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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2886-2895
A Novel Mutant Gene Involved in T-Lymphocyte-Specific Homing Into
Peripheral Lymphoid Organs on Mouse Chromosome 4
By
Hideki Nakano,
Shigeyuki Mori,
Hiromichi Yonekawa,
Hideo Nariuchi,
Akio Matsuzawa, and
Terutaka Kakiuchi
From the Department of Immunology, Toho University School of
Medicine, Tokyo, Japan; the Laboratory Animal Research Center and
Department of Allergology, Institute of Medical Science, University of
Tokyo, Tokyo, Japan; and the Department of Laboratory Animal Science,
The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
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ABSTRACT |
Previously, we have shown a mutant mouse DDD/1 with T-cell-specific
homing defect that is regulated by an autosomal recessive gene,
plt (paucity of lymph node T cells), and seems to be caused by
lymph node (LN) stromal cells. In the present study,
immunohistochemical analysis showed unusual distribution of T cells in
LN, Peyer's patches (PP), and spleen from plt/plt, probably
due to the failure of T cells to migrate from blood into the T-cell
zone in LN or PP, or into the spleen white pulp across high endothelial
venule or marginal zone, respectively, based on the experiments in
which labelled T cells were injected intravenously and detected in the tissues. Analysis of surface L-selectin and CD44 suggested that T cells
with memory phenotype, probably from afferent lymphatics, recruit into
plt/plt LN. Linkage mapping by simple-sequence length polymorphism of genomic DNA from 190 backcross progenies produced by
intercrossing with MSM/Ms, linked plt most closely with
D4Mit237, and localized at 24.7 cM from cetromere on chromosome 4. We
discuss the possibility that a wild-type gene on plt locus
encodes a chemokine inducing T-cell-specific homing into peripheral
lymphoid tissues.
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INTRODUCTION |
LYMPHOCYTES THAT have matured and
differentiated in the primary lymphoid organs, thymus and bone marrow,
are released into peripheral blood and circulate to secondary lymphoid
organs such as lymph nodes (LN), Peyer's patches (PP), and spleen, in
which they respond to antigens.1-4 They recirculate from
lymph to blood and the recirculation seems to be essential for the
immune surveillance. The lymphocyte homing into secondary lymphoid
organs is initiated by the interaction of the homing receptors on the
lymphocyte surface, such as molecules of the selectin and integrin
families, and their ligand on the surface of the high endothelial
venule (HEV).1-7 L-selectin and
 4 7 integrin induce rolling of lymphocytes
on HEV,5,6 followed by their arrest and firm adhesion
through the interaction of L2
integrin/LFA-1 on lymphocytes with ICAM-1 on HEV.3,4,7
Then, the lymphocytes transmigrate and home into the lymphoid tissues.
Recently, we have shown a mutant mouse in which the T-cell number in
LN, but not B-cell number, is extremely small. The mutant mouse bears a
T-cell specific homing deficiency into LN, which is regulated by a
single autosomal recessive gene, plt (paucity of lymph node
T cells).8 The mutation seems to be expressed on LN stromal
cells, not on T cells themselves.8 T cells can bind to HEV
in peripheral LN (PLN) from plt/plt, as well as from +/+, in Stamper-Woodruff assay, and the binding is inhibited by a monoclonal antibody (MoAb) specific for L-selectin or its ligand peripheral node addressin (PNAd), suggesting that these molecules are
functional in plt/plt.8 Thus, some other factor(s)
might be required for homing of T cells into PLN. Based on these
findings, it is quite possible that plt is a mutant of the gene
that encodes a factor to allow T cells to home into PLN.
In contrast with PLN, PP and spleen in plt/plt contain as many
or more T cells than those in +/+.8 In this report
we examined T-cell-homing deficiency on the functional and
histological basis in PLN, mesenteric LN (MLN), PP, and spleen in
plt/plt. In addition, as an approach to identify the
plt gene, we mapped the gene on chromosome by the analysis with
simple-sequence length polymorphism (SSLP) of genomic
DNA9,10 in the intercrossing progeny between DDD/1 mouse
(Mus musculus domesticus)
[plt/plt]11 and Japanese wild-mouse-derived
inbred strain MSM/Ms (Mus musculus molossinus)
[+/+].12
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MATERIALS AND METHODS |
Mice.
DDD/1 (DDD/1-plt/plt) and DDD/1-Mtv-2/Mtv-2
(DDD/1-+/+) were maintained in the specific pathogen-free
conditions in animal facilities in the Institute of
Medical Science, University of Tokyo (Tokyo, Japan).8,11,13
MSM/Ms (MSM)12 were kindly provided by Dr Toshihiko
Shiroishi (National Institute of Genetics, Mishima, Shizuoka, Japan).
BALB/c mice were purchased from SLC (Hamamatsu, Shizuoka,
Japan). BALB/c-plt/plt were produced by backcrossing of mice
expressing plt phenotype that was detected with low content of
T cells in biopsied inguinal LN by flow cytometry.8,14 Sixth-backcrossed mice were used for experiments.
Antibodies.
Anti-TCR C (H57-597), -CD3 (145-2C11), -Thy1.2 (30H12), -B220
(RA3-3A1/6.1), -CD4 (GK1.5), -CD8 (53-6-7.2), -L-selectin (MEL-14), -CD44 (KM201), and -FcR II (2.4G2) MoAbs were used. Purified H57-597, 145-2C11, RA3-3A1/6.1, MEL-14, and KM201 were conjugated with fluorescein isothiocyanate (FITC) (Sigma, St Louis, MO) or with biotin-NHS (Vector, Burlingame, CA). FITC-conjugated anti-B220 MoAb
(RA3-6B2) was purchased from Pharmingen (San Diego, CA). Antimetallophilic macrophage MoAb (MOMA-1)15 and
Cy5-conjugated goat antirat IgG antibodies (Ab) were purchased from BMA
Biomedicals Ltd (Augst, Swizerland) and Amersham (Buckinghamshire,
England), respectively.
In vivo homing assay.
LN cells were collected from PLN and MLN of DDD/1-+/+. T cells
were enriched from DDD/1-+/+ or DDD/1-plt/plt spleen
cells through nylon wool column, followed by panning with goat
antimouse IgG (Cappel, Durham, NC)-coated dish, and B cells by panning
with anti-CD4 and -CD8 MoAb-coated dish after removal of adherent
cells. T-cell- or B-cell-enriched preparation always contained more
than 90% CD3+ or B220+ cells, respectively.
These cells (4 × 107/mL) were labeled with 3 µmol/L
3'-acetyl-2'-carboxyethyl-6',7'-(dihydropyran-2"-one)-5 or
6-carboxyfluorescein diacethoxymethyl ester (BCECF-AM) (Dojindo, Kumamoto, Japan) at 37°C for 1 minute in Hank's balanced salt solution (HBSS) containing 5% fetal calf serum (FCS).8
After washing, 1 × 107 or 5 × 107
labeled cells were injected intravenously into mice.
Fluorecence-positive cells in PLN, MLN, PP, and spleen were detected
with flow cytometry or histology.
Assessment of leukocyte accumulation into peritoneum.
Mice were intraperitonealy injected with 1 mL of 2% thioglycollate
(Nissui, Tokyo, Japan) to induce peritonitis.16 They were
killed 24 or 48 hours later, intraperitonealy injected with 5 mL of
phosphate-buffered saline (PBS) containing 2% FCS, 0.5 mmol/L EDTA,
and 10 U/mL heparin (Takeda, Osaka, Japan), and massaged extensively.
PBS was recovered from the peritoneum, and the cells were washed once,
pelleted by centrifugation, resuspended in the buffer described above,
and counted under a microscope. After washing, 5 × 105 cells in 5 µL FCS were smeared onto a glass slide.
The cells stained with Giemsa's solution (Merck Japan, Tokyo, Japan)
were examined morphologically under a microscope. The percentages of neutrophils and macrophages were calculated from more than 200 cells.
Immunohistochemical staining.
Cryostat sections (8 µm) from the lymphoid organs were dried and
fixed with acetone. After washing with PBS, the section was incubated
with 2% normal rabbit serum in PBS, containing 1% bovine serum
albumin (BSA) to prevent nonspecific reactions, and then sequentially
treated with 30H12 or RA3-3A1/6.1 MoAb with biotinylated rabbit antirat
IgG polyclonal Ab (mouse serum adsorbed, Vector), and with horseradish
peroxidase-conjugated streptavidin (Zymed, SanFrancisco, CA).
Peroxidase activity was visualized with 3, 3'-diaminobenzidine
tetrahydrochloride (Dojindo). The sections were counterstained with
Gill's hematoxylin (Polysciences, Warrington, PA) and examined under a
microscope.8 To study homing ability of BCECF-labeled
lymphocytes injected into mice, a cryostat section from the spleen was
stained with MOMA-1 MoAb that detects marginal zone macrophage
(MZM)15 and Cy5-conjugated antirat IgG, then examined with
a confocal laser microscope system (Bio-Rad Laboratories, Tokyo,
Japan).
Flow cytometry.
Single-cell suspensions were prepared from PLN, PP, and spleen and were
depleted of red blood cells by hemolysis. One million cells in each
sample were stained with FITC-conjugated MoAb in PBS containing 3% FCS
and 0.1% NaN3, or biotinylated MoAb followed by phycoerythrin-conjugated streptavidin (SA-PE) (Becton Dickinson, Mountain View, CA).17 Nonspecific reaction was blocked by
preincubation with mouse IgG and 2.4G2 MoAb if necessary. The stained
cells were analyzed on a FACScan (Becton Dickinson). Lymphocytes
recognized with the forward and sideward scatter were analyzed. Dead
cells positively stained with 7-aminoactinomycin D (Sigma) were gated out.14
Phenotype assessement.
(DDD/1-plt/plt × MSM) F1 hybrid and
F2 progeny were produced. Backcross (BC) progeny were
produced by mating male F1 to female DDD/1-plt/plt
or female F1 to male DDD/1-plt/plt. Mice were
phenotyped at 5 to 6 weeks of age. Lymphocytes of PLN (axillary,
brachial, and inguinal) were counted, dual-stained with FITC-conjugated anti-TCR and biotinylated anti-L-selectin MoAb, followed by SA-PE, and
analyzed in flow cytometry. Phenotype was determined based on the PLN
cell count and the percentage of
L-selectin+TCR+ cells. A section of spleen was
immunohistochemically analyzed with anti-Thy1.2 or anti-B220 MoAb, and
plt/plt showed the characteristic localization of T cells
(Fig 1).
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| Fig 1.
Histological analysis of T and B cells in PLN, PP, and
spleen in +/+ and plt/plt. PLN (a-d), PP (e-h) and
spleens (i-l) were from DDD/1-+/+ and
DDD/1-plt/plt. Spleens from BALB/c-+/+ (m,n) and
BALB/c-plt/plt (o, p) were also examined. Cryostat section was
treated sequentially with 30H12 (anti-Thy1.2) or RA3-3A1/6.1 (anti-B220) MoAb, biotinylated rabbit antirat IgG (Vector) polyclonal Ab, and horse radish peroxidase-conjugated streptavidin (Zymed), and
visualized with 3, 3'-diaminobenzidine tetrahydrochloride (Dojindo).
The sections were counterstained with Gill's hematoxylin (Polysciences) and photographed under a microscope. Bar indicates 400 µm.
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Genotype analysis.
For assessment of SSLP, 100 ng of genomic DNA from liver of
DDD/1-plt/plt, MSM, and BC progenies were amplified in a 10 µL polymerase chain reaction (PCR) with MapPairs primer (Genetic Research Inc, Huntsville, AL).9,10 Information for
microsatellite markers was obtained from the database of the Genetic
and Physical Map of the Mouse Genome published by Massachusetts
Institute of Technology (http://www.genome.wi.mit.edu./). The
conditions for PCR were 30 seconds at 96°C (5 minutes for the first
cycle), 1 minute at 55°C, and 1.5 minutes at 72°C for 35 cycles
in 10 mmol/L Tris-HCl (pH9.0) containing 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L deoxynucleotide triphosphates, 0.1%
Triton X-100, 0.2 µmol/L each primer, and 0.05 U rTaq DNA polymerase
(Toyobo, Tokyo, Japan).18 In amplification for D4Mit237,
concentration of MgCl2 was 2 mmol/L. To detect polymorphism
in the CD72 gene, the following primers were used for PCR amplification
of a simple sequence repeat found in genomic CD72 DNA
sequence19: left, 5'-ATGGGAGATGCTGGATGGAGAT-3'; and
right, 5'-CAGACCTAATTCCAACACTCAG-3'. The amplified products were
subjected to electrophoresis on a 3% NuSieve 3:1 agarose gel (FMC
Bioproducts, Rockland, ME), visualized with ethidium bromide staining,
and photographed. The size of each PCR product from BC progeny was
compared with that from parental strains, DDD/1-plt/plt and
MSM. Individuals with wild-type phenotype (plt/+) and
homogenous DDD/1/DDD/1 genotype for each marker, or with a mutant
phenotype (plt/plt) and heterogenous DDD/1/MSM genotype, were
assessed as a recombinant. Recombination distance between plt
and each marker or between markers was analyzed by computer by using
Map Manager v2.6.4 software provided by Drs Kenneth F. Manly and Robert
Cudmore (Roswell Park Cancer Institute, State University of New York,
Buffalo, NY). Information for chromosomal mapping was obtained from the
database of the Genetic and Physical Map of the Mouse Genome by
Massachusetts Institute of Technology or from National Center for
Biothechnology Information by the National Institutes of Health
(http://www.ncbi.nlm.nih.gov./).
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RESULTS |
Unusual distribution of T cells in PLN, PP, and spleen in
plt/plt.
To analyze the effect of the plt mutation on peripheral
lymphoid organs, the distribution of T and B cells in PLN, PP, and spleen from plt/plt was immunohistochemically compared with
that from +/+. In PLN of +/+, T cells were densely
distributed in the subcortical T-cell zones (Fig 1a). In PLN of
plt/plt, however, the cellularity of T cells was extremely low,
and the stroma was much more dominant in the subcortical zone than in
+/+ (Fig 1c). In PP of +/+, T cells were densely
observed in the T-cell zones between B-cell zones filled with
B220high and B220low B cells (Fig 1e, f), and
few T cells were found in the B-cell zones. In plt/plt, the
number and the size of PP were varied from mouse to mouse and from PP
to PP, and most of PP were smaller than those in +/+. T cells
were hardly observed in the distinct T-cell zones in PP from
plt/plt, but a lot of T cells were observed in the area
occupied by B cells (Fig 1g, h). Also in spleen, the T-cell
distribution was quite different between plt/plt and
+/+. In +/+, spleen T cells colonized densely at the
periarterial lymphatic sheath in the white pulp and spread sparsely in
the red pulp (Fig 1i). In plt/plt spleen, however, few T cells
were found in the white pulp, whereas numerous T cells distributed
around the vascular sinusoid in the red pulp (Fig 1k). The unusual
T-cell distribution in the spleen was observed not only in
DDD/1-plt/plt but also in BALB/c-plt/plt (Fig 1o). In
contrast, the B-cell distribution in PLN (Fig 1b, d), PP (Fig 1f, h),
and spleen (Fig 1j, l, n, p), in plt/plt was not different from
that in +/+. These results strongly suggest that the
plt gene is involved in T-cell homing not only in PLN but also
in PP and spleen.
Defect in T-cell migration into PLN, PP, and spleen white pulps in
plt/plt.
Defective recruitment of T cells into PLN was previously found in
plt/plt.8 To examine whether the defect was also
observed in other peripheral lymphoid organs, lymphocyte homing into
PLN, PP, and spleen was assessed in plt/plt. Lymphocytes from
+/+ PLN were labelled with fluorescent BCECF, intravenously
injected into plt/plt and +/+, and the labelled
lymphocytes were detected at 2 and 48 hours, and 6 days after the
injection. The number of fluorescence-positive cells in PLN, MLN, or PP
in plt/plt was always significantly smaller than that in
+/+. In spleen, however, fluorescence-positive cells in
plt/plt were more than those in +/+ throughout the
observation period (data not shown). To compare T-cell homing with
B-cell homing, fluorescence-labeled T or B cells enriched from
+/+ spleen cells were injected
into mice, and detected in PLN, PP, and spleen 48 hours after the
injection. The labelled T cells in PLN and PP in plt/plt were
fewer than those in +/+, but labelled B cells were detected in
plt/plt as well as in +/+
(Fig 2). In spleen, in contrast, the
labelled T cells in plt/plt were more than in +/+ (Fig
2). These results suggest a possibility that the homing deficiency in
plt/plt results from some defect(s) in secondary lymphoid
organs.
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| Fig 2.
Homing of T and B cells into PLN, PP, and spleen in vivo.
T and B cells were enriched from spleen of DDD/1-+/+,
labeled with BCECF-AM (Dojindo) and 1 × 107 of the cells
were intravenously injected. Fluorescence-positive cells in PLN, PP,
and spleen of DDD/1-+/+ (filled column) or
DDD/1-plt/plt (crosshatched column) were detected with flow
cytometry at 48 hours after the injection. Cell number was calculated
from the total cell number in each tissue and percentage of
fluorescence-positive cells. Mean and SD from two mice is indicated.
*P < .072, ** P < .05, *** P < .001.
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There was another possibility, however, that plt/plt T cells
themselves had a homing deficiency into secondary lymphoid organs. To
examine this possibility, plt/plt T cells were compared with those from +/+ for the migration into secondary lymphoid
organs. T cells were prepared from +/+ or plt/plt
spleen, labelled, intravenously injected into +/+ and
plt/plt mice, and detected in PLN, PP, and spleen 40 hours
after the injection. Labelled T cells from +/+ migrated into
PLN, PP, and spleen in +/+ recipients
(Fig 3), as described above. When those
from plt/plt were injected into +/+ recipients, they
migrated into these organs as well as, or rather better than those from
+/+, whereas their homing into these organs was deficient when
injected into plt/plt recipients (Fig 3). The deficiency was
similar to that observed when +/+ T cells were intravenously
injected into plt/plt recipients (Figs 2 and 3). Thus, the
defect in the migration ability was not detected in T cells from
plt/plt. These results strongly support our previous suggestion
that homing deficiency in plt/plt is caused by the defect in
the stromal cells in secondary lymphoid organs, but not in T cells
themselves.
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| Fig 3.
Comparison of plt/plt T cells with those from
+/+ for the migration into PLN, PP, and spleen. T cells
were enriched from DDD/1-plt/plt or DDD/1-+/+
spleen cells, and labelled with BCECF-AM. Ten million cells were
intravenously injected into DDD/1-+/+ and
DDD/1-plt/plt recipient mice. Forty hours after the injection,
fluorescence-positive cells were detected in PLN, PP, and spleen in
DDD/1-+/+ (filled columns) and in DDD/1-plt/plt
(crosshatched columns) recipients. Cell number was calculated from the
total cell number in each tissue and percentage of
fluorescence-positive cells. Mean and SD from two recipient mice is
indicated. *P < .053, ** P < .05, *** P < .005.
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The distribution of fluorescence-labelled T and B cells in spleen 48 hours after the injection was also examined in plt/plt and in
+/+. To identify the white pulp, the marginal zone was stained
with MoAb specific for metalofilic macrophages. In plt/plt, few
T cells labelled with fluorescence were detected in white pulp, whereas
many labelled T cells were observed outside the marginal zone, ie, the
red pulp (Fig 4). In +/+ spleen,
the accumulation of labelled T cells was observed at the periarterial
lymphatic sheath in the white pulp, as expected. Labelled B cells in
the white pulp were detected in plt/plt as well as in
+/+ (Fig 4). These results indicate that T cells migrate into
the white pulp in +/+, but do not in plt/plt, although
B cells are able to migrate into the white pulp in plt/plt as
well as in +/+.
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| Fig 4.
Localization of T and B cells homing into spleen. T and B
cells were enriched from spleen of DDD/1-+/+, labeled with
BCECF-AM (Dojindo), and 5 × 107 of the cells
were intravenously injected into DDD/1-+/+ and
DDD/1-plt/plt. Spleens from recipients were frozen at 48 hours
after the injection. Cryostat section of the spleen was stained with
MOMA-1 MoAb (BMA Biomedicals Ltd) and Cy5-conjugated antirat IgG
(Amersham) to identify MZM, then examined under a confocal laser
microscope system (Bio-Rad). The region sorrounded by MZM (red,
indicated by arrowheads) is white pulp. Injected T or B cells (green)
were identified in white pulp (thick arrow) and in red pulp (thin
arrow). Bar at the lower right corner indicates 250 µm.
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Increase in the proportion of PLN T cells with memory phenotype in
plt/plt.
Although the T-cell-specific defect in plt/plt was found in
the migration into PLN, PP, or spleen white pulp (Figs 2-4), some T
cells were still detected in these lymphoid organs. To characterize these T cells, the expression of L and 4
integrins, L-selectin, and CD44 was examined. L and
4 integrins were similary expressed on T cells from PLN,
PP, and spleen in plt/plt and +/+ (data not shown). In
PLN, the percentage of L-selectin+ T cells in
plt/plt was smaller than in +/+, but that of T cells highly expressing CD44 (CD44high) in plt/plt was
larger than in +/+ (Fig 5),
suggesting that the frequency of memory T cells in plt/plt PLN
was higher than in +/+, whereas predominant T cells expressed
naive phenotype in +/+ PLN, as reported
previously.20-22 In PP, although majority of T cells were
L-selectin both in plt/plt and in +/+,
L-selectin+ T cells were less frequent in plt/plt
than in +/+. In plt/plt, the percentage of
CD44high T cells was similar to, and that of those slightly
expressing CD44 (CD44low) was larger than that in
+/+ (Fig 5). The smaller percentage of L-selectin+
T cells might suggest that the proportion of memory T cells in plt/plt was larger also in PP than in +/+. In spleen,
L-selectin+ T cells were more frequent in plt/plt
than in +/+, but the obvious difference in the expression of
CD44 was not observed among them. These L-selectin+ T cells
might represent naive T cells. The expression of these molecules on B
cells in PLN, PP, and spleen from plt/plt was not different
from that from +/+ (data not shown).
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| Fig 5.
Expression of L-selectin and CD44 on T-cell surface.
Lymphocytes from LN, PP, and spleen of DDD/1-+/+ (dotted
line) or DDD/1-plt/plt (solid line) were stained with
FITC-conjugated H57-597 (anti-C TCR) and biotinylated MEL-14
(anti-L-selectin) or KM201 (anti-CD44) MoAb followed by
phycoerythrin-conjugated streptavidin (Becton Dickinson). The stained
cells were analyzed in a FACScan (Becton Dickinson). T cells expressing
TCR were gated and their expression of L-selectin or CD44 molecules
(bold line) were analyzed. Cells stained only with FITC-conjugated
H57-597 and phycoerythrin-conjugated streptavidin as a negative control
(fine line) were also shown. Each line indicates the result as follows:
bold dotted line: L-selectin or CD44 expression in +/+;
bold solid line: L-selectin or CD44 expression in plt/plt; fine
dotted line: negative control in +/+; fine solid line:
negative control in plt/plt. The percentage of T cells
expressing L-selectin or highly expressing CD44 in +/+ or
plt/plt was indicated in the each panel. Reproducible results were obtained from three independent similar analyses.
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Accumulation of neutrophils and macrophages into inflammatory site in
plt/plt.
To examine the ability of plt/plt to recruit neutrophils and
macrophages into inflammatory site, thioglycollate was intraperitonealy injected and the number of neutrophils and macrophages in the peritoneum was determined 12, 24, and 48 hours after injection. The
number of neutrophils and macrophages in plt/plt peritoneum increased rapidly as in +/+ (data not shown), suggesting that the ability of plt/plt to recruit these cells was not affected.
plt gene locates on chromosome 4.
In our previous report, plt has been shown to be a autosomal
recessive gene.8 For further analysis, we intercrossed
DDD/1-plt/plt with MSM, an inbred strain derived from a
Japanese wild mouse,12 to produce F1, BC, and
F2 progeny. The phenotype of the progeny was assessed by
PLN cell count and flow cytometric analysis of PLN cells, and
plt/plt phenotype was distingushed from wild-type by paucity of
T cells and by the low percentage of
L-selectin+TCR+ cells
(Fig 6A). The phenotype of individual mouse
in MSM-+/+, DDD/1-plt/plt, F1, and BC was
plotted against PLN cell number and percentage of
L-selectin+TCR+ cells, as shown in Fig 6B. In
plt/plt phenotype, PLN cell number was less than 1 × 107, and the frequency of
L-selectin+TCR+ cells was less than 31%. Mice
with plt/plt phenotype, determined on this criteria, showed the
characteristic distribution of T cells in spleen as shown in Fig 1k, o.
Although the percentage of L-selectin+TCR+
cells in PLN from MSM-+/+ was clearly higher than that from
DDD/1-plt/plt, the number of PLN cells in MSM-+/+ was
as small as DDD/1-plt/plt (Fig 6B), which was probably due to
the small body weight of the former (11.82 ± 0.65 g) as compared
with the latter (22.87 ± 1.48 g). One exceptional mouse in BC had
only 26% L-selectin+TCR+ cells in PLN, but
this mouse was identified as a heterozygote based on the large number
of PLN cells and the quite similar distribution of T cells in spleen to
that in +/+ (data not shown). Forty-eight F1 mice
examined exclusively expressed wild-type phenotype.
Wild-type:plt/plt ratio was 104:86 in BC. In the similar
assessment, wild-type:plt/plt ratio was 72:28 in
F2. Similar ratios were obtained in male and female
progenies. These results confirmed that plt is an autosomal recessive gene, as previously reported.8
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| Fig 6.
Phenotypic analysis of lymphocytes in PLN. Lymphocytes
from PLN were counted and stained with FITC-conjugated H57-597
(anti-C TCR) and biotinylated MEL-14 (anti-L-selectin) MoAb
followed by phycoerythrin-conjugated streptavidin and analyzed in a
FACScan (Becton Dickinson). (A) Total 10,000 cells of PLN from
MSM-+/+ or DDD/1-plt/plt were analyzed and
percentage was indicated at the corner of each quadrant.
plt/plt was characteristically distinguished by small content
of T cells, especially of L-selectin+TCR+
cells (15.0%) compared with those of +/+ (73.0%). (B) PLN
cell number and the percentage of
L-selectin+TCR+ cells in PLN lymphocytes of
individual mouse in MSM-+/+, DDD-plt/plt, F1, and BC progeny were assessed and represented with a
dot. BC was pool of 101 (DDD/1-plt/plt × F1)BC
and 89 (F1 × DDD/1-plt/plt)BC. plt/plt
was distinguished by paucity of PLN cell number (less than 1 × 107) and small content of
L-selectin+TCR+ cells (less than 31%). The
percentage of L-selectin+TCR+ cells of
wild-type (+/+ or plt/+) were more than 32%.
Some mice ( or  ) in BC were decided their phenotype by the T-cell
distribution in the spleen in immunohistochemical analysis as shown in
Fig 1i, k. Wild-type or plt/plt were indicated with black or
white, respectively. One exceptional mouse ( ) in BC showed 26%
L-selectin+TCR+ cells in PLN, but this was
classified as a heterozygote based on large number of PLN cells and the
quite similar distribution of T cells in spleen to that in
+/+. The number of wild-type versus that of plt/plt
is indicated at the upper corner of each group.
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We mapped plt gene on chromosome by analyzing 190 BC progenies,
which was a pool of 101 (DDD/1-plt/plt × F1)BC and 89 (F1 × DDD/1-plt/plt)BC mice. Genotype was determined with SSLP in genomic DNA.9,10 When three markers on each chromosome were analyzed in 22 BC progenies expressing plt/plt phenotype,
markers on only chromosome 4 showed high linkage with the phenotype
(D4Mit4: 100%; D4Mit9: 77.3%; and D4Mit16: 54.5%). To obtain precise
linkage map of plt locus, further markers around D4Mit4
(Dietrich et al10, and Genetic and Physical Map of the
Mouse Genome) were used for the analysis of 190 BC progenies.
plt showed high linkage with D4Mit4, D4Mit286, and CD72, and
was not segregated from D4Mit237 (Table 1).
plt was mapped on 24.7 cM from the centromere when D4Mit149 was
used as a centromere marker (Fig 7). CD72,
which is the ligand for CD5 on T cells23 and expressed on B
cells, linked with plt. CD72, however, locates on a distinct
locus from plt, because a recombinant was obtained in BC (Table
1, Fig 7).
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
Linkage map of mouse chomosome 4 including plt
locus. Distance ± SE (cM) between each locus is expressed at left
side. plt is located on 24.7 cM from cetromere and most closely
linked with D4Mit237. Corresponding human chromosomes with mouse
chromosome 4 are indicated.38
|
|
| |
DISCUSSION |
Previously, we have shown the T-cell-specific paucity in PLN in
plt/plt mutant mice,8,17 and the paucity seems to
result from the inability of PLN stroma to allow T cells to migrate
from blood into PLN.8 In the present study, we investigated
whether the plt mutation affected other peripheral lymphoid
organs as well as PLN. Although the T-cell frequency in plt/plt
PP is comparable with that in +/+ PP in our previous
study,8 the distribution of T cells in plt/plt PP
is different from that in +/+ PP. In plt/plt PP, T
cells were unusually detected in the area occupied by B cells rather
than in the distinct T-cell zone, and the T-cell zone and B-cell zones
were unable to be distinguished (Fig 1). The unusual distribution of T
cells in plt/plt PP might be due to the failure for T cells to
migrate from blood into PP, because the labelled T cells injected
intravenously were almost undetectable in plt/plt PP, whereas
labelled and IV injected B cells were detected in plt/plt pp
as well as in +/+ PP (Figs 2 and 3). Similar
failure was observed in the T-cell migration into PLN in
plt/plt. Thus, the T-cell migration from blood into PP is
severely affected in plt/plt, as well as into PLN, which
suggests that most of T cells in plt/plt PP might migrate from
the surrounding submucosal tissue. The distribution of B cells in
plt/plt PP was similar to that in +/+, except for the
colocalization with T cells in plt/plt PP. Thus, plt
mutation specifically affects the distribution of T cells not only in
PLN, but also in PP.
It has been shown that T cells migrating from blood into PLN through
HEV predominantly express a naive phenotype, whereas those from the
afferent lymphatics express a memory phenotype.20 In PLN
from plt/plt, the proportion of T cells with
L-selectin- and CD44high, which are
phenotypically memory T cells,22 is larger than in that
from +/+, suggesting that most of T cells in plt/plt PLN migrated from the afferent lymphatics, whereas those in +/+ PLN migrated either from blood or afferent lymphatics, as
described.24 Consistently, few labelled T cells
intravenously injected were detected in plt/plt PLN, but many
in +/+ PLN. In plt/plt PP, however, the proportion of T
cells with this phenotype is not larger than that in +/+ PP. If
most of T cells in PP were memory cells, as suggested,25
their phenotype might be different from that in PLN.
The distribution of T cells in plt/plt spleen is quite
different from that in +/+, although the T-cell content is never
smaller than in +/+. In plt/plt spleen, few T cells
were found in the white pulp but a lot of T cells were found in the red
pulp (Fig 1). The number of the labelled T cells that were injected
intravenously and migrated into plt/plt spleen was greater than
that into +/+ spleen (Figs 2 and 3), but most of them were
localized in the red pulp in plt/plt, whereas the major part of
them in +/+ were detected in the periarterial lymphatic sheath
in the white pulp (Fig 4). Characteristically, the labelled T cells in
plt/plt spleen hardly migrated into the white pulp across the
marginal zone (Fig 4). The labelled B cells injected intravenously,
however, localized mainly in the white pulp even in plt/plt
spleen. Thus, plt mutation selectively affects T-cell migration
from blood into the white pulp in spleen, which might result in the
unusual distribution of T cells in plt/plt spleen (Fig 1). The
subpopulations of T cells in plt/plt spleen might be different
from those of +/+ because the majority of T cells in
plt/plt spleen expressed L-selectin, whereas about a half of T
cells in +/+ spleen did not (Fig 5). This finding might suggest
that naive T cells were dominant in plt/plt spleen. The
expression of CD44, however, was quite similar on T cells in spleens
from plt/plt and from +/+.
The defect of T-cell homing and the relative increase in memory-type T
cells has been shown also in L-selectin-deficient
mice.16,26 Although the T-cell-specific paucity in
plt/plt seems to be due to the failure of PLN or spleen stromal
cells to allow T-cell homing across the HEV or the marginal
zone8 (Figs 3 and 4), respectively, low frequency of
L-selectin+ T cells in plt/plt PLN and PP (Fig 5)
raises a possibility that L-selectin+ T cells might be
fewer in plt/plt than in +/+, resulting in a smaller
number of L-selectin+ T cells that migrate into these
lymphoid tissues in plt/plt than in +/+. It might be
also possible that the paucity was due to the deficient expression of
or dysfunction of L-selectin in plt/plt. These possibilities
are unlikely, however, based on the following reasons: (1) The
propotion of L-selectin+ T cells in spleen (Fig 5) and in
peripheral blood (data not shown) is higher in plt/plt than in
+/+, suggesting that the T-cell-specific paucity is not caused
by a smaller number of L-selectin+ T cells; (2) T cells
from plt/plt spleen are able to migrate into +/+ PLN or
PP (Fig 3), as well as those from +/+; (3) B-cell distribution in
plt/plt is quite similar in plt/plt and in +/+, but is impaired in L-selectin-deficient mice26; (4) The
accumulation of neutrophiles and macrophages into the thioglycollate-stimulated peritoneum is not affected in plt/plt (data not shown), whereas that has been reported not to accumulate in
L-selectin-deficient mice16; (5) In addition, the
expression or function of L-selectin ligand is not affected in
plt/plt.8 Thus, our findings suggest a factor(s) distinct from L-selectin, which is selectively required for T-cell homing into T-cell zone in PLN or PP, or spleen white pulp.
As described above, T-cell homing is strongly suggested to be regulated
distinctly from B-cell homing. Consistently, Förster et
al27 have reported a GTP-binding protein
(G-protein)-coupled chemokine receptor, BLR1, which is selectively
expressed on B cells and mediates B-cell homing into the B-cell zone of
PP or into the white pulp in spleen. Although the mechanisms for the selective regulation of the B-cell homing is presently unknown, a
signal(s) delivered through G-proteins might be included, because the
treatment with pertussis toxin has been shown to induce the homing
defect of T and B cells into PLN, PP, and spleen white pulp.28-33 It is well known that the activation of
L 2 integrin/LFA-1 on lymphocytes is
required for binding to ICAM-1 on endothelial cells, which results in
the adhesion of the lymphocytes to and their transmigration through the
endothelial cells.7,34,35 A cytoplasmic protein,
Cytohesin-1, might be involved for the activation of L
2 integrin/LFA-1, because Cytohesin-1 has been shown to
bind to the intracellular domain of 2 integrin and to induce its adhesiveness to ICAM-1.36 Taken together, a
certain chemokine(s) might bind to BLR1 and transduces signals to
activate integrins on B cells through the BLR1-coupled G-protein and
Cytohesin-1.
In this context, it might be also possible that the T-cell homing is
mediated by a distinct chemokine(s) from that for the B-cell homing,
which binds to the specific receptor on T cells and triggers a
signal(s) through a G-protein(s) for the T-cell homing. The involvement
of a signal(s) through G-protein in the homing defect of T cells in
plt/plt might be supported by the findings that pertussis
toxin-treated T cells retain the ability to bind to LN HEV in
vitro,28 but are unable to home into PLN, PP, and spleen
white pulp in vivo.28-33 This is quite similar to our
findings that plt/plt LN HEV keep the ability to bind to T cells in vitro, but the stromal cells are unable to allow them to home
into the lymphoid organs in vivo (Nakano et al8 and this
report). Wild-type gene on plt locus might encode the chemokine for the T-cell homing in soluble or membrane-bound form, or regulate its function. The possibility is now under investigation in our laboratory.
Recently, CD43 on T cells has been shown to mediate their homing into
PLN, PP, and spleen.37 Although it is possible that CD43 or
its ligand(s) is affected in plt/plt, the possibility seems
unlikely because of some differences among these homing defects. For
example, anti-CD43 antibody inhibits the lymphocytes binding to PLN HEV
in in vitro binding assay and the migration into spleen,37
whereas those in plt/plt are not affected (Nakano et
al8 and this report).
We have performed chromosomal mapping of plt gene with SSLP.
Recombination between plt and each microsatellite as a locus marker showed the linkage of plt with the microsatellites on
chromosome 4 (Table 1). A detailed analysis on chromosome 4 mapped
plt on 24.7 cM from the centromere and linked it most closely
with D4Mit237 (Fig 7). No recombination was observed between
plt and D4Mit237 in 190 BC progenies examined. The locus
occupied by D4Mit237 on mouse chromosome 4 corresponds to that on human
chromosome 9p (Lyon et al,38 Genetic and Physical Map of
the Mouse Genome, and National Center for Biotechnology Information).
Around plt locus, there is no gene reported that encodes a
molecule selectively affecting T cells, as far as we searched. Thus,
plt seems to be a mutant of a novel gene whose product
participates selectively in T-cell homing into peripheral lymphoid
organs. Very recently, a novel chemokine with CX3C motif
was reported.39 This chemokine is expressed as a
membrane-bound form on TNF- or IL-1-activated, but not unstimulated,
human umbilical vein endothelial cells, and serves as a chemoattractant
for T cells and monocytes, suggesting that this chemokine is expressed
on the endothelial cells at an inflammatory site. The wild-type gene of
plt locus is unlikely to be the CX3C chemokine,
because the plt product functions in uninflammatory lymphoid
organs in a T-cell-specific manner. In addition, the CX3C
chemokine locates on human chromosome 16,39 whereas
plt gene was mapped on mouse chromosome 4 corresponding to
human chromosome 9.
Only lymphocytes among leukocytes can migrate from blood into lymph,
but the mechanisms for the selective regulation remain to be
elucidated. The investigation of the mechanisms for the function of the
plt gene product will help us to elucidate the selective
migration of lymphocytes and their recirculation.
| |
FOOTNOTES |
Submitted June 26, 1997;
accepted November 25, 1997.
Supported by Grant-in-Aids for Scientific Research (No. 08770344) from
the Ministry of Education, Science, Sports, and Culture, Japan; and by
the Uchida grant from the Japan Foundation of Cardiovascular Research
(1996).
Address correspondence to H. Nakano, Department of
Immunology, Toho University School of Medicine, Omori-nishi 5-21-16, Ota-ku, Tokyo 143, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr T. Shiroishi (National Institute of Genetics, Mishima,
Shizuoka, Japan) for providing MSM; Drs Y. Ishii, K. Yamaguchi, H. Hemmi (Toho University), and T. Yoshimoto (University of Tokyo) for
their encouragement; and K. Nakano for her technical asistance.
| |
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Y. K. Choi, B. A. Fallert, M. A. Murphey-Corb, and T. A. Reinhart
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S.-C. Chen, G. Vassileva, D. Kinsley, S. Holzmann, D. Manfra, M. T. Wiekowski, N. Romani, and S. A. Lira
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G. Henning, L. Ohl, T. Junt, P. Reiterer, V. Brinkmann, H. Nakano, W. Hohenberger, M. Lipp, and R. Forster
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C. Ploix, D. Lo, and M. J. Carson
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K. W. Christopherson II, J. J. Campbell, and R. A. Hromas
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R. Forster, L. Ohl, and G. Henning
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H. Nakano, M. Yanagita, and M. D. Gunn
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W. Weninger, M. A. Crowley, N. Manjunath, and U. H. von Andrian
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J. Lieberman, P. Shankar, N. Manjunath, and J. Andersson
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K. Christopherson II and R. Hromas
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S.-A. Kellermann and L. M. McEvoy
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G. Chen, P. Shankar, C. Lange, H. Valdez, P. R. Skolnik, L. Wu, N. Manjunath, and J. Lieberman
CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection
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C. H. Kim, L. S. Rott, I. Clark-Lewis, D. J. Campbell, L. Wu, and E. C. Butcher
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R. Abe, S. C. Donnelly, T. Peng, R. Bucala, and C. N. Metz
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P. Hjelmström
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S. Mori, H. Nakano, K. Aritomi, C.-R. Wang, M. D. Gunn, and T. Kakiuchi
Mice Lacking Expression of the Chemokines CCL21-Ser and CCL19 (plt Mice) Demonstrate Delayed but Enhanced T Cell Immune Responses
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H. Nakano and M. D. Gunn
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P. Schaerli, K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, and B. Moser
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S. A. Luther, H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster
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M.-C. Dieu-Nosjean, C. Massacrier, B. Homey, B. Vanbervliet, J.-J. Pin, A. Vicari, S. Lebecque, C. Dezutter-Dambuyant, D. Schmitt, A. Zlotnik, et al.
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T. M. Engeman, A. V. Gorbachev, R. P. Gladue, P. S. Heeger, and R. L. Fairchild
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A. M. Norment, L. Y. Bogatzki, B. N. Gantner, and M. J. Bevan
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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
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J. G. Cyster
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M. D. Gunn, S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and H. Nakano
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K. Uchimura, K. Kadomatsu, H. Nishimura, H. Muramatsu, E. Nakamura, N. Kurosawa, O. Habuchi, F. M. El-Fasakhany, Y. Yoshikai, and T. Muramatsu
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Abstract
Introduction
Abstract