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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2869-2874
IMMUNOBIOLOGY
From 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.
Interleukin (IL)-16 is a chemoattractant cytokine for
CD4+ leukocytes. Because delayed-type hypersensitivity
(DTH) reaction is mediated by T helper 1 (Th1) cells and
CD4+ T cells can be chemoattracted by IL-16, we have
investigated the involvement of IL-16 in the DTH reaction.
Immunohistochemical analysis revealed the IL-16 expression in
infiltrating cells and epithelial cells in the DTH footpads. The IL-16
expression was also detected intracellularly in the infiltrating cells.
In addition, markedly increased production of IL-16 was detected in the
DTH footpad extracts, but not in the control footpad extracts, by an
enzyme-linked immunosorbent assay and also by Western blot analysis.
The DTH footpad extracts exhibited a strong chemoattractant activity
toward splenic T cells, which was significantly inhibited by the
inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in
the migration assay. Furthermore, treatment of sensitized mice in vivo
with the anti-IL-16 neutralizing mAb significantly suppressed the
footpad swelling induced by an antigen challenge, together with
decreased infiltration of leukocytes including not only
CD4+ T cells but also CD8+ T cells and
macrophages into the DTH footpads. Decreased production of
macrophage inflammatory protein 1
Interleukin (IL)-16 is a chemoattractant cytokine for
CD4+ leukocytes1,2 and has been shown to
up-regulate IL-2 receptor 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 (Th1) cells secreting predominantly
interferon- Considering that the DTH reaction is thus mediated by Th1 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.
Mice
DTH response
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.28Intracellular 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.7 Briefly, infiltrating cells in staining buffer (PBS containing 2% fetal calf serum and 0.1% sodium azide) were blocked with anti-Fc II/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)6
in 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.30 Briefly, 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  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 <.05 were considered statistically significant.
To explore whether IL-16 expression is induced by challenge with Ag
in footpads of sensitized mice for the elicitation of a DTH response,
we first analyzed immunohistochemically the IL-16 expression in the
footpads 24 hours after the Ag challenge. IL-16 immunoreactivity was
detected in the mass of infiltrating cells and also in the epithelium
in footpads challenged with mBSA (Figure 1A), but it was hardly or much less
detected in footpads challenged with PBS (Figure 1B) or in footpads of
nontreated mice (data not shown). No staining with control mAb was
detected in footpads challenged with mBSA (data not shown). Then, the
infiltrating cells into DTH footpads were isolated 24 hours after the
Ag challenge and stained intracellularly for IL-16. Most infiltrating
cells, including mainly Mac-1+ cells and also
CD4+ and CD8+ T cells (data not shown), were
positively stained with PE-conjugated anti-IL-16 but not with
PE-conjugated control mAb (Figure 2). The
intracellular detection of IL-16 was completely blocked by preincubating PE-conjugated anti-IL-16 with recombinant
IL-16 (Figure 2). These results suggest the induction of
IL-16 expression in the footpads of sensitized mice by Ag challenge in
the elicitation phase of the DTH response.
In the present study, we have investigated the involvement of IL-16
in the DTH response. First of all, immunohistochemical analysis
revealed the expression of IL-16 in infiltrating cells and epithelial
cells in footpads challenged with mBSA (Figure 1). Similar findings are
reported in the airways after repeated ovalbumin inhalation in
sensitized mice.9 In addition, the intracellular staining
for IL-16 elucidated that most infiltrating cells such as, mainly,
Mac-1+ and, also, CD4+ and CD8+ T
cells into DTH footpads were positive for the IL-16 expression (Figure
2). IL-16 was first reported to be produced by CD8+ T
cells33,34 and subsequently by eosinophils,35
mast cells,36 epithelial cells,9
CD4+ T cells,8 and recently by dendritic
cells.37 Therefore, the results of intracellular analysis
imply that macrophages may be another candidate for producer cells of
IL-16. Moreover, these results could suggest the possible secretion of
IL-16 protein from infiltrating cells and epithelial cells, potentially
including Langerhans cells, which are premature dendritic cells resting in the skin,37 in the DTH footpads. Indeed, we observed
increased production not only of pro-IL-16 but also its processed forms and mature IL-16 in the DTH footpad extracts by ELISA (Figure 3B) and
Western blot analysis (Figure 4). Although we could detect the mature
IL-16 protein in the DTH footpad extracts in a few experiments,
including this one (Figure 4), we could not detect it in most
experiments. This is because that mature IL-16 protein seems to be
difficult to detect by physicochemical techniques, probably due to the
low production even when its bioactivity is detected by a migration
assay.7 A strong chemoattractant activity toward splenic T
cells was constantly detected in the DTH footpad extracts in vitro, and
approximately 50% of the activity was inhibited by the inclusion of
anti-IL-16 neutralizing mAb (Figure 5B). These results suggest that
bioactive IL-16 was produced in the DTH footpads and that it could be
involved in the recruitment of CD4+ T cells to the DTH
reaction site. Because MIP-1 Submitted May 24, 1999; accepted January 5, 2000.
Supported by a Grant-in-Aid for Scientific Research on Priority Areas
from the Ministry of Education, Science, Sports and Culture, Japan, and
from the Japanese Ministry of Public Health and Welfare.
W.W.C. is a recipient of a Clinical Investigator Award from the
American Lung Association.
T.Y.'s present address is Intractable Disease Research Center, Tokyo
Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan;
e-mail: yoshimot{at}tokyo-med.ac.jp.
Reprints: Takayuki Yoshimoto, Department of
Allergology, Institute of Medical Science, The University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail:
yoshimot{at}ims.u-tokyo.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
was also observed in the DTH
footpad extracts by the mAb treatment. These results suggest that IL-16
plays an important role in the recruitment of leukocytes
presumably including antigen-specific Th1 cells, which secrete cytokines and
chemokines mediating the following hypersensitivity reaction after
activation by the interaction with Langerhans cells carrying the
antigen
, IL-6, and tumor necrosis factor-
