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Originally published online as doi:10.1189/jlb.0304193 on August 24, 2004

Published online before print August 24, 2004
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(Journal of Leukocyte Biology. 2004;76:941-949.)
© 2004 by Society for Leukocyte Biology

The intensity of neutrophil infiltration controls the number of antigen-primed CD8 T cells recruited into cutaneous antigen challenge sites

Tara Engeman*, Anton V. Gorbachev*, Danielle D. Kish* and Robert L. Fairchild*,{dagger},{ddagger},1

* Department of Immunology and
{dagger} The Glickman Urological Institute, The Cleveland Clinic Foundation, Ohio; and
{ddagger} Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio

1 Correspondence: NB3-79. Dept. Immunology, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195-0001. E-mail: fairchr{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recruitment of antigen-specific T cells into the skin is a critical initiating event during immune responses to many parasites and tumors as well as T cell-mediated, cutaneous, allergic responses and autoimmune diseases. Mechanisms directing T cell trafficking into skin remain largely undefined. Here, we show that cutaneous contact with reactive antigen induces KC/CXC chemokine ligand 1 production and neutrophil infiltration in an antigen, dose-dependent manner. The intensity of neutrophil infiltration into cutaneous antigen challenge sites, in turn, controls the number of antigen-primed T cells recruited into the site and the magnitude of the immune response elicited. The absence of responses in immune animals challenged with suboptimal doses of antigen is overcome by manipulating neutrophil infiltration that then directs antigen-primed T cell infiltration into the challenge site. This inflammation also directs T cells primed to one antigen (dinitrofluorobenzene) into the site when challenged with a completely different antigen (oxazolone). These results identify the intensity of neutrophil infiltration into cutaneous, antigen-deposition sites as a critical parameter for the level of antigen-primed T cell recruitment to mediate the adaptive immune response. This interplay between the innate and adaptive responses suggests a strategy to modulate, in a positive or negative manner, antigen-primed T cell infiltration into cutaneous inflammation sites.

Key Words: chemokines • delayed-type hypersensitivity • leukocyte trafficking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell trafficking into peripheral tissues during the elicitation of immune responses requires the coordinated functions of adhesion molecules, chemokines, and their respective ligand/receptors [1 ]. Antigen-primed T lymphocyte recruitment into the skin is a critical event initiating protective as well as harmful immune responses in the dermis and epidermis [2 3 4 ]. During the development of the immune system, the skin of neonatal mice is accessible to CD8+ T cells in the absence of overt inflammatory stimuli, but this trafficking is not observed in the skin of adult animals [5 ]. Similarly, studies following the trafficking of antigen-primed T cells have indicated the absence of trafficking through the skin without antigen challenge or appropriate inflammatory signaling [5 6 7 ]. These studies indicate the presence of inflammatory mechanisms regulating the entry of T cells into the adult skin.

Allergic contact dermatitis or contact hypersensitivity (CHS) is a T cell-mediated immune response to hapten sensitization and challenge of the epidermis that is the most frequently observed dermatosis in industrialized countries [8 ]. During sensitization of the epidermis, the reactive hapten covalently couples to cell-surface proteins, and the haptenated proteins are acquired and processed by the epidermal dendritic cells (DC), Langerhans cells, which then migrate to the skin-draining lymph nodes and prime hapten/major histocompatibility complex (MHC)-specific T cell populations [9 10 11 ]. Hapten-specific CD8+ T cells are the primary effector cells mediating CHS responses to many haptens, including dinitrofluorobenzene (DNFB), oxazolone (Ox), fluorescein isothiocyanate, and urushiol, the reactive agent in poison ivy [12 13 14 15 ]. Studies from this laboratory have demonstrated that sensitization with DNFB or Ox induces polarized populations of hapten-specific T cells: CD8+ T cells producing interferon-{gamma} (IFN-{gamma}), which are the effector cells of the response and interleukin (IL)-4/IL-5-producing populations of CD4+ T cells [16 ]. Subsequent challenge with the hapten results in the cutaneous infiltration of the hapten-primed CD8+ T cells and their activation to produce proinflammatory cytokines including IFN-{gamma} and tumor necrosis factor {alpha}, which cause the characteristic edema/spongiosis of the CHS response [17 ].

Chemokines produced by epidermal keratinocytes (CTACK; cutaneous T cell-attracting chemokine) and vascular endothelium (TARC; thymus and activation-regulated chemokine) have recently been shown to play a role in directing hapten-primed T cell trafficking into the skin following hapten challenge to mediate CHS [18 , 19 ]. In response to hapten application, keratinocytes also produce chemokines for other leukocyte populations including the neutrophil chemoattractants IL-8 and KC/CXC chemokine ligand 1 (CXCL1). Antibodies to KC, when given at the time of challenge, inhibit the elicitation of CHS responses to DNFB and Ox in sensitized mice [20 ]. The inhibitory effect of KC-specific antibodies was circumvented by delivery of hapten-primed T cells to the site followed by challenge, indicating that KC/CXCL1 has a role in directing hapten-primed T cell trafficking to the skin challenge site during elicitation of CHS responses. However, CXCL1 is a neutrophil chemoattractant and does not have direct chemotactic properties for T cells, suggesting that the role of CXCL1 in this response may be indirectly mediated through neutrophils. This possibility prompted investigation into the ability of neutrophils to control T cell-mediated responses to epidermal sensitization and challenge with hapten and to test if the level of innate immunity expressed at the time of antigen challenge correlated with the number of antigen-primed T cells recruited into the challenge site.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
BALB/c (H-2d) and C57BL6 (H-2b) mice were obtained through Dr. Clarence Reeder (National Cancer Institute, Frederick, MD). Adult females, 6–8 weeks of age, were used in all experiments.

Antibodies and cytokines
Purified monoclonal antibodies (mAb) YTS 191.1.2 and GK1.5 (anti-mouse CD4) and YTS 169 and TIB-150 (anti-mouse CD8) were purchased from Ligocyte (Bozeman, MT). RB6.8C5 anti-mouse Ly6G mAb was purified from culture supernatants using protein G chromatography. Capture and detection mAb for IFN-{gamma} were purchased from PharMingen (San Diego, CA). Recombinant KC (rKC)/CXCL1 was purchased from R&D Systems (Minneapolis, MN).

Hapten sensitization and elicitation of CHS
Mice were sensitized to DNFB by painting the shaved abdomen with 25 µl 0.25% DNFB (Sigma Aldrich, St. Louis, MO) and with 10 µl on each footpad on days 0 and +1. On day +5, hapten-sensitized and control, unsensitized mice were challenged with 10 µl 0.2% DNFB on both sides of each ear. The increase in ear swelling was measured at 24 h intervals after challenge using an engineer’s micrometer (Mitutoyo, Elk Grove Village, IL) and expressed in units of 10–4 inches as previously reported [16 ]. The ear-swelling response is presented as the mean increase of each group of four sensitized or nonsensitized mice (i.e., eight ears) ± SEM. The statistical significance of ear-swelling responses between experimental groups of mice was determined using Student’s t-test.

Transfer of neutrophils to ear tissue
Neutrophils were elicited by intraperitoneal (i.p.) injection of 1 ml sterile thioglycollate (Difco Laboratories, Detroit, MI), and 4 h later, the cells were harvested by washing the peritoneal cavity with Hanks’ balanced saline solution (HBSS) plus heparin. Wright’s stain of aliquots of the peritoneal wash cells indicated >70% neutrophils. The peritoneal exudate cells were washed and resuspended at 2 x 107 cells/ml in HBSS, and 50 µl was injected intradermally (i.d.) into the pinnae of each ear. Immediately following cell transfer, ears were challenged with hapten, and the increase in ear swelling was measured 24 and 48 h later. Previous results have demonstrated that removal of neutrophils from peritoneal exudate cells abrogated reconstitution of CHS responses [20 ].

Quantification of KC/CXCL1 protein in ear tissue
Production of CXCL1 was determined by enzyme-linked immunosorbent assay (ELISA). Mice were sensitized with 0.25% DNFB on days 0 and +1. On day +5, the ears were challenged with various concentrations of DNFB, and 24 h later, the ears were excised and homogenized in 500 µl phosphate-buffered saline (PBS) with 0.01 M EDTA and a proteinase inhibitor cocktail (10 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 100 µg/ml Pefabloc SC, and 100 µg/ml chymostatin) and 1 ml 1.5% Triton X-100 in PBS. Samples were gently shaken at 4°C for 30 min, then centrifuged at 12,000 g for 10 min, and the supernatants collected. Total protein concentration for each sample was quantified using the Coomasie Plus protein assay reagent kit (Pierce, Rockford, IL). All samples were diluted to 2 mg/ml, and the concentrations of tissue KC were tested using a KC/CXCL1 immunoassay (R&D Systems).

Immunohistochemistry to detect infiltrating neutrophils in ear tissue
Mice were sensitized with 0.25% DNFB as above. On day +5, ears of sensitized and naïve mice were challenged with various concentrations of DNFB, and 24 h later, the ears were excised and fixed with 10% buffered formalin. Paraffin-embedded, 8 µm sections were prepared and cut on edge. Slides were fixed in acetone for 10 min and air-dried. Slides were immersed in PBS for 10 min and in 0.03% H2O2 in PBS for 10 min at room temperature (RT) to eliminate endogenous peroxidase activity. Endogenous biotin activity was blocked with the biotin-blocking system (DAKO, Carpinteria, CA). Slides were stained for 1 h at RT with RB6-8C5 diluted at 10 µg/ml in 0.05% Tris-HCl buffer with 1% bovine serum albumin (BSA). Control slides were incubated with rat immunoglobulin G (IgG) as the primary Ab. After three washes in PBS for 5 min each, slides were incubated for 20 min with biotinylated rabbit anti-rat IgG, diluted 1:300 in the same buffer. After three washes in PBS, slides were incubated with streptavidin-horseradish peroxidase (DAKO) for 20 min. The substrate-chromagen solution was prepared by dissolving a 3,3'-diaminobenzidine (DAB), 10 mg tablet (Sigma Chemical Co., St. Louis, MO) in 15 mL PBS, and 12 µl 30% H2O2 was added just before use. After three washes in PBS for 5 min each, the DAB solution was applied to the slides and incubated for 2–3 min. After a wash in dH2O, slides were counterstained with hematoxylin and eosin (H&E), rinsed with dH2O, and immersed in 37 nM NH4OH for 10 s. Finally, the slides were dehydrated, coverslipped, and viewed with a light microscope. Images were captured using Image Pro Plus (Media Cybernetics, Silver Spring, MD). The number of neutrophils was counted in five random fields/slide and three slides/ear for three different ears at x40 magnification.

Enzyme-linked immunospot (ELISPOT) assays for enumeration of hapten-specific, IFN-{gamma}-producing T cells infiltrating hapten-challenged ear tissue
Groups of sensitized and unsensitized mice were challenged with the indicated dose of DNFB, 10 µl on each side of both ears, and 24 h later, the ears were excised and cut into small pieces and placed in Petri dishes containing 2.77 mg/ml collagenase (Sigma Chemical Co.), 1 mg/ml hyaluronidase (Sigma Chemical Co.), and 0.1 mg/ml DNase-I (Roche Diagnostics, Indianapolis, IN) in RPMI 1640 supplemented with 1 mM sodium pyruvate and 20 mM HEPES buffer. Ears were incubated in the enzyme solution for 2 h at 37°C in 5% CO2. After, the incubation ears were pressed through nylon mesh bags using a syringe plunger. The cell suspensions were collected and washed. The cells were resuspended in 4 ml RPMI, placed over 2 ml Lympholyte-M gradient (Cedarlane, Hornby, Canada), and centrifuged at 2000 rpm for 30 min. Interface cells were collected and washed in RPMI 1640 two times, and the cells were cultured in ELISPOT assays to determine the number of hapten-specific, IFN-{gamma}-producing T cells per ear. It should be noted that small numbers of cells are isolated from the digested ear tissue during elicitation of CHS responses requiring the pooling of cells from five mice for one individual sample to test the number of infiltrating cells producing IFN-{gamma} in response to hapten stimulation.

ELISPOT assays were performed as described previously [21 , 22 ]. Briefly, ELISPOT plates (Unifilter 350, Polyfiltronics, Rockland, MA) were coated with 4 µg/ml IFN-{gamma}-specific mAb and incubated overnight at 4°C. The plates were blocked with 1% BSA in PBS and washed four times with PBS. Syngeneic spleen cells from naïve mice were labeled with 100 µg/ml DNBS and treated with 50 µg/ml mitomycin C before use as stimulator cells, which were plated at 5 x 105 cells/well with 2 x 105 or 5 x 105 responder cells/well in serum-free HL-1 medium (BioWhittaker, Walkersville, MD), supplemented with 1 mM L-glutamine and 1 mM antibiotic, as described previously. Responder cells plated with unlabeled splenocytes were used as a negative (hapten-specificity) control. After 24 h of cell culture at 37°C in 5% CO2, cells were removed from the plate by extensive washing with PBS/0.05% Tween-20 (PBS-T). Biotinylated anti-IFN-{gamma} (2 µg/ml) was added, and the plate was incubated overnight at 4°C. The following day, the plate was washed three times with PBS-T, and conjugated streptavidin-alkaline phosphatase was added to each well. After 2 h at room temperature, the plates were washed with PBS-T and nitroblue tetrazolium/5-bromo-4-cholor-30-indolyl substrate (Bio-Rad Laboratories, Hercules, CA). The resulting spots were counted with an ImmunoSpot Series I analyzer (Cellular Technology Ltd., Cleveland, OH), which was designed to detect ELISA spots with predetermined criteria for spot size, shape, and colorimetric density. The mean number ± SEM of cytokine-producing cells in triplicate cultures for two individual mice is shown after subtraction of spots from control wells containing T cells with unlabeled stimulator cells. All experiments were repeated at least two times with similar results observed each time, and results from a representative experiment are shown.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increasing hapten challenge dose results in neutrophil-dependent increases in T cell recruitment and ear-swelling responses in sensitized animals
Challenge of hapten-sensitized animals with increasing doses of hapten resulted in increased immune responses, which were dependent on neutrophils (Fig. 1 ). Groups of mice were sensitized with an optimal dose of DNFB (0.25%) on 2 consecutive days (days 0 and +1). On day +5, mice were challenged on the ears with doses of DNFB ranging from 0.05% to 0.5%, and the increase in swelling was assessed 24 h later. In contrast to unsensitized mice, sensitized mice challenged with the dose of DNFB normally used for challenge, 0.2%, displayed an easily detectable increase in ear swelling (Fig. 1 , Group 3 vs. 4). Challenge with a higher dose of DNFB (0.5%) increased the magnitude of ear-swelling responses in sensitized mice but also increased the ear thickness of unsensitized mice (Fig. 1 , Groups 6 and 8 vs. 3 and 5). Challenge with a suboptimal dose, 0.05% DNFB, did not elicit a CHS response (Fig. 1 , Groups 1 and 2).



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  		Figure 1. Increased CHS response is induced by increasing challenge (chlg) hapten dose and is dependent on neutrophils. Groups of four BALB/c mice were sensitized with 0.25% DNFB on days 0 and +1, and on day +5, each animal was challenged by applying 10 µl of the indicated dose of DNFB to each side of both ears. As indicated, DNFB-sensitized (DNFB sens) animals were treated with 100 µg control, rat IgG, or depleted of neutrophils ({alpha}nØ) by giving 100 µg aliquots of RB6-8C5 i.p. on days +4 and +5 after sensitization. This treatment (Tx) resulted in <5% neutrophils in the peritoneal wash of mice 4 h after thioglycollate injection, as assessed by staining the peritoneal cells with Wright’s stain. Depletion of neutrophils at the time of ear challenge with 0.2% or 0.5% DNFB resulted in significantly decreased CHS responses (*, P<0.005).

 
To test the requirement of neutrophils for the elicitation of these swelling responses, groups of the sensitized mice were depleted of neutrophils by treatment with anti-Ly6G mAb RB6.8C5 [16 ] on the day before and the day of hapten ear challenge (i.e., days +4 and +5). The ear-swelling response in mice elicited by challenge with 0.2% DNFB was almost completely eliminated by neutrophil depletion prior to hapten challenge (Fig. 1 , P<0.005, Group 3 vs. 4). Responses of sensitized mice to challenge with 0.5% DNFB was also decreased by neutrophil depletion to near the level observed in unsensitized animals challenged with the high hapten dose (Fig. 1 , P<0.005, Group 6 vs. 7). Differences in ear-swelling responses between sensitized mice depleted of neutrophils before challenge with 0.5% DNFB and challenge of unsensitized/nonimmune mice with 0.5% DNFB were not significantly different.

In CHS responses to the model haptens DNFB and Ox as well as to urushiol, the reactive hapten of poison ivy, the primary effector cells are IFN-{gamma}-producing CD8+ T cells [13 14 15 16 ]. Associated with the increased immune responses, challenge of hapten-sensitized animals with increasing doses of hapten also resulted in increased, hapten-specific, IFN-{gamma}-producing T cell infiltration into the challenge site, which was entirely dependent on neutrophils (Fig. 2 ). At 24 h after challenge of sensitized mice with 0.2% or 0.5% DNFB, the ear-infiltrating leukocytes were isolated, and the number of IFN-{gamma}-producing, hapten-specific T cells in the infiltrate was tested by ELISPOT assay [22 ]. IFN-{gamma}-producing T cells were detected following retrieval from the hapten-challenged ears of sensitized mice when cocultured with DNFB-labeled, but not unlabeled, stimulator cells. IFN-{gamma}-producing T cells were not detected in the ears of unsensitized mice challenged with 0.2% or 0.5% DNFB. Consistent with the increased ear-swelling responses observed when sensitized animals were challenged with higher doses of hapten, the number of hapten-specific, IFN-{gamma}-producing, T cell-infiltrating ears challenged with 0.5% DNFB was threefold greater than with 0.2% challenge. Regardless of the challenge dose, depletion of neutrophils prior to hapten challenge abrogated T cell infiltration into the ears, indicating the requirement for these leukocytes to direct hapten-primed effector T cell recruitment into hapten challenge sites for the elicitation of CHS responses.



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  		Figure 2. Increased numbers of antigen-primed T cells infiltrating the challenge site induced by increasing challenge hapten dose are dependent on neutrophils. Groups of DNFB-sensitized and unsensitized BALB/c mice were challenged on day +5 with the indicated dose of DNFB, and 24 h later, the ear-infiltrating leukocytes were isolated from groups of five animals and tested for the number of hapten-specific T cells producing IFN-{gamma} by performing ELISPOT assays using DNFB-labeled and unlabeled stimulator cells. The indicated groups were depleted of neutrophils (nØ depl) during challenge by treatment with RB6-8C5 as above. The number of IFN-{gamma}-producing T cells per ear is shown after subtraction of spots from control wells containing T cells with unlabeled cells (less than five spots per well). ND, Not detectable.

 
The neutrophil-regulated recruitment of antigen-primed, effector T cells into the site suggested an antigen-nonspecific component of this response. To test this, the ability of a different antigen to induce the recruitment of antigen-primed T cells into the ear challenge site was investigated. Mice were sensitized with DNFB on days 0 and +1, and on day +5, the ears were challenged with DNFB or with Ox, and 24 h later, the number of DNFB-specific CD8+ T cells infiltrating the ear tissue was examined using ELISPOT assays (Fig. 3 ). Challenge of naïve mice with DNFB or Ox resulted in the ear tissue infiltration of low to undetectable numbers of T cells producing IFN-{gamma} in response to culture with DNP-labeled stimulator cells. As observed above, challenge of DNFB-sensitized mice with 0.2% DNFB resulted in the infiltration of DNP-reactive T cells producing IFN-{gamma} during culture with DNP-labeled stimulator cells. Similarly, challenge of DNFB-immune mice with Ox resulted in the infiltration of DNP-reactive T cells producing IFN-{gamma} during culture with DNP-labeled cells. As previously observed [16 , 23 ], challenge of DNFB-sensitized mice with Ox did not elicit an ear-swelling response (data not shown).



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  		Figure 3. Antigen-nonspeciifc infiltration of antigen-primed T cells into skin challenge sites. Groups of DNFB-sensitized and unsensitized BALB/c mice were challenged on day +5 with 0.2% DNFB or 1% Ox, and 24 h later, the ear-infiltrating leukocytes were isolated and tested for the number of dinitrophenol (DNP)-specific T cells producing IFN-{gamma} by performing ELISPOT assays using DNFB-labeled and unlabeled stimulator cells. The number of IFN-{gamma}-producing T cells per ear is shown after subtraction of spots from control wells containing T cells with unlabeled cells (less than five spots per well).

 
Increased production of KC/CXCL1 and neutrophil infiltration with increasing hapten challenge dose
Hapten application with 0.2% DNFB quickly induces KC expression of KC/CXCL1 with peak expression levels observed between 6 and 9 h after contact with the hapten [12 , 20 ]. The effect of increasing hapten challenge dose on production of CXCL1 and neutrophil infiltration into the challenge site of sensitized animals were tested. Challenge site production of CXCL1 production was low to absent 6 h after challenge with 0.05% DNFB but was readily apparent following challenge with 0.2% and increased further with increasing challenge doses (Fig. 4 ). Similarly, increasing challenge doses resulted in increased amounts of CXCL1 mRNA in the challenged tissue (data not shown). Correlating with the increased production of CXCL1, immunohistochemical analyses indicated increased intensity of neutrophil infiltration into the DNFB-challenged ears with an increasing hapten challenge dose (Fig. 5a and 5b ).



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  		Figure 4. Increasing hapten challenge dose induces increased production of KC/CXCL1. Groups of BALB/c mice were sensitized with 0.25% DNFB. On day +5, each animal was challenged by applying 10 µl of the indicated dose of DNFB to each side of both ears, and 6 h later, the ears were excised, and tissue homogenates were prepared and standardized to 2 mg total protein/ml. The level of CXCL1 protein was tested by sandwich ELISA.

 


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  		Figure 5. Increasing hapten challenge dose induces increased neutrophil infiltration into the challenge site. (a) Comparison of neutrophil infiltration into hapten challenge sites of sensitized mice 6 h after hapten challenge with the indicated challenge dose of DNFB by immunohistochemistry using anti-Ly6G mAb and RB6.8C5 and by H&E staining of formalin-fixed sections. (b) The number of positively staining cells, neutrophils, was counted in five random 40x fields for three different tissue sections, and the mean number of cells per microscopic field (mF) following challenge is indicated.

 
Promotion of primed T cell recruitment in the absence of an immune response by manipulation of innate immunity
The increased production of neutrophil chemoattractants and neutrophil infiltration following challenge with higher doses of hapten led to greater numbers of hapten-primed T cells infiltrating hapten challenge sites and greater CHS responses. This suggested that absent CHS responses to a low hapten challenge dose in sensitized animals might be overcome by manipulating neutrophil infiltration into the challenge site. On day +5, neutrophils were delivered directly into the ear pinnae of DNFB-sensitized mice, which were then challenged with suboptimal doses of DNFB (0.04% or 0.008%) that did not induce KC expression. In contrast to challenge with 0.2% DNFB, challenge with suboptimal DNFB doses, 0.04% and 0.008%, did not elicit a CHS response in DNFB-sensitized mice (Fig. 6a ). However, injecting neutrophils directly into the challenge site at the time of low-dose hapten challenge induced a readily observable CHS response in the sensitized mice (P<0.005, when compared with responses without neutrophil injection). Injection of neutrophils into unsensitized mice did not induce an ear-swelling response to challenge with the optimal challenge dose, 0.2% DNFB (P<0.005, Group 1 vs. 2), indicating that neutrophils and hapten in the absence of hapten-primed T cells did not mediate the ear-swelling response.



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  		Figure 6. The absence of contact sensitivity in immune animals following suboptimal hapten challenge is circumvented by delivery of neutrophils to the challenge site. (a) Groups of sensitized BALB/c mice were challenged with an optimal, 0.2%, or suboptimal, 0.04%, dose of DNFB, and the increase in ear thickness was measured 24 h post-challenge. Neutrophils (nØ) were elicited by injecting thioglycollate i.p. and 4 h later, washing the peritoneal cavity with HBSS plus heparin. The peritoneal exudate cells were washed and resuspended in HBSS, and 50 µl was injected i.d. into the ears of the indicated groups. Immediately following cell transfer, ears were challenged with hapten, and the increase in ear swelling was measured 24 h later (*P<0.005, Groups 1 vs. 2, 5 vs. 4, and 7 vs. 6). (b) Neutrophils direct the recruitment of primed T cells to sites of low antigen challenge. Groups of DNFB-sensitized and unsensitized BALB/c mice were challenged on day +5 with the indicated dose of DNFB. Just before challenge, the ear pinnae of the indicated groups were injected with 50 µl PBS with or with out neutrophils, and 24 h later, the ear-infiltrating leukocytes were isolated and tested for the number of hapten-specific T cells producing IFN-{gamma} by ELISPOT.

 
Consistent with the appearance of ear-swelling responses induced to the low hapten challenge dose by delivering neutrophils directly to the challenge site, the injection of these leukocytes into the ears of DNFB-sensitized mice just prior to hapten challenge with 0.04% DNFB restored the infiltration of hapten-specific, IFN-{gamma}-producing T cells into the ear tissue (Fig. 6b) . These results indicate that the absence of immune T cell recruitment and immune response in antigen-primed animals to low amounts of antigen challenge can be overcome by delivery of neutrophils to the challenge site.

In place of neutrophil delivery, injection of KC/CXCL1 into the site also directed T cell recruitment into the hapten-challenged ears to mediate CHS to low-dose hapten challenge. The ear pinnae of groups of DNFB-sensitized mice were injected with 50 µl PBS (Fig. 7a , Group 3) or 10 or 100 ng rKC (Groups 4 and 5) and were then challenged with 0.04% DNFB. Additional groups of sensitized mice (Group 1) and unsensitized mice (Group 2) were challenged with 0.2% DNFB as positive and negative control groups, respectively. Delivery of KC/CXCL1 to the challenge site induced a low but detectable CHS response to low hapten challenge dose at 24 and 48 h after challenge. Injection of CXCL1 into the ears of immune animals just before challenge with a suboptimal dose of hapten also restored recruitment of T cells to the ear, as indicated in ELISPOT assays, to detect the number of hapten-specific, IFN-{gamma}-producing T cells (Fig. 7b , Group 2 vs. 3). The effect of CXCL1 on this recruitment and the resulting ear-swelling response were circumvented by depletion of neutrophils just prior to hapten challenge (Group 4). Although injection of CXCL1 induced a threefold greater number of hapten-specific CD8+ T cells into the ear during challenge with 0.04% DNFB when compared with challenge with 0.2% (Fig. 7b , Group 3 vs. 1), the disparity in the ear-swelling responses elicited (Fig. 7a , Group 1 vs. 4) is likely to reflect the lower amount of hapten available to activate the recruited T cells to mediate the ear-swelling response in the former group. Collectively, these results demonstrate that delivery of CXCL1 to the challenge site elicits antigen-primed T cell recruitment and the elicitation of CHS in response to suboptimal hapten challenge through a neutrophil-mediated mechanism.



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  		Figure 7. The absence of contact sensitivity in immune animals following suboptimal hapten challenge is circumvented by delivery of KC/CXCL1 and is dependent on neutrophils. (a) Groups of sensitized BALB/c mice were challenged with an optimal, 0.2%, or suboptimal, 0.04%, dose of DNFB. Just before challenge, the ear pinnae of the indicated groups were injected with 50 µl PBS containing 10 or 100 ng rKC. The increase in ear thickness was measured 24 and 48 h post-challenge (*, P<0.0075, Group 4 vs. Group 3; **, P<0.03, Group 5 vs. Group 3). (b) Groups of DNFB-sensitized and unsensitized BALB/c mice were challenged on day +5 with an optimal, 0.2%, or suboptimal, 0.04%, dose of DNFB. The indicated group was depleted of neutrophils (nØ depl) during challenge by treatment with RB6-8C5 as above. Just before challenge, the ear pinnae of the indicated groups were injected with 50 µl PBS containing 100 ng rKC, and 24 h later, the ear-infiltrating leukocytes were isolated and tested for the number of hapten-specific T cells producing IFN-{gamma} by ELISPOT.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Considerable interest has been raised recently concerning the ability of innate immune mechanisms to regulate antigen-specific, T cell-mediated responses [24 , 25 ]. Much of this interest has focused on the roles of pattern recognition receptors including Toll like receptors (TLR) expressed on myeloid and DC. Engagement of TLR on antigen-presenting cells during the initiation of immune responses influences the phenotype of responding T cells as well as the magnitude of the response. The data in this report demonstrate a unique level of control that innate immunity imposes on T cell-mediated immune responses in the skin: strict control of antigen-primed T cell recruitment into sites of antigen challenge during the elicitation of an immune response. This control is mediated at the level of neutrophil chemoattractant production and subsequent neutrophil infiltration into the site.

KC/CXCL1 is likely to be the primary neutrophil chemoattractant directing neutrophil recruitment to hapten challenge sites during elicitation of CHS in mice. Higher levels of CXCL1 production lead to more intense neutrophil infiltration that subsequently leads to increased numbers of hapten-primed CD8+ T cells into the site. Consistent with the proposed role of this chemokine in this response, our previous results indicated the ability of KC-specific antibodies to inhibit the elicitation of CHS in sensitized mice. Studies in other laboratories have indicated a requirement for C5a for the optimal elicitation of CHS responses to picryl chloride [26 ]. C5a is a potent neutrophil chemoattractant and the possible link between C5a, and neutrophil infiltration in elicitation of CHS is intriguing and warrants further investigation.

Neutrophils are the first leukocytes infiltrating inflammatory sites, including epidermal hapten challenge sites, and are followed by the primed T cells into the tissue. Previous studies in this CHS model and in heart allograft rejection models have indicated the rapid production of KC/growth-related oncogene-{alpha} followed by neutrophil infiltration into parenchymal tissues [20 , 27 ]. The question that arises from these studies is what factors produced by neutrophils or what functions expressed by neutrophils are required for the infiltration of the T cells into the challenge site to mediate the characteristic spongiosis of the response. It is becoming clear that neutrophils can process and present antigens as peptide/class I MHC complexes to CD8+ T cells [28 ]. However, our previous studies indicated that delivery of antigen-primed T cells directly to the ear tissue followed by antigen challenge resulted in restored CHS responses in mice treated with anti-KC or neutrophil-depleting antibodies at the time of challenge [20 ]. These results are indicative that infiltrating neutrophil processing and presentation of haptenated peptides in the challenge site are not required functions for elicitation of CHS. Neutrophils produce many chemokines and other chemoattractants including those for antigen-activated T cells [29 ]. For example, in situ hybridization studies have shown cardiac allograft-infiltrating neutrophils expressing monokine induced by IFN-{gamma} as early as day 3 after transplantation in a mouse model [30 ]. Recently, the role of neutrophil-derived {alpha}-defensins in directing human leukocyte tissue infiltration has been shown [31 ]. However, mouse neutrophil granules do not contain these defensins. In addition to chemoattractant factors, neutrophils produce a large number of proteases, and the activity of these enzymes may mediate hapten-primed T cell infiltration into the hapten challenge site to elicit the CHS response. These facets of neutrophil function in the CHS response remain unclear at this time.

The recruitment of antigen-primed CD8+ T cells into the challenge site is not directed by the specific antigen but by the inflammation induced by antigen application/challenge to the challenge site. Challenge with a nonrelevant antigen induced the recruitment of DNFB-primed T cells into the challenge site. Although these T cells were detectable using hapten-specific ELISPOT assays, the elicitation of the immune response, as indicated by an increase in ear swelling, was not observed. Therefore, trafficking of T cells to the dermal microvasculature, through the vascular endothelium barrier and possibly through the parenchymal tissue of the challenge site, may be antigen-independent processes, all dependent on the level of inflammation. It may not be until the hapten-primed T cells engage presented antigen in the epidermis that they are activated to express the effector functions resulting in the tissue edema that is the feature of CHS responses.

It is not clear if these results are applicable to the recruitment of antigen-primed CD4+ T cells to the site. Hapten sensitization results in the polarized development of hapten-specific CD8+ T cells producing IFN-{gamma} and CD4+ T cells, producing IL-4 and IL-5 [16 ]. Although these CD4+ T cells infiltrate the ear tissue following hapten challenge and can be detected using ELISPOT assays to enumerate IL-4-producing CD4+ T cells, we have not yet tested the cellular and molecular components directing the tissue infiltration of these hapten-specific T helper cell type 2 cells. Studies in a rat model of delayed-type hypersensitivity (DTH) to sheep red blood cells have indicated the required role of neutrophils for elicitation of the response [32 ]. Similarly, depletion of neutrophils reduced DTH responses to viral antigen challenge in sensitized mice [33 ]. In a rabbit model, treatment of tuberculin-sensitized animals with anti-IL-8 mAb at the time of challenge decreased neutrophil infiltration into the challenge site and the level of the DTH response elicited [34 ]. Whether neutrophils direct the recruitment of CD4+ and/or CD8+ T cells to the challenge site or perform other functions in these responses has not been addressed, and the role of neutrophils in DTH remains unclear.

The current study has demonstrated the feasibility and efficacy of a strategy promoting enhanced neutrophil tissue infiltration to elicit greater T cell responses. The ability to increase or decrease T cell-mediated immune responses in the skin through manipulation of neutrophil recruitment may be useful in producing better outcomes in skin disease. For example, the absence of responses may be overcome by using neutrophils to promote T cell recruitment to a specific site of antigen deposition such as a tumor. Similarly, inhibiting neutrophil infiltration into a cutaneous site may attenuate elicitation of pathogenic, T cell-mediated, immune responses in the skin. It will be equally important to determine if such a strategy would be applicable only to the manipulation of CD8+ T cells or would extend to antigen-primed CD4+ T cells.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant AI45888. We thank Drs. Peter Heeger and Charles Bevins for critical reading of this manuscript and the Cleveland Clinic Foundation Biological Resources Unit for excellent animal care.

Received March 24, 2004; revised July 7, 2004; accepted July 16, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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