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Regulation of T Lymphocyte Trafficking into
Lymph Nodes During an Immune Response
by the Chemokines Macrophage
Inflammatory Protein (MIP)-1 α and MIP-1β
Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick
Di Girolamo, Taline Hampartzoumian, Denis Wakefield and
Andrew Lloyd
J Immunol 1998; 161:5663-5672; ;
http://www.jimmunol.org/content/161/10/5663
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Copyright © 1998 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Regulation of T Lymphocyte Trafficking into Lymph Nodes
During an Immune Response by the Chemokines Macrophage
Inflammatory Protein (MIP)-1a and MIP-1b1
Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick Di Girolamo,
Taline Hampartzoumian, Denis Wakefield, and Andrew Lloyd2
T
he migratory properties of leukocytes have evolved to allow efficient surveillance of tissues for infectious pathogens and rapid accumulation at sites of injury or infection.
In contrast to neutrophils and monocytes, T lymphocytes may exit
from the vascular compartment via specialized high endothelial
venules (HEV)3 in lymphoid organs and recirculate many thousands of times during their life span (1, 2). It has been estimated
that one in every four lymphocytes leaves the circulation by crossing the HEV (3). After encountering Ag in lymph nodes, memory
T lymphocytes continually patrol the body for that Ag by recirculating from the blood, through tissues, into the lymphatic system,
and back to the blood (1– 4). T lymphocytes thus acquire a predilection, based on the environment in which they first encounter Ag,
to home to, or recirculate through, that same environment (5, 6).
Hence, relatively distinct subsets of T lymphocytes extravasate
through the microvasculature in tissues such as skin and gut and
across HEV in lymph nodes (4 – 6).
The arrival of Ag, and hence the induction of an immune response in the node, greatly increases blood flow and traffic of lymphocytes across HEV, coupled with a transient, sharp decrease in
recirculating lymphocyte output from the efferent lymphatics (7–
9). In lymph nodes undergoing an immune response, lymphocyte
Inflammation Research Unit, School of Pathology, University of New South Wales,
Sydney, Australia
Received for publication April 14, 1998. Accepted for publication July 13, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported by the National Health and Medical Research Council of
Australia.
2
Address correspondence and reprint requests to Dr. Andrew Lloyd, Inflammation
Research Unit, School of Pathology, University of New South Wales, Sydney, NSW,
2052, Australia.
3
Abbreviations used in this paper: HEV, high endothelial venules; VLA, very late
Ag; DNFB, 2,4-dinitrofluorobenzene; MIP, macrophage inflammatory protein; MCP,
monocyte chemotactic protein; MMCP-5, mouse mast cell protease-5; PCNA, proliferating cell nuclear Ag; TBS, Tris-buffered saline; R-PE, R-phycoerythrin.
Copyright © 1998 by The American Association of Immunologists
traffic across the HEV may increase substantially within 3 h after
antigenic stimulation and by as much as 10-fold over the first 48 h
of the response (8, 9). This effect is not caused by lymphocyte
proliferation within the node nor by increased numbers of cells
entering the lymph nodes from the lymphatics; instead, .95% of
the effect is due to trafficking of cells from blood (8, 9).
At the molecular level, the leukocyte adhesion molecules, Lselectin, LFA-1 (CD11a/CD18), and VLA-4 (CD49d/CD29), mediate T lymphocyte binding to peripheral lymph node HEVs by
interaction with glycosylation-dependent cell adhesion molecule-1
(GlyCAM-1) (10) or CD34 (11) for L-selectin, with ICAM-1 and
ICAM-2 for LFA-1 (12, 13), and potentially with fibronectin for
VLA-4 (14). Neutralizing antisera to L-selectin (15, 16), LFA-1
(17), or VLA-4 (14) markedly reduce lymphocyte migration into
peripheral lymph nodes. Thus, lymphocyte recruitment into lymph
nodes is likely to be a multistep process (similar to the processes
of neutrophil and monocyte localization in inflammation) that requires L-selectin molecules to allow lymphocytes to “tether and
roll’’ via low affinity interactions and LFA-1 or VLA-4 to induce
firm adhesion to their counterreceptors (4, 18, 19). Activation of
L-selectin alone has been shown to trigger the high affinity state of
integrins on naive T lymphocytes in vitro (20, 21), thus providing
a potential mechanism for the preferential recirculation of these
cells through peripheral lymph nodes. However, pertussis toxin
treatment abrogates LFA-1-dependent arrest of lymph node-derived lymphocytes (22) and may inhibit lymphocyte entry into secondary lymphoid organs (23), suggesting a requirement for G protein-linked signaling events in T lymphocyte trafficking into
lymph nodes. Lymphocyte chemoattractants secreted from within
peripheral lymph nodes and their G protein-coupled receptors expressed on lymphocyte subpopulations may provide this signal to
stimulate integrin-dependent recruitment of lymphocyte subsets.
Members of the b-chemokine family are known to direct T lymphocyte migration along a protein gradient (chemotaxis) (24, 25)
and to induce adhesion to extracellular matrix proteins (26). The
b-chemokines MIP-1a, MIP-1b, monocyte chemotactic protein
0022-1767/98/$02.00
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By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of
inflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune response
has not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene
(DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulation
of CD81 and CD41 T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophage
inflammatory protein (MIP)-1a and MIP-1b mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensitization were the predominant source of MIP-1b, whereas MIP-1a was expressed by multiple cell types. Neutralization of these
chemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challenge
to DNFB. Thus, b-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immune
response and contribute significantly to the immunologic outcome. The Journal of Immunology, 1998, 161: 5663–5672.
5664
CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES
Lymph node excision
Both right and left inguinal lymph nodes were removed by carefully dissecting the tissue to a radius of approximately 1 cm from the center of the
node. The right inguinal lymph node of each animal was used to prepare a
single-cell suspension, and the left inguinal lymph node was fixed in 10%
buffered formalin (pH 7.0) and embedded in paraffin. Additional mice were
used to harvest the right inguinal node for extraction of protein from tissue
lysates, and the left inguinal node was snap frozen by embedding the tissue
in Tissue-Tek (OCT compound, Miles, Elkhart, IN) before storage at
270°C for subsequent immunohistochemical studies.
Preparation of the single-cell suspension
Materials and Methods
A cell suspension from each right inguinal lymph node was prepared using
a modification of a previously published method (34). In brief, the lymph
node was weighed and washed twice in RPMI 1640/10% FCS supplemented with penicillin/streptomycin and L-glutamine. The tissue was
minced to nearly complete dissociation using scissors and forceps in a
6-well cell culture dish (Costar, Cambridge, MA) and resuspended in 4 ml
of the above-mentioned medium. One mg/ml of collagenase type H
(Boehringer Mannheim, Berlin, Germany) containing 1 mM calcium chloride was added to the mince, which was then incubated at 37°C for 60 min.
Digestion was then stopped by adding 1 ml of RPMI 1640/20% FCS and
the suspension filtered through a 40-mm nylon cell strainer (Becton Dickinson, Mountain View, CA) into a 50-ml Falcon tube. A tuberculin syringe
plunger was used to tease cells from tissue on top of the nylon mesh with
repeated rinsing in PBS. The cells were washed twice with PBS and resuspended in this medium at 2 3 106/ml. The total cell count was enumerated after assessment of viability with trypan blue.
Mice
Measurement of differential leukocyte counts in the lymph nodes
Six- to eight-week-old male and female C3H/HeN mice were bred in specific pathogen free (SPF) conditions in the Animal Breeding and Holding
Unit at the University of New South Wales. SPF conditions were maintained throughout the experimental phase of the study. For the experiments
described, each sampling point consisted of four mice.
From each single-cell suspension, four air-dried smears were made. One
slide from each mouse was then stained with Giemsa-Grunwald to determine a differential leukocyte count. The remaining three slides were fixed
in acetone and stored at 220°C for immunohistochemical staining.
Sensitization of contact hypersensitivity
Twenty-five microliters of 0.5% 2,4-DNFB (Sigma, Sydney, Australia) in
4/1 diluted acetone/olive oil (Sigma) was applied to the freshly shaved
abdominal surface of mice on day 0 and day 1 for sensitization (33). Control mice were painted on the shaved abdomen with 25 ml of the vehicle
alone.
Elicitation of contact hypersensitivity and evaluation of ear
swelling
Five days after sensitization, mice were challenged by applying 10 ml of
0.2% DNFB in acetone/olive oil to one ear. Control mice were similarly
painted with the vehicle alone. The degree of ear swelling was measured
before challenge and 24 h postchallenge using an engineer’s micrometer
(Mitutoyo, Tokyo, Japan). Each earlobe was measured twice and contact
hypersensitivity determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear, expressed in micrometers (mean 6 SD). Mice that were challenged with the
hapten without previous sensitization served as an additional negative control.
Antichemokine treatment in vivo
Four hours before DNFB sensitization, neutralizing goat anti-mouse polyclonal Abs directed against murine MIP-1a and/or MIP-1b were injected
i.p., diluted in sterile endotoxin-free saline, and control animals were injected with control goat IgG (R&D Systems, Minneapolis, MN) in comparable doses to the experimental animals. Mice were coded so that cell
counts obtained during the sensitization phase and ear thickness measurements after challenge were made by an independent observer without
knowledge of the status of the mouse.
Peripheral blood collection
Anticoagulated blood (200 –500 ml) was collected from the heart of each
mouse 1–2 min after the animals were sacrificed by CO2 inhalation, and a
vertical neck-to-tail skin incision was performed. Two sets of thin blood
films were made for differential counting after Giemsa-Grunwald staining,
and the total cell count was measured by automated cell counter (Sysmex
NE 800, Australian Diagnostic Services, Sydney, Australia). Blood (50 ml)
was used to stain cell surface markers with mAbs in three-color flow cytometry (see below).
Three-color flow cytometry
The Abs use in flow cytometry included: anti-CD3-FITC, anti-CD4-Rphycoerythrin (R-PE), and anti-CD8a-RED163, which were rat anti-mouse
mAbs purchased from Life Technologies (Victoria, Australia). An irrelevant negative control IgG with subclasses g1 and g2a (IgG1-FITC/IgG2aPE) and the FACS lysing solution were purchased from Becton Dickinson.
The wash solution consisted of PBS containing 2% BSA and 0.2% sodium
azide. A 1% paraformaldehyde solution in PBS was used to fix cells after
staining.
mAb (4 ml) and ;2 3 105 cells in 100 ml were added to each labeling
tube. After mixing, tubes were incubated in the dark at 4°C for at least 30
min, then FACS lysing solution was added and tubes were further incubated at room temperature for 10 min followed by centrifugation. The
supernatant was decanted and the cell pellet washed and then fixed in
paraformaldehyde solution. A total of 10,000 events were acquired using a
FACScan flow cytometer and data analysis performed with PC LYSIS II
software (Becton Dickinson).
Analysis of proliferating lymphocytes in vivo
To determine the proportion of proliferating lymphocytes in response to
DNFB sensitization, as opposed to the cells recruited into the lymph nodes,
a mAb against proliferating cell nuclear Ag (PCNA) conjugated to FITC
was purchased from Boehringer Mannheim (Mannheim, Germany).
Lymph node-derived single-cell suspensions (106/ml) were fixed for 2 min
in 1% paraformaldehyde, followed by washing in cold PBS. Cells were
then incubated in 100% methanol at 220°C for 10 min, centrifuged again,
and washed in PBS containing 0.1% Triton X100 (Serva, Heidelberg, Germany). Subsequently, cells were incubated with anti-PCNA Ab (1.25 mg in
50 ml of 2% BSA in PBS) for 15 min at room temperature. After washing
in PBS supplemented with 2% BSA, the cells were spun down and incubated with anti-CD4-R-PE and anti-CD8a-RED163 Abs for at least 30 min
at 4°C in the dark. Cells were then washed twice in PBS/2% BSA and
resuspended in PBS. As a positive control for this analysis, lymph nodederived mononuclear cells were stimulated in vitro with 10 mg/ml of phytohemagglutinin (Wellcome Diagnostics, Charlotte, NC) before staining as
described above. Samples were analyzed on a FACScan (Becton Dickinson) equipped with LYSIS II software.
In situ hybridisation
Single-stranded cRNA probes of 350 bp in both the antisense and sense
orientation were prepared by in vitro transcription from murine MIP-1a
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(MCP)-1, MCP-2, MCP-3, and RANTES reportedly show chemotactic activity for T cells in vitro (27). Of these chemokines, some
in vitro studies suggest that MIP-1a and MIP-1b preferentially
attract CD81 and CD41 T cells, respectively (24, 25, 28).
We have recently reported the first evidence for a potential role
of chemokines in the regulation of lymphocyte traffic into lymph
nodes in studies of nodes taken from HIV-1-infected patients and
control subjects (29). Strong expression of MIP-1a in the HIV
lymph nodes was associated with the accumulation of CD81 T
cells in these tissues. The present experiments were designed to
further define the potential in vivo role of MIP-1a and MIP-1b in
recruitment of T cell subsets to lymph nodes during an immune
response. The well-characterized murine model of contact hypersensitivity induced by dinitrofluorobenzene (DNFB) was chosen
for this study. It is believed that both CD41 and CD81 T cell
subsets are involved in the development of contact hypersensitivity
mediated by DNFB, because studies to define the specific role of
either subset have provided conflicting results (30, 31, 32). The
pattern of expression of MIP-1a and MIP-1b mRNAs and proteins
in draining lymph nodes of DNFB-painted mice was examined,
and the kinetics of accumulation of T lymphocyte subsets in the
nodes was determined.
The Journal of Immunology
5665
and MIP-1b plasmid cDNAs using a nonisotopic probe-labeling technique
(digoxigenin; Boehringer Mannheim). After a 2-h prehybridization at
42°C, hybridization was performed overnight at the same temperature using 100 ng of probe in 25 ml of prehybridization solution on 4-mm thick,
formalin-fixed, paraffin-embedded sections. This was followed by repeated
stringency washing at 42°C in 0.53 SSC. These hybridization and washing
conditions were empirically determined to give optimal signal with the
antisense probes, but minimal nonspecific signal with the control (sense
strand) probes. Probe detection was conducted according to the manufacturer’s directions with an anti-digoxigenin mAb conjugated to alkaline
phosphatase followed by an appropriate substrate (nitro blue tetrazolium 1
59-bromo-4-chloro-3-indolyl phosphate (NBT 1 BCIP); Boehringer
Mannheim). Control samples included sections of lymph nodes of the
DNFB-treated mice hybridized without probe or without Ab detection, as
well as sections obtained from the lymph nodes of acetone-treated mice.
Immunohistochemical staining
Quantitative evaluation of MIP-1a and MIP-1b mRNA
expression
After a systemic sampling procedure as described elsewhere (29), computer-assisted morphometric analysis of lymph node sections was used to
quantitate the number of cells expressing chemokine mRNAs as well as the
total number of mast cells in the nodes (36). In brief, contiguous fields
across the whole section (on average, 10 fields) at a final magnification of
2503 were assessed. After ensuring that the sections hybridized with the
sense probes exhibited no significant signal, the number of positive cells
(cytoplasmic blue staining) per field was enumerated. Although significant
regional variations in staining were observed, the mean count for the whole
section is reported as a conservative measure of the signal for each probe.
Morphologic analyses and immunohistochemical staining of adjacent sections were used to determine the cellular sources of MIP-1a and MIP-1b.
Analysis of MIP-1a and MIP-1b production by Western blotting
Cell lysates from groups of three mice sacrificed 4, 24, and 96 h after DNFB
repainting, and 24 h after acetone/olive oil repainting (control mice), were
prepared using a standard method. To ensure equal loading of protein, the
amount of total protein in the cell lysate was measured using the bicinchonic
acid protein assay kit (Pierce, Rockford, IL). Lymph node-derived cell lysates
were electrophoretically separated on a 4% stacking and 10% resolving acrylamide gel under nonreducing conditions and then transferred to Immobilon-P
membranes (Millipore, Sydney, Australia) using a Trans-blot SemiDry Electrophoretic Transfer Cell (Bio-Rad, Sydney, Australia). After protein transfer,
the membranes were washed twice in Tris-buffered saline (TBS) for 15 min at
room temperature. Membranes were then incubated overnight at 4°C with 50
ml of blocking solution containing 3% BSA and 5% skim milk powder in
TBS. The membranes were then rinsed twice with TBS for 5 min at room
temperature and incubated with the primary goat anti-mouse MIP-1a or
MIP-1b Ab (R&D Systems) for 1 h at room temperature with continuous
gentle shaking. Primary Abs were reconstituted in sterile PBS at a concentration of 1 mg/ml and used at a 1:500 dilution. After incubation with the primary
Ab, the membrane was washed four times for 15 min with TBS and incubated
for 1 h at room temperature with a 1:500 dilution of donkey anti-goat second-
FIGURE 1. DFNB sensitization produces a rapid increase in the weight
and cellularity of inguinal lymph node (A) and a decrease in the peripheral
blood leukocyte count (B). The right inguinal lymph node collected from
animals sensitized by painting with 0.5% DNFB and control animals
treated with vehicle alone (acetone) were weighed, minced, and digested
with collegenase. Single-cell suspensions prepared from each node were
counted both manually and with an automated cell counter (Sysmex NE
800). Cell smears prepared from the single-cell suspension were used for
differential cell counting after Giemsa-Grunwald staining. A total leukocyte count was also performed, using the automated cell counter, in peripheral blood collected from each animal. Each experimental group consisted of four animals, and each experiment was performed twice.
“Acetone” on the x-axis represents pooled data from two sets of four control animals that were sacrificed 4 and 24 h after repainting with vehicle
alone. Results are presented as mean 6 SD.
ary Ab (Serotec), which was directly conjugated to horseradish peroxidase.
Membranes were then washed four times for 15 min in TBS, followed by the
addition of a chemiluminescence reagent (Renaissance, DuPont, Sydney, Australia). The membranes were finally exposed to X-OMAT-AR scientific imaging film (Kodak, Sydney, Australia).
Metachromatic staining
After dewaxing and a 10-min incubation with 0.5 N hydrochloric acid,
overnight staining with 1% toluidine blue in 0.5 N HCL was used as a
metachromatic stain for mouse mast cells in the formalin-fixed lymph node
tissue sections. A standard hematoxylin and eosin staining method was
used for evaluation of the histopathologic changes in the lymph node.
Statistical analysis
A computer software program, Microsoft EXCEL, version 5.0 (Microsoft,
Seattle, WA), was used to calculate means and SD. To assess the level of
statistical significance, a two-tailed Mann-Whitney U test was performed
using a statistical software program, SPSS for Windows, version 6.0
(SPSS, Chicago, IL). Parameters of interest in each group of DNFB-treated
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A standard two-step streptavidin-horseradish peroxidase staining technique
was performed to identify cell types on frozen sections and smears using
primary rat mAbs against mouse T cell Ags CD3, CD4, CD8; a B cell
marker, CD40; and a marker for tissue macrophages (Mac-1); as well as an
isotype-matched negative control Ab. A biotinylated rabbit anti-rat secondary Ab was used. All of the above Abs were purchased from Serotec
(Australian Laboratory Services, Sydney, Australia). A polyclonal rabbit
anti-mouse Ab directed against mast cell protease-5 (MMCP-5; Ref. 35)
was used to stain mast cells in formalin-fixed lymph node tissues after an
enzymatic digestion and Ag retrieval by microwave treatment of the sections in 0.01 M sodium citrate buffer, pH 6.0. A biotinylated goat antirabbit secondary Ab was used for this staining (Dako, Glostrup, Denmark).
Immunohistochemical staining using primary goat anti-mouse polyclonal
Abs directed against MIP-1a and MIP-1b (R&D Systems) and secondary
donkey anti-goat IgG directly conjugated to horseradish peroxidase (Serotec) were used to detect MIP-1a and MIP-1b proteins in formalin-fixed
sections after a similar enzymatic digestion and Ag retrieval by microwave
of the sections in citrate buffer. An isotype-matched goat IgG (R&D Systems) was used as a negative control.
A standard two-step streptavidin-horseradish peroxidase staining technique was performed on formalin-fixed, paraffin-embedded lymph node
sections to localize proliferating cells using primary anti-PCNA mAb
(Dako) and a biotinylated goat anti-mouse secondary Ab (Serotec).
5666
CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES
Table I. Induction of cell proliferation in draining lymph nodes of
mice treated with DNFB a
Treatment
Mean
Lymphocyte
Count per
Lymph Node
(3106)
CD41
CD81
Acetone
4 h after DNFB
24 h after DNFB
96 h after DNFB
PHA-stimulated lymphocytes
1.5 6 0.2
3.4 6 0.5
7.9 6 1.0
3.2 6 0.5
—
1.5 6 0.4
2.4 6 0.5
2.4 6 0.2
14.3 6 2.7
99.2 6 5.8
1.8 6 0.6
2.7 6 0.8
2.2 6 0.7
10.1 6 0.4
98.4 6 5.5
% PCNA1b
FIGURE 2. DFNB sensitization produces a rapid increase in CD41 and
CD81 T lymphocytes in lymph nodes (A) and an associated decrease in the
peripheral blood (B). Cells (2 3 105) of the single-cell suspension from the
inguinal lymph node and heparinized whole blood obtained from each
animal were stained with rat anti-mouse CD3-FITC, CD4-R-PE, and CD8RED613 mAbs. To obtain the proportions of the T cell subsets, three-color
flow cytometric analysis was performed on the lymphocyte gate based on
forward and side scatter as well as the T cell (CD3) gate. The absolute
values for the T cell subsets were then obtained from the total leukocyte
count, the proportions of total lymphocytes (obtained from the differential
count), and the proportions of the T cell subsets (obtained from the flow
cytometry). Isotype-matched irrelevant rat IgG Abs conjugated to the three
different fluorochromes were used as negative controls. Each experimental
group consisted of four animals, and each experiment was performed
twice. “Acetone” on the x-axis represents pooled data from two sets of four
control animals that were sacrificed 4 and 24 h after repainting with vehicle
alone. Results are presented as mean 6 SD.
animals were analyzed for statistical significance in comparison with control animals. As several comparisons were performed, Bonferoni adjustments for statistical significance were made.
Results
DNFB sensitization causes a rapid increase in cellularity and
weight of inguinal lymph nodes
The lymph node weight increased significantly within 30 min of
repainting with DNFB ( p , 0.01), peaking at 12 h and gradually
decreasing thereafter (Fig. 1A). The lymph node weight remained
mildly increased above that of the control animals 1 mo after repainting. The enlargement of the lymph nodes was accompanied
by a .10-fold increase in the number of nucleated cells in the
nodes (Fig. 1A). Differential cell counting after Giemsa-Grunwald
staining of the cell smears showed that .90% of the cells in lymph
nodes of DNFB-painted mice were lymphocytes, confirming that
these cells represented the predominant leukocyte subpopulation
accumulating in the nodes. A substantial proportion of the increase
in lymphocyte numbers was evident within 30 min, with ;85% of
the peak increase detected within 12 h (Fig. 1A). This rapid accumulation was statistically significant ( p , 0.01) when compared
with the control animals. The brisk kinetics indicate increased lymphocyte traffic into the nodes (and perhaps reduced output) rather
than in situ proliferation of lymphocytes.
DNFB repainting induces a rapid drop in PBL counts
Further evidence in support of the notion of increased trafficking of
lymphocytes into lymph nodes was obtained from the rapid decrease in lymphocyte numbers in peripheral blood coincident with
their accumulation in the lymph nodes. Sensitization with DNFB
produced an ;50% reduction in the total number of PBL within
the first 30 min after repainting (Fig. 1B). The count gradually
returned to normal values within 48 h after the treatment.
CD41 and CD81 T lymphocytes accumulate in lymph nodes
after repainting with DNFB
Three-color flow cytometric analysis of the draining lymph nodes
of DNFB-treated mice showed a significant increase in the total
(CD31) T lymphocytes in lymph nodes, which peaked within 12 to
24 h after repainting (Fig. 2A). Both CD41 and CD81 T lymphocyte subsets showed a similar rapid increase ( p , 0.01 for each)
following repainting with DNFB. After reaching a peak at 24 h, the
CD41 and CD81 lymphocyte numbers in the nodes of DNFBtreated mice remained significantly higher than those of control
lymph nodes 28 days after the repainting. The proportions of
PCNA-positive CD41 and CD81 T cell subsets, however, remained ,2.5% (Table I) over the first 24 h, suggesting that the
marked increase of both T cell subpopulations was predominantly
attributable to recruitment rather than proliferation in situ.
In the peripheral blood, there was a rapid and marked drop in the
number of total T lymphocytes (CD31), .50% in the first 30 min,
which gradually recovered within 24 to 48 h posttreatment (Fig.
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a
Sensitization was performed on day 0 and 1 by painting hapten or acetone on the
shaved abdomen as described elsewhere (see Materials and Methods). Lymphocytederived single-cell suspensions (1 3 106/ml) were fixed in 1% paraformaldehyde for
2 min. After washing in cold PBS, cells were incubated in 100% methanol at 220°C
for 10 min and washed again with cold PBS containing 0.1% Triton X-100. Subsequently, cells were incubated with FITC-conjugated anti-PCNA Ab (1.25 mg in 50 ml
of 2% BSA in PBS) for 15 min at room temperature. After washing in PBS supplemented with 2% BSA, cells were further incubated with anti-CD4-R-PE and antiCD8-RED613 Abs at 4°C for 30 min in the dark. Cells were then washed twice in
PBS/2% BSA and resuspended in 0.5 ml of PBS. As a positive control for this
analysis, lymph node-derived mononuclear cells (.70% T lymphocytes) were stimulated for 3 days in vitro with 10 mg/ml of PHA. Samples were analysed on a
FACScan equipped with LYSIS II software. Each group consisted of four animlas.
Results are expressed as mean 6 SD.
b
Represents the proportion of PCNA-positive cells (mean 6 SD) of the total
CD41 or CD81 T cell populations.
The Journal of Immunology
5667
2B). This reduction included both the CD41 and CD81 populations (Fig. 2B).
the paracortex was also noted. Increased activity of germinal centers was visible in some nodes (data not shown).
DNFB sensitization induces parafollicular hyperplasia and sinus
histiocytosis of draining lymph nodes
MIP-1a and MIP-1b are rapidly induced during DNFB
sensitization
During the period of lymph node enlargement after DNFB repainting (0 – 4 days), histologic examination revealed prominent subcapsular and medullary sinus expansion (data not shown). These
regions were filled with a large number of leukocytes, predominantly lymphocytes, macrophages, and mast cells, but also polymorphonuclear cells. At later time points, there was also a significant enlargement of the hilar region and a slight expansion the
medullary regions of the nodes. A less pronounced expansion of
In situ hybridization and immunohistochemical staining of the
draining lymph nodes demonstrated the induction of MIP-1a and
MIP-1b mRNAs (Fig. 3) and abundant production of these proteins in animals sensitized with DNFB (Fig. 3). By contrast, lymph
nodes obtained from acetone-painted (control) mice showed no
expression of these chemokine mRNAs (Figs. 3 and 5). Computerassisted morphometric analysis of lymph node sections to quantitate the number of cells expressing chemokine mRNA, confirmed
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FIGURE 3. DFNB sensitization rapidly induces expression of MIP-1a and MIP-1b mRNAs in draining lymph nodes. A and B, Sections (1603
magnification) from animals sacrificed 4 and 24 h after DNFB repainting, showing intense blue staining of MIP-1b mRNA-expressing cells located in the
subcapsular and hilar regions. The background is lightly counterstained with neutral red. C, Lymph node section from a mouse sacrificed 4 h after repainting
with DNFB, hybridized with the MIP-1a probe, showing abundant expression of the chemokine mRNA (blue staining) in the hilar and medullary regions.
D, Section obtained 24 h after repainting with DNFB, showing substantial parafollicular staining of MIP-1a mRNA. Hybridization of adjacent sections to
those shown in A–D with the sense MIP-1b amd MIP-1a probes showedno signal (data not shown). E, A lymph node section from an acetone-painted
(control) mouse hybridized with the atisense MIP-1a probe showing no mRNA signal. No positive signal was found in control lymph node sections
hybridized with the antisense MIP-1b probe (data not shown). F, Immunohistochemical stain (positive cells stained red) with anti-MIP-1a Ab on a section
from an animal sacrificed 24 h after repainting with DNFB.
5668
CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES
the maximal induction of both chemokines at 4 h (Fig. 4A). As
expected, the kinetics of induction of the chemokine proteins in
Western blot analyses was slightly delayed in comparison with the
mRNA. MIP-1b expression was faintly evident in control tissues
and was relatively constant from 4 –96 h after DNFB treatment
(Fig. 4B, upper panel), whereas MIP-1a expression appeared maximal at 24 h (Fig. 4B, lower panel). Thus, the expression of these
chemokine proteins was coincident with the accumulation of
CD41 and CD81 T lymphocyte subsets, suggesting a functional
role for these chemokines in the regulation of T lymphocyte
recruitment.
MIP-1b is expressed predominantly by mast cells, whereas
MIP-1a is expressed by a wide range of lymph node cells
Treatment with anti-chemokine Abs abrogates T cell recruitment
to the draining lymph nodes
To test whether T lymphocyte recruitment in this model was dependent on MIP-1a and MIP-1b, mice were given an i.p. injection
of neutralizing anti-chemokine Abs 4 h before the first painting
with DNFB (37, 38). The anti-chemokine Abs caused a dose-dependent inhibition of the recruitment of T lymphocytes to the
draining lymph nodes. Administration of 50, 200, or 500 mg of
anti-MIP-1a Ab reduced CD31 T cell numbers, evaluated at 24 h
after repainting, by 16% ( p , 0.05), 31% ( p , 0.01), or 48%
( p , 0.01), respectively, of the cell numbers in animals injected
with 1000 mg of control Ab. Similarly, anti-MIP-1b Ab treatment
in the same doses produced 10, 20, and 49% inhibition. The inhibition produced by pretreatment of mice with 500 mg of either
anti-MIP-1a or anti-MIP-1b Abs was significant for both CD41
and CD81 T cells ( p , 0.01 for all four comparisons; Fig. 6).
Treatment with anti-chemokine Abs modifies a subsequent
delayed-type hypersensitivity response
To assess the effect of chemokine inhibition on the outcome of the
DNFB-induced immune response, mice treated with a combination
of anti-MIP-1a (500 mg) and anti-MIP-1b (500 mg) Abs before
DNFB sensitization were challenged 5 days later with 0.2% DNFB
applied to the left ear. Mice that were injected with control Ab
exhibited a good ear swelling response, comparable with that of
positive control animals, whereas mice pretreated with a combination of anti-MIP-1a and anti-MIP-1b Abs before hapten appli-
FIGURE 4. Chemokine mRNA and protein expression in draining
lymph nodes increases dramatically during DNFB sensitization. A, In situ
hybridization was performed using MIP-1a or MIP-1b riboprobes. Contiguous high power fields across each whole section were examined before
the mean number of cells per high power field (6SEM) was enumerated
with a computer-assisted analysis system. Two lymph node sections from
each mouse (four mice per time point) were analysed. B, Immunoblotting
from chemokines in cell lysates prepared from mouse lymph nodes using
anti-MIP-1b Ab (upper panel) demonstrated the production of an ~8.5-kDa
MIP-1b protein at 4, 24, and 96 h after repainting with DNFB. The 8.3-kDa
band of the standard m.w. marker (Bio-Rad) is indicated on the left side of
the membrane. Immunoblotting of cell lysates derived from mouse lymph
nodes using an anti-MIP-1a Ab (lower panel) demonstrated the production
of an ~8.5-kDa protein at 4 h (lane 2) and 24 h (lane 3) but not at 96 h (lane
4) after repainting with DNFB. Lysates from acetone-painted (control)
mice (lane 1) showed no detectable MIP-1a. Each lane was loaded with an
equal amount (20 mg) of cell lysate derived from a pool of four to five
animals.
cation showed a modest, but significant, 19% inhibition of the
contact hypersensitivity reaction elicited by ear lobe challenge
(Table II).
Discussion
A considerable body of in vitro and in vivo data highlights the role
of chemokines in the regulation of leukocyte emigration from the
vascular compartment to sites of inflammation (27). This chemokine regulation hinges upon the ability of these cytokines to induce
affinity modulation of integrin adhesion molecules expressed on
leukocytes (4, 13, 27, 39) and upon the target cell specificity of the
individual chemokines (27, 39 – 41). Like other leukocyte subpopulations, T lymphocytes appear to depend on chemokine-regulated mechanisms for recruitment across vascular endothelium to
sites of inflammation (23, 27).
The present findings provide clear in vivo evidence for an association between the production of the b-chemokines MIP-1a
and MIP-1b and the recruitment of CD41 and CD81 T lymphocytes into peripheral lymph nodes during an immune response. In
this model of contact hypersensitivity, T lymphocytes accumulated
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Histomorphologic and immunohistochemical studies of lymph
node sections revealed the cellular sources of MIP-1b mRNA were
significantly different from those of MIP-1a. At all time points, a
range of cell types in the nodes was found to express MIP-1a,
including macrophages, lymphocytes, and endothelial cells (figure
not shown). By contrast, a striking and novel finding was the demonstration that mast cells were the predominant source of MIP-1b
mRNA in the lymph nodes of DNFB-treated mice (Fig. 5). Furthermore, computer-assisted quantitation in the nodes confirmed a
significant increase in the number of mast cells in the subcapsular
and hilar regions of the draining nodes (54). This increase was
concurrent with a decrease in the number of mast cells at the site
of sensitization in the skin, thus suggesting that mast cells travel
from the skin via afferent lymphatics. Immunohistochemical detection of MIP-1b protein confirmed the mast cell localization of
this chemokine. Furthermore, prominent staining of MIP-1b was
found on endothelial cells (Fig. 5D), despite the absence of detectable mRNA in these cells. As the accumulation of MIP-1bexpressing mast cells coincided with the accumulation of T lymphocytes in the nodes, this pathway may be critical to the observed
rapid recruitment of T lymphocytes into the nodes after DNFB
repainting.
The Journal of Immunology
5669
very rapidly within the nodes and were predominantly PCNA negative, thereby precluding any significant role for lymphocyte proliferation as an explanation for the 10-fold increase in T lymphocyte numbers observed during sensitization.
The notion of altered trafficking of leukocytes into lymph nodes
during the induction of contact hypersensitivity was supported by
the rapid decrease in T lymphocyte numbers in the peripheral
blood coincident with their accumulation in the lymph nodes. In a
similar fashion, a significant decrease in the PBL count coincident
with accumulation of these cells in the lymph nodes was observed
in mice that had received repeated i.p. injections of Corynebacterium granulosum (42).
Three-color flow cytometric analysis of peripheral blood and
draining lymph node cells suggested that both CD41 and CD81 T
cell subsets had migrated into the nodes. This finding is consistent
with previous adoptive cell transfer studies indicating that both
CD41 and CD81 T lymphocyte subsets are mediators of DNFBinduced contact hypersensitivity (30, 31). By contrast, after stimulation with purified protein derivative of tuberculin, the large increase in
T cell traffic through lymph nodes during the recruitment phase was
mostly due to CD41 memory phenotype T lymphocytes (8).
During the induction of contact hypersensitivity, abundant MIP-1a
and MIP-1b mRNA and protein expression in the draining lymph
nodes was detected. The kinetics of the protein expression of MIP-1a
and MIP-1b was coincident with the recruitment of CD41 and CD81
T lymphocyte subsets, suggesting a functional role for these chemokines in the regulation of T cell recruitment. Although both chemokines were produced as early as 4 h after repainting, there was a
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FIGURE 5. Mast cells are the predominant source of MIP-1b in draining lymph nodes during DNFB sensitization. A and B, Adjacent sections of the
subcapsular region (2503 magnification) of a lymph node from a mouse sacrificed 4 h after DNFB treatment, showing expression of MIP-1b mRNA (blue
staining with background neutral red counterstaining; A), which colocalized with cells immunostained for MMCP-5, indicative of mast cells (red staining
with hematoxylin counterstaining; B). The arrows indicate at least two of the mast cells, which can confidently be identified in both sections. C and F,
Adjacent sections of the subcapsular region of a lymph node from an acetone-treated (control) mouse illustrating the absence of MIP-1b mRNA expression
(F), despite the presence of MMCP-5-positive mast cells (red staining; C) in the adjacent section. D, Lymph node section from a mouse sacrificed 24 h
after DNFB treatment, immunostained with an anti-MIP-1b Ab showing widespread staining (in red) of the lumenal surface od endothelial cells (arrows).
E, An adjacent section to D, stained with control goat IgG (negative control).
5670
CHEMOKINE REGULATION OF T CELL RECRUITMENT INTO LYMPH NODES
difference in the kinetics of expression, with MIP-1b protein expression being maximal at 4 h following DNFB repainting and persisting
at high levels beyond 96 h, which was earlier and more prolonged
than the expression of MIP-1a. Chemokine expression in the lymph
nodes was not examined at time points in between the first and second
paintings with DNFB; however, given the rapid kinetics of mRNA
expression, it is likely that some induction of chemokine gene expression occurs before the second painting.
The prominent expression of the protein, but not mRNA, of
MIP-1b by endothelial cells in the DNFB-sensitized lymph nodes
suggests translocation of this chemokine protein. This may occur
via the recently described transendothelial chemokine transport
mechanism (43), following initial synthesis by mast cells and macrophages in the nodes. In addition, the protein may be immobilized
by proteoglycans on the surface of the endothelial cells to facilitate
interaction with circulating lymphocytes (25, 44).
Table II. Effect of neutralizing anti-chemokine Abs administered before sensitzation on the subsequent
elicitation of contact hypersensitivity a
Sensitization (%)
Acetone
DNFB
DNFB
DNFB
DNFB
(0.5)
(0.5)
(0.5)
(0.5)
Challenge (%)
Acetone
DNFB (0.2)
DNFB (0.2)
DNFB (0.2)
DNFB (0.2)
DNFB (0.2)
Treatment Before
Sensitization
Ear Thickness (mm),
Mean 6 SD
Reduction (%)
Anti-MIP-1a and 1b
Goat IgG
Sodium chloride
60 6 0.2
80 6 0.3
380 6 1.2
305 6 0.3*
365 6 0.2 NS
370 6 0.3 NS
19.7
3.9
2.6
a
Five days after pretreatment with a combination of anti-MIP-1a and anti-MIP-1b or irrelevant Abs and sensitization with
DNFB, mice were challenged by application of 10 ml of 0.2% DNFB in acetone olive oil to one ear. Control mice were similarly
painted with the vehicle alone. The degree of ear swelling was measured before challenge and 24 h postchallenge by an
independent observer, using an engineer’s micrometers (Mitutoyo). Each ear lobe was measured twice and contact hypersensitivity determined as the amount of swelling of the DNFB-challenged ear compared with the thickness of the vehicle-treated
ear expressed in micrometers. Mice that were challenged with the hapten without previous sensitization served as an additional
negative control. Each experimental and control group consisted of four animals. Results are expressed as mean 6 SD.
*p , 0.05 vs positive control; NS, not significant (Two-tailed Mann-Whitney U test of the row data).
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FIGURE 6. Chemokine inhibition reduces recruitment of T lymphocytes in the lymph nodes. Four hours before sensitization with DNFB,
experimental animals were pretreated by i.p. injection of 500 mg of antiMIP-1a and/or anti-MIP-1b neutralizing Abs. Control animals were injected with control goat IgG in doses comparable to those given the experimental animals. Pretreatment with anti-MIP-1a and/or anti MIP-1b
Abs significantly abrogated the recruitment of T lymphocytes into the
draining lymph nodes. Mice similarly treated with the maximum amount (1
mg) of control goat IgG showed no inhibition of T cell accumulation after
DNFB sensitization. Each experimental and control group consisted of four
animals. Results are presented as mean 6 SD.
The kinetics and magnitude of lymphocyte accumulation in the
regional lymph nodes demonstrated following Ag challenge in this
study were very similar to previously published reports (8, 9, 42).
Although prior studies have indicated the involvement of draining
lymph nodes in both the sensitization and elicitation phases of
DNFB-induced contact hypersensitivity (30 –33), there are no reports on the histologic changes in the lymph nodes in this model.
At the early time points, the nodes showed marked expansion of
the subcapsular and medullary sinuses, which were filled with a
large number of cells, predominantly lymphocytes, macrophages,
and mast cells. This extensive expansion of the sinuses may reflect
an increased flow of lymph and inflammatory cells from the skin
via the afferent lymphatic vessels into the draining nodes. At later
time points, increased activity of germinal centers was visible in
some nodes, indicating a proliferative response to the Ag.
The significance of MIP-1a and MIP-1b in this model was further confirmed by substantial inhibition of T lymphocyte recruitment into draining lymph nodes following administration of either
anti-MIP-1a or anti-MIP-1b Abs. The reported preferential activity of these chemokines on CD41 or CD81 T lymphocyte subset
was not observed, as the accumulation of both T cell subsets was
similarly inhibited by either anti-MIP-1a or anti-MIP-1b Abs.
However, the combination of both Abs produced a significantly
greater reduction in CD81 ( p , 0.05) than CD41 T lymphocyte
recruitment. This suggests that in CD41 T lymphocytes the two
chemokines may act via a receptor (such as CCR5) that binds both
ligands, whereas in CD81 T lymphocytes, additional receptors
(such as CCR1, which is not responsive to MIP-1b) may also be
utilized (27).
Other recently identified b-chemokines such as secondary lymphoid tissue chemokine (SLC) and EBI1-ligand chemokine (ELC)
have been proposed to be relevant to T lymphocyte recruitment
into lymphoid organs (40, 45). These chemokines are constitutively expressed in lymphoid tissues and are chemotactic for lymphocytes (45). However, there are no in vivo data regarding the
function of these molecules. Nevertheless, it is likely that several
chemokines, in addition to MIP-1a and MIP-1b, have the capacity
to regulate both the homing of T lymphocytes in the physiologic
state and the dramatically enhanced recruitment events that occur
during the development of an immune response. This notion is
consistent with the incomplete inhibition of T lymphocyte trafficking demonstrated in this contact hypersensitivity model.
Animals pretreated with a combination of anti-MIP-1a and antiMIP-1b Abs before hapten application showed a modest but significant 19% inhibition of the contact hypersensitivity reaction
The Journal of Immunology
Acknowledgments
We thank Ms. Angelina Enno for processing and cutting the histologic
sections.
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