1995. Major histocompatibility higher IFN-g and IL-17 and equivalent CXCL1 and CXCL2 and + + complex class I-restricted CD8 T cells and class II-restricted CD4 T cells, neutrophil infiltration were observed when compared with wild- respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. type mice. Higher levels of the IFN-g induced chemokines Eur. J. Immunol. 25: 3006–3010. 7. Gocinski, B. L., and R. E. 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