Which of the following hypersensitivity reactions are mediated by sensitized t helper-1 cells?

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IMMUNOBIOLOGY| May 1, 2000

Takayuki Yoshimoto,

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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Chrong-Reen Wang,

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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Toshihiko Yoneto,

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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Akio Matsuzawa,

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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William W. Cruikshank,

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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Hideo Nariuchi

1From the Department of Allergology and Animal Laboratory Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Intractable Disease Research Center, Tokyo Medical University, Tokyo, Japan; and The Pulmonary Center, Boston University School of Medicine, Boston, MA.

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Blood [2000] 95 [9]: 2869–2874.

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Interleukin [IL]-16 is a chemoattractant cytokine for CD4+ leukocytes1,2 and has been shown to up-regulate IL-2 receptor α chain [CD25] and major histocompatibility complex class II on T cells1 and also induce the transient loss of responsiveness via the T-cell receptor [TCR] stimulation.3,4 IL-16 is first synthesized as an 80-kd precursor, pro-IL-16, which is constitutively and most exclusively expressed in lymphoid organs, such as spleen, lymph node, and thymus, including both CD4+ and CD8+ T cells,5-8 and also expressed in activated epithelium.9 The amino terminus of the pro-IL-16 is proteolytically processed by caspase-3 upon cell activation to become a 14-kd mature IL-16, which then autoaggregates to form a bioactive homotetramer.5,7,8,10 IL-16 has been reported to play a critical role in diseases characterized by CD4+T-cell involvement, such as allergic asthma,9,11-13rheumatoid arthritis,14 multiple sclerosis,15,16 and acquired immunodeficiency syndrome.17-20 Thus, IL-16 is postulated to be a proinflammatory and immunoregulatory molecule playing an important role in recruitment and activation of CD4+ T cells at the site of inflammation.

One of the important mechanisms for the clearance of pathogen from infected host is the cell-mediated response that potentiates the infiltration of leukocytes, especially lymphocytes and macrophages, into infected tissues. Delayed-type hypersensitivity [DTH] is the typical in vivo manifestation of the cell-mediated immunity, and the response can be measured easily and semiquantitatively. A typical DTH reaction is characterized by activation and recruitment predominantly of T cells and macrophages and by resultant swelling at 24 to 48 hours at the site of intradermal antigen [Ag] injection in previously sensitized hosts. DTH is well known to be mediated by T helper 1 [Th2] cells secreting predominantly interferon-γ and IL-2 and provides a useful model to study the role of putative inflammatory mediators in leukocyte recruitment in vivo. However, how leukocytes are attracted to the Ag site in a sensitized host has not been fully elucidated yet, although a cascade of multiple cytokines and chemokines has been considered to be involved in the DTH reaction. Interferon-γ was previously demonstrated to be a major mediator of lymphocyte recruitment into the DTH reaction site.21 Chemokines such as IL-8,22monocyte chemoattractant protein 1 [MCP-1]23 and macrophage inflammatory protein 1α [MIP-1α],24 and macrophage migration inhibitory factor [MIF]25 were demonstrated to be involved in the recruitment of leukocytes to the DTH reaction site. Moreover, the expression of interferon-inducible protein 10 [IP-10]26 and RANTES27 [regulated on activation normal T expressed and secreted] was reported to be detected in a contact sensitivity reaction and in DTH granulomas, respectively.

Considering that the DTH reaction is thus mediated by Th2 cells and CD4+ T cells can be chemoattracted by IL-16, we have reasonably investigated the involvement of IL-16 in the DTH reaction. In the present study, we have found that bioactive IL-16 is produced by an Ag challenge in the DTH footpads and that treatment of sensitized mice with neutralizing monoclonal antibody [mAb] against IL-16 immediately before the Ag challenge suppresses significantly the DTH response, suggesting that IL-16 plays an important role for the development of the DTH reaction in the elicitation phase. This is the first report on the detection of IL-16 in the DTH footpads and the involvement of IL-16 in the DTH reaction.

Materials and methods

Mice

Female C57BL/6 mice [6-8 weeks of age] were purchased from Japan SLC [Hamamatsu, Japan].

DTH response

DTH response to methylated bovine serum albumin [mBSA, Sigma, St. Louis, MO] was estimated by footpad swelling as described.28 Briefly, mice were sensitized to mBSA by an intradermal injection of 50 μL of 2.5-mg/mL mBSA emulsified with complete Freund's adjuvant [Difco, Detroit, MI] at 2 sites on the abdomen. Seven days after the immunization, the mice were challenged by an injection of 30 μL of 5-mg/mL mBSA in phosphate-buffered saline [PBS] into 1 rear footpad, while the other rear footpad received a comparable volume of PBS as a control. Footpad swelling was measured using a dial caliper [Mitutoyo Corp, Tokyo, Japan] at a given time after the challenge. The magnitude of the DTH response was determined as the difference in footpad thickness between mBSA- and PBS-injected footpads.

Immunohistochemical examination

Histologic examination of the DTH footpad was carried out as described.28 Soft tissue samples from each footpad were collected 24 hours after the challenge with mBSA, immersed in OCT embedding medium, and snap-frozen in liquid nitrogen. Endogenous peroxidase activity in tissue section was blocked by treatment with 0.6% H2O2 and 0.2% sodium azide for 10 minutes. Tissue section was then incubated for 20 minutes at 37°C in PBS containing 2% normal mouse or rabbit serum and 1% BSA to block nonspecific immunoglobulin [Ig] G binding. For detection of IL-16, tissue section was incubated with biotinylated antihuman/mouse IL-16 [clone 17.1, mouse IgG1]7 or isotype-matched control mAb [MOPC-21, mouse IgG1, Pharmingen, San Diego, CA] [5 μg/mL] in PBS containing 1% BSA overnight at 4°C. For detection of CD4+ T cells, CD8+ T cells, and macrophages, tissue section was incubated with antimouse primary rat mAb to the respective markers [5 μg/mL] in PBS containing 1% BSA for 1 hour at room temperature, and the bound primary rat mAb was then labeled with rabbit antirat IgG conjugated with biotin for 1 hour at room temperature. The following primary mAbs were used for the immunostaining: antimouse CD4 [GK1.5, rat IgG2b, American Type Cell Culture [ATCC], Rockville, MD], antimouse CD8 [53.6.7, rat IgG2a, ATCC], and antimouse macrophage [F4/80, rat IgG2b, Pharmingen]. The isotype-matched control mAbs of rat IgG2a and IgG2b used were clone R35-95 and R35-38 [Pharmingen], respectively. Detection was performed with a streptavidin-horseradish peroxidase conjugate and diaminobenzidine substrate. The section was counterstained with hematoxylin to detect cell nuclei, dehydrated in graded ethanol solutions, equilibrated with xylene, and coverslipped. Positively stained cells were counted in 10 randomly selected fields [each [100 μm]2] as described.28 

Intracellular staining

Infiltrating cells into the DTH footpad were isolated as described.29 Pooled infiltrating cells prepared from 10 mice 24 hours after the Ag challenge were stained intracellularly with phycoerythrin [PE]-conjugated anti-IL-16 as described.7Briefly, infiltrating cells in staining buffer [PBS containing 2% fetal calf serum and 0.1% sodium azide] were blocked with anti-FcγII/III receptor mAb [2.4G2, Pharmingen], incubated with fluorescein isothiocyanate–conjugated mAb to the desired surface Ag, and fixed in PBS containing 1% paraformaldehyde for 20 minutes. After washing, the cells were incubated in 50 μL of permeabilization buffer [0.1% saponin in the staining buffer] containing 0.1 μg of PE-conjugated antihuman/mouse IL-16 [clone 14.1, mouse IgG2a, Pharmingen] or isotype-matched control mAb [G155-178, mouse IgG2a, Pharmingen] for 30 minutes. For blocking, PE-conjugated anti-IL-16 [0.1 μg] preincubated with recombinant human IL-16 [1 μg]6in 50 μL of permeabilization buffer for 30 minutes at 4°C was incubated with the cells. Then, the cells were washed with permeabilization buffer and analyzed by flow cytometry using a FACScan [Becton Dickinson, Mountain View, CA].

Footpad cytokine extraction

Footpad cytokine extraction was prepared as described.30Briefly, at a given time after the Ag challenge into the footpad of sensitized mice, their footpads were cut off, minced in cold PBS [1 mL], and incubated on ice for approximately 1 hour to release soluble materials from the tissue. The supernatant was collected after centrifugation and frozen at −80°C until used.

Detection of IL-16 and MIP-1 by enzyme-linked immunosorbent assay

The sandwich enzyme-linked immunosorbent assay [ELISA] for mouse IL-16 was carried out using anti-IL-16 [14.1]9 as capture mAb, biotinylated anti-IL-16 [17.1]7 as detection mAb, and recombinant IL-166 as a standard. The ELISA for mouse MIP-1α was performed by using a kit [Genzyme-Techne, Minneapolis, MN] according to the manufacturer's instructions.

Immunoprecipitation and Western blot analyses

Protein G Sepharose beads preincubated with anti-IL-16 [14.1] or an isotype-matched control mAb [MOPC-173, mouse IgG2a, Pharmingen] were added to an aliquot [approximately 0.4 mL] of each footpad extract and incubated overnight at 4°C. The beads were washed, resuspended in sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS-PAGE] sample buffer, and boiled, and released materials were electrophoresed on an SDS-polyacrylamide [10-20% gradient] gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride microporous membrane [PVDF, Immobilon, Millipore, Bedford, MA], and the membrane was probed with biotinylated anti-IL-16 [17.1] or isotype-matched control mAb [MOPC-21] followed by incubation with a streptavidin-horseradish peroxidase conjugate. Visualization of the signal was by electrochemiluminescence [Amersham, Oakville, Canada].

Migration assay

Migration assay was performed using a modified Boyden chemotaxis chamber as described.9 Briefly, T cells were prepared by passing spleen cells through a nylon wool column. A total of 107 T cells in 50 μL of Medium 199 enriched with 0.4% BSA were loaded into the upper well of the chamber, and 30 μL of the sample to be tested were placed in the lower well. The upper and lower well were separated by a nitrocellulose filter with a pore size of 8 μm. The chamber was incubated for 3 hours, and afterward the filter was fixed and stained with hematoxylin. Migration was quantified by counting the number of cells that migrated beyond a depth of 50 μm using an Optomax automated image analyzer [Burlington, MA]. All migration data are expressed as the number of cells per high-power field [hpf]. All samples were performed in triplicate. On average, 14 to 16 cells/hpf were counted under control conditions. Counts were compared with control [medium alone] migration, which was normalized to 100%. For blocking experiments, 5 μg/mL of anti-IL-16 neutralizing mAb [14.1]9 or the control mAb [MOPC-173] were added to the lower well.

Neutralization of IL-16 in vivo with monoclonal antibodies

To neutralize endogenous IL-16, sensitized mice were injected intraperitoneally with 0.5 mg/injection of anti-IL-16 [14.1] twice at 15 to 18 hours and immediately before the Ag challenge.9 As an isotype-matched control mAb, anti-SRBC [S-S.1, mouse IgG2a, ATCC] was similarly injected. These mAbs were purified from ascites on a protein G column.

Statistical analysis

Statistical analysis was performed by Student t test.P values