| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
and MIP-1ß1
Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and MIP-1ß mRNAs and proteins. Mast cells
accumulating in the nodes during DNFB sensitization were the
predominant source of MIP-1ß, whereas MIP-1 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, ß-chemokines
regulate T lymphocyte emigration from the circulation into lymph nodes
during an immune response and contribute significantly to the
immunologic outcome. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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, 8, 9). In lymph nodes undergoing an immune response, lymphocyte 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, L-selectin, 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 ß-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 ß-chemokines
MIP-1, MIP-1ß, monocyte chemotactic protein (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-1
and MIP-1ß preferentially attract CD8+ and
CD4+ 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-1 in the HIV lymph nodes was
associated with the accumulation of CD8+ T cells in these
tissues. The present experiments were designed to further define the
potential in vivo role of MIP-1 and MIP-1ß 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 CD4+ and CD8+ 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-1 and MIP-1ß 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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.
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 µl of the vehicle alone.
Elicitation of contact hypersensitivity and evaluation of ear swelling
Five days after sensitization, mice were challenged by applying 10 µl 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 ± 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-1 and/or
MIP-1ß 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 µl) 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 µl) was used to stain cell surface markers with mAbs in three-color flow cytometry (see below).
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 -70°C for subsequent immunohistochemical studies.
Preparation of the single-cell suspension
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-µm 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 x 106/ml. The total cell count was enumerated after assessment of viability with trypan blue.
Measurement of differential leukocyte counts in the lymph nodes
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 -20°C for immunohistochemical staining.
Three-color flow cytometry
The Abs use in flow cytometry included: anti-CD3-FITC,
anti-CD4-R-phycoerythrin (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 
1 and 2a (IgG1-FITC/IgG2a-PE) 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 µl) and 
2 x 105 cells in 100 µl 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 -20°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 µg in 50 µl 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 node-derived mononuclear cells were stimulated in vitro with 10 µg/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-1 and MIP-1ß 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 µl of
prehybridization solution on 4-µm thick, formalin-fixed,
paraffin-embedded sections. This was followed by repeated stringency
washing at 42°C in 0.5x 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 + 5'-bromo-4-chloro-3-indolyl phosphate (NBT +
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
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; 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 anti-rabbit secondary
Ab was used for this staining (Dako, Glostrup, Denmark).
Immunohistochemical staining using primary goat anti-mouse
polyclonal Abs directed against MIP-1 and MIP-1ß (R&D Systems) and
secondary donkey anti-goat IgG directly conjugated to horseradish
peroxidase (Serotec) were used to detect MIP-1 and MIP-1ß 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).
Quantitative evaluation of MIP-1 and MIP-1ß 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 250x 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-1 and MIP-1ß.
Analysis of MIP-1 and MIP-1ß 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-1 or MIP-1ß 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 secondary 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-WhitneyU 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 animals were analyzed for statistical significance in comparison with control animals. As several comparisons were performed, Bonferoni adjustments for statistical significance were made.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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. 1
A). 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.
|
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. 1
B). The count gradually returned
to normal values within 48 h after the treatment.
CD4+ and CD8+ 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
(CD3+) T lymphocytes in lymph nodes, which peaked within 12
to 24 h after repainting (Fig. 2A). Both CD4+ and
CD8+ 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 CD4+ and
CD8+ lymphocyte numbers in the nodes of DNFB-treated mice
remained significantly higher than those of control lymph nodes 28 days
after the repainting. The proportions of PCNA-positive CD4+
and CD8+ 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.
|
|
B). This reduction included both the CD4+
and CD8+ populations (Fig. 2B). DNFB sensitization induces parafollicular hyperplasia and sinus histiocytosis of draining lymph nodes
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 the paracortex was also noted. Increased activity of germinal centers was visible in some nodes (data not shown).
MIP-1 and MIP-1ß are rapidly induced during DNFB sensitization
In situ hybridization and immunohistochemical staining of the
draining lymph nodes demonstrated the induction of MIP-1 and
MIP-1ß 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).
Computer-assisted morphometric analysis of lymph node sections to
quantitate the number of cells expressing chemokine mRNA, confirmed 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-1ß expression was
faintly evident in control tissues and was relatively constant from
4–96 h after DNFB treatment (Fig. 4B, upper
panel), whereas MIP-1 expression appeared maximal at
24 h (Fig. 4B, lower panel). Thus, the expression of
these chemokine proteins was coincident with the accumulation of
CD4+ and CD8+ T lymphocyte subsets, suggesting
a functional role for these chemokines in the regulation of T
lymphocyte recruitment.
|
|
|
is expressed by a wide range of lymph node cells
Histomorphologic and immunohistochemical studies of lymph node
sections revealed the cellular sources of MIP-1ß mRNA were
significantly different from those of MIP-1. At all time points, a
range of cell types in the nodes was found to express MIP-1,
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-1ß 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-1ß protein confirmed
the mast cell localization of this chemokine. Furthermore, prominent
staining of MIP-1ß was found on endothelial cells (Fig. 5D), despite the absence of detectable mRNA in these cells.
As the accumulation of MIP-1ß-expressing 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.
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-1 and MIP-1ß, 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 µg of
anti-MIP-1 Ab reduced CD3+ 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 µg of control Ab. Similarly,
anti-MIP-1ß Ab treatment in the same doses produced 10, 20, and
49% inhibition. The inhibition produced by pretreatment of mice with
500 µg of either anti-MIP-1 or anti-MIP-1ß Abs was
significant for both CD4+ and CD8+ T cells
(p < 0.01 for all four comparisons; Fig. 6).
|
To assess the effect of chemokine inhibition on the outcome of the
DNFB-induced immune response, mice treated with a combination of
anti-MIP-1 (500 µg) and anti-MIP-1ß (500 µg) 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-1 and anti-MIP-1ß Abs before hapten application
showed a modest, but significant, 19% inhibition of the contact
hypersensitivity reaction elicited by ear lobe challenge (Table II).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present findings provide clear in vivo evidence for an association
between the production of the ß-chemokines MIP-1 and MIP-1ß and
the recruitment of CD4+ and CD8+ T lymphocytes
into peripheral lymph nodes during an immune response. In this model of
contact hypersensitivity, T lymphocytes accumulated 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 CD4+ and CD8+ T cell subsets had migrated into the nodes. This finding is consistent with previous adoptive cell transfer studies indicating that both CD4+ and CD8+ T lymphocyte subsets are mediators of DNFB-induced 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 CD4+ memory phenotype T lymphocytes (8).
During the induction of contact hypersensitivity, abundant
MIP-1 and MIP-1ß mRNA and protein expression in the draining lymph
nodes was detected. The kinetics of the protein expression of MIP-1
and MIP-1ß was coincident with the recruitment of CD4+
and CD8+ 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 difference in the kinetics of expression, with MIP-1ß
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-1. 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-1ß 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).
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, 31, 32, 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-1 and MIP-1ß in this model was further
confirmed by substantial inhibition of T lymphocyte recruitment into
draining lymph nodes following administration of either
anti-MIP-1 or anti-MIP-1ß Abs. The reported preferential
activity of these chemokines on CD4+ or CD8+ T
lymphocyte subset was not observed, as the accumulation of both T cell
subsets was similarly inhibited by either anti-MIP-1 or
anti-MIP-1ß Abs. However, the combination of both Abs produced a
significantly greater reduction in CD8+
(p < 0.05) than CD4+ T lymphocyte
recruitment. This suggests that in CD4+ T lymphocytes the
two chemokines may act via a receptor (such as CCR5) that binds both
ligands, whereas in CD8+ T lymphocytes, additional
receptors (such as CCR1, which is not responsive to MIP-1ß) may also
be utilized (27).
Other recently identified ß-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-1 and MIP-1ß, 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-1 and
anti-MIP-1ß Abs before hapten application showed a modest but
significant 19% inhibition of the contact hypersensitivity reaction
elicited by ear lobe challenge (Table II). Therefore, pretreatment of
animals with a combination of anti-MIP-1 and anti-MIP-1ß
before hapten application not only significantly abrogates T cell
recruitment to the draining nodes, but also partially blocks the
subsequent delayed-type hypersensitivity reaction. This finding
suggests that mice injected with anti-MIP-1 and
anti-MIP-1ß Abs followed by hapten application became partially
tolerant to this hapten, perhaps as a result of decreased recruitment
of hapten-primed T cells into the regional lymph nodes and thus a
decrease in the expansion of hapten-specific T cells responsible for
mounting a strong response upon subsequent challenge.
A striking finding in this work is the demonstration of
mast cells as the predominant cellular source of MIP-1ß in the lymph
nodes of DNFB-treated mice. These cells were distinctively located in
the subcapsular and hilar regions of the nodes. Previous in vitro
experiments have described the expression of MIP-1ß mRNA by mast cell
lines (46, 47). However, the abundant expression of this chemokine by
mast cells observed in this in vivo model of contact hypersensitivity
is novel. The brisk appearance of mast cells in a distinctive location
in the subcapsular and hilar regions of the nodes, in association with
the decrease in their number in the affected skin shortly after
repainting with DNFB, strongly suggests that mast cells travel from the
skin via the afferent lymphatic system. As expected, mast cell
accumulation in the draining nodes was unaffected by neutralization of
MIP-1 and MIP-1ß activity,4 indicating that movement
of these cells into the nodes is independent of a concentration
gradient of these chemokines. The novel observation of mast cells as
the dominant source of MIP-1ß indicated in this work clarifies the
controversy regarding the role of these cells in contact
hypersensitivity (48, 49, 50, 51). In particular, mast cells are not only a
source of histamine, serotonin, and other vasoactive amines that are
believed to control vascular tone and permeability (52, 53), but also
act as a key early regulator of T cell recruitment into draining lymph
nodes.
These findings provide the first direct in vivo evidence of chemokine regulation of trafficking of T lymphocyte subpopulations into lymph nodes during the induction of an immune response. Mast cells accumulating in the nodes have been identified as the principal source of MIP-1ß in this model of contact hypersensitivity. Further studies examining additional chemokines and other models of immune response are warranted, as interventions to manipulate this chemokine-dependent T lymphocyte trafficking pathway may have profound influences on the outcome of host responses to infection or autoimmune triggers.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
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.
Received for publication April 14, 1998. Accepted for publication July 13, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4 ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
and MIP-1ß. Science 260:355. (MIP-1) and MIP-1ß chemokines attract distinct populations of lymphocytes. J. Exp. Med. 177:1821.-producing (Tc1) effector CD8+ T cells and interleukin (IL) 4/IL-10 producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001.This article has been cited by other articles:
|
|
 |
|
  B. K. Masouleh, G. B. Ten Dam, M. K. Wild, R. Seelige, J. van der Vlag, A. L. Rops, F. G. Echtermeyer, D. Vestweber, T. H. van Kuppevelt, L. Kiesel, et al. Role of the Heparan Sulfate Proteoglycan Syndecan-1 (CD138) in Delayed-Type Hypersensitivity J. Immunol., April 15, 2009; 182(8): 4985 - 4993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Furuta, T. Kikuchi, Y. Iwakura, and N. Watanabe Protective Roles of Mast Cells and Mast Cell-Derived TNF in Murine Malaria. J. Immunol., September 1, 2006; 177(5): 3294 - 3302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Willard-Mack Normal Structure, Function, and Histology of Lymph Nodes Toxicol Pathol, August 1, 2006; 34(5): 409 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. G. Diacovo, A. L. Blasius, T. W. Mak, M. Cella, and M. Colonna Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes J. Exp. Med., September 6, 2005; 202(5): 687 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-i. Kashiwakura, H. Yokoi, H. Saito, and Y. Okayama T Cell Proliferation by Direct Cross-Talk between OX40 Ligand on Human Mast Cells and OX40 on Human T Cells: Comparison of Gene Expression Profiles between Human Tonsillar and Lung-Cultured Mast Cells J. Immunol., October 15, 2004; 173(8): 5247 - 5257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. LaFranco-Scheuch, K. Abel, N. Makori, K. Rothaeusler, and C. J. Miller High Beta-Chemokine Expression Levels in Lymphoid Tissues of Simian/Human Immunodeficiency Virus 89.6-Vaccinated Rhesus Macaques Are Associated with Uncontrolled Replication of Simian Immunodeficiency Virus Challenge Inoculum J. Virol., June 15, 2004; 78(12): 6399 - 6408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Grimm, R. Newman, Z. Hassim, N. Cuan, S. J. Connor, Y. Le, J. M. Wang, J. J. Oppenheim, and A. R. Lloyd Cutting Edge: Vasoactive Intestinal Peptide Acts as a Potent Suppressor of Inflammation In Vivo by Trans-Deactivating Chemokine Receptors J. Immunol., November 15, 2003; 171(10): 4990 - 4994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Tanzola, M. Robbie-Ryan, C. A. Gutekunst, and M. A. Brown Mast Cells Exert Effects Outside the Central Nervous System to Influence Experimental Allergic Encephalomyelitis Disease Course J. Immunol., October 15, 2003; 171(8): 4385 - 4391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Helmby and R. K. Grencis IFN-{gamma}-Independent Effects of IL-12 During Intestinal Nematode Infection J. Immunol., October 1, 2003; 171(7): 3691 - 3696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Li, C.-S. Tsai, P.-C. Hsu, C.-T. Wu, C.-S. Wong, and S.-T. Ho Dobutamine Modulates Lipopolysaccharide-Induced Macrophage Inflammatory Protein-1{alpha} and Interleukin-8 Production in Human Monocytes Anesth. Analg., July 1, 2003; 97(1): 210 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Kohler, A. C. Caon, D. O. Willenborg, I. Clark-Lewis, and S. R. McColl A Role for Macrophage Inflammatory Protein-3{alpha}/CC Chemokine Ligand 20 in Immune Priming During T Cell-Mediated Inflammation of the Central Nervous System J. Immunol., June 15, 2003; 170(12): 6298 - 6306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Nakae, Y. Komiyama, S. Narumi, K. Sudo, R. Horai, Y.-i. Tagawa, K. Sekikawa, K. Matsushima, M. Asano, and Y. Iwakura IL-1-induced tumor necrosis factor-{alpha} elicits inflammatory cell infiltration in the skin by inducing IFN-{gamma}-inducible protein 10 in the elicitation phase of the contact hypersensitivity response Int. Immunol., February 1, 2003; 15(2): 251 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nakajima, N. Inagaki, H. Tanaka, A. Tanaka, M. Yoshikawa, M. Tamari, K. Hasegawa, K. Matsumoto, H. Tachimoto, M. Ebisawa, et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison Blood, December 1, 2002; 100(12): 3861 - 3868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Wood, R. Sehmi, S. Dorman, Q. Hamid, M. K. Tulic, R. M. Watson, R. Foley, P. Wasi, J. A. Denburg, G. Gauvreau, et al. Allergen-induced Increases in Bone Marrow T Lymphocytes and Interleukin-5 Expression in Subjects with Asthma Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 883 - 889. [Abstract] [Full Text]
|
|
|
|
|
|
M. K. Park, D. Amichay, P. Love, E. Wick, F. Liao, A. Grinberg, R. L. Rabin, H. H. Zhang, S. Gebeyehu, T. M. Wright, et al. The CXC Chemokine Murine Monokine Induced by IFN-{gamma} (CXC Chemokine Ligand 9) Is Made by APCs, Targets Lymphocytes Including Activated B Cells, and Supports Antibody Responses to a Bacterial Pathogen In Vivo J. Immunol., August 1, 2002; 169(3): 1433 - 1443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. King, R. Anderson, and J. S. Marshall Dengue Virus Selectively Induces Human Mast Cell Chemokine Production J. Virol., July 17, 2002; 76(16): 8408 - 8419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Borchers, T. Ansay, R. DeSalle, B. L. Daugherty, H. Shen, M. Metzger, N. A. Lee, and J. J. Lee In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration J. Leukoc. Biol., June 1, 2002; 71(6): 1033 - 1041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Mellor, K. F. Austen, and J. A. Boyce Cysteinyl Leukotrienes and Uridine Diphosphate Induce Cytokine Generation by Human Mast Cells Through an Interleukin 4-regulated Pathway that Is Inhibited by Leukotriene Receptor Antagonists J. Exp. Med., March 4, 2002; 195(5): 583 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Ebert and S. R. McColl Up-Regulation of CCR5 and CCR6 on Distinct Subpopulations of Antigen-Activated CD4+ T Lymphocytes J. Immunol., January 1, 2002; 168(1): 65 - 72. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. S. Baekkevold, T. Yamanaka, R. T. Palframan, H. S. Carlsen, F. P. Reinholt, U. H. von Andrian, P. Brandtzaeg, and G. Haraldsen The Ccr7 Ligand ELC (Ccl19) Is Transcytosed in High Endothelial Venules and Mediates T Cell Recruitment J. Exp. Med., May 7, 2001; 193(9): 1105 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fiorentini, S. Licenziati, G. Alessandri, F. Castelli, S. Caligaris, M. Bonafede, M. Grassi, E. Garrafa, A. Balsari, A. Turano, et al. CD11b Expression Identifies CD8+CD28+ T Lymphocytes with Phenotype and Function of Both Naive/Memory and Effector Cells J. Immunol., January 15, 2001; 166(2): 900 - 907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Andrew, N. Ruffing, C. H. Kim, W. Miao, H. Heath, Y. Li, K. Murphy, J. J. Campbell, E. C. Butcher, and L. Wu C-C Chemokine Receptor 4 Expression Defines a Major Subset of Circulating Nonintestinal Memory T Cells of Both Th1 and Th2 Potential J. Immunol., January 1, 2001; 166(1): 103 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Shang, B. Qiu, K. A. Frait, J. S. Hu, J. Sonstein, J. L. Curtis, B. Lu, C. Gerard, and S. W. Chensue Chemokine Receptor 1 Knockout Abrogates Natural Killer Cell Recruitment and Impairs Type-1 Cytokines in Lymphoid Tissue during Pulmonary Granuloma Formation Am. J. Pathol., December 1, 2000; 157(6): 2055 - 2063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Gretz, C. C. Norbury, A. O. Anderson, A. E.I. Proudfoot, and S. Shaw Lymph-Borne Chemokines and Other Low Molecular Weight Molecules Reach High Endothelial Venules via Specialized Conduits While a Functional Barrier Limits Access to the Lymphocyte Microenvironments in Lymph Node Cortex J. Exp. Med., November 20, 2000; 192(10): 1425 - 1440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Exton, A. Elfers, W.-Y. Jeong, D. F. Bull, J. Westermann, and M. Schedlowski Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1310 - R1315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Dawson, M. A. Beck, W. A. Kuziel, F. Henderson, and N. Maeda Contrasting Effects of CCR5 and CCR2 Deficiency in the Pulmonary Inflammatory Response to Influenza A Virus Am. J. Pathol., June 1, 2000; 156(6): 1951 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Lee, M. E. Brummet, S. Shahabuddin, T. G. Woodworth, S. N. Georas, K. M. Leiferman, S. C. Gilman, C. Stellato, R. P. Gladue, R. P. Schleimer, et al. Cutaneous Injection of Human Subjects with Macrophage Inflammatory Protein-1{alpha} Induces Significant Recruitment of Neutrophils and Monocytes J. Immunol., March 15, 2000; 164(6): 3392 - 3401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Stein, A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian The Cc Chemokine Thymus-Derived Chemotactic Agent 4 (Tca-4, Secondary Lymphoid Tissue Chemokine, 6ckine, Exodus-2) Triggers Lymphocyte Function-Associated Antigen 1-Mediated Arrest of Rolling T Lymphocytes in Peripheral Lymph Node High Endothelial Venules J. Exp. Med., January 3, 2000; 191(1): 61 - 76. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. H. Tesch, S. Maifert, A. Schwarting, B. J. Rollins, and V. R. Kelley Monocyte Chemoattractant Protein 1-Dependent Leukocytic Infiltrates Are Responsible for Autoimmune Disease in Mrl-Faslpr Mice J. Exp. Med., December 20, 1999; 190(12): 1813 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamagami, D. Miyazaki, S. J. Ono, and M. R. Dana Differential Chemokine Gene Expression in Corneal Transplant Rejection Invest. Ophthalmol. Vis. Sci., November 1, 1999; 40(12): 2892 - 2897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Zabel, W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. Human G Protein-Coupled Receptor Gpr-9-6/Cc Chemokine Receptor 9 Is Selectively Expressed on Intestinal Homing T Lymphocytes, Mucosal Lymphocytes, and Thymocytes and Is Required for Thymus-Expressed Chemokine-Mediated Chemotaxis J. Exp. Med., November 1, 1999; 190(9): 1241 - 1256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Krzysiek, E. A. Lefevre, W. Zou, A. Foussat, J. Bernard, A. Portier, P. Galanaud, and Y. Richard Antigen Receptor Engagement Selectively Induces Macrophage Inflammatory Protein-1{alpha} (MIP-1{alpha}) and MIP-1{beta} Chemokine Production in Human B Cells J. Immunol., April 15, 1999; 162(8): 4455 - 4463. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
