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In vivo homing of thymus-enriched bone marrow cells.

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THE ANATOMICAL RECORD 221:714-719 (1988)
In Vivo Homing of Thymus-Enriched
Bone Marrow Cells
KURT G. KLUSSMANN AND JACK L. HAAR
Department of Anatomy, Division of Immunology, Medical College of Virginia, Virginia
Commnwealth University, Richmond, Virginia 23298
ABSTRACT
A population of adult CBNJ mouse bone marrow (BM)cells enriched
by in vitro migration to supernatant prepared from neonatal thymus was labeled
with a DNA-binding fluorochrome, Hoechst dye No. 33342 (H33342). Labeled cells
were injected into irradiated recipients in order to compare the in vivo localization
of the migration-enrichedBM (MEBM)cells to the localization of injected nonenriched
BM (NEBM) cell controls. A characteristic difference in the distribution of localized
cells was observed in the spleen but not in other lymphoid organs. At 2 hr after
injection the MEBM cells were located in the marginal zones surrounding the periarterial lymphoid sheaths (PALS)of the splenic white pulp. At 6 hr after hjection
the MEBM cells were seen distributed between marginal zones and the PALS and
by 16 hr they had localized almost exclusively in the white pulp. In contrast, the
NEBM cells were located in the marginal zones or red pulp for the duration of the
experiment. These observations show that the MEBM cells home selectively to T-cell
areas of the spleen. Direct immunofluorescentmonoclonal antibody staining of H33342labeled cells obtained from the recipient spleens at 16hr demonstrated that the MEBM
cells were negative for Thy-1 antigen, indicating that acquisition of Thy-1 was not
prerequisite to the observed homing. The results are compared to known localization
patterns of mature lymphocytes.
The lymphoid system of mammals is an orderly arrangement of cells and tissues distributed throughout
the body. The lymphoid organs, interconnected with
cells circulating through blood, lymph, and connective
tissue, are composed mainly of bone marrow (BM), thymus, spleen, lymph nodes, and subepithelial tissues of
the gastrointestinal and respiratory tracts. Considerable information exists concerning the origin and distribution of cells in lymphoid organs, including the areas
occupied by lymphocyte subsets (reviewed in Osmond,
1985). For example, mature T and B cells can be located
in splenic periarterial lymphoid sheaths (PALS) and
marginal zones, respectively, by a variety of methods
(Jonjic et al., 1987; Rolstad et al., 1986; Tabibzadeh and
Gerber, 1986; Brenan et al., 1985; Timens and Poppema, 1985; Murray et al., 1984; Rouse et al., 1984;
Witmer and Steinman, 1984; Kumararante and
MacLennon, 1981; Veerman and van Ewijk, 1975).
Questions that naturally arise in the study of lymphocyte localization address specific homing mechanisms and the developmental stages a t which a homing
capacity is acquired. In the case of T cells it is known
that prethymic cells, derived during embryogenesis from
fetal yolk sac and liver (Hemmingson and Alm, 1973;
Moore and Metcalf, 1970) and then BM in the developing mammal (Rosenthal et al., 1983; Ford et al., 1966;
Moore and Owen, 1967b),enter the thymus, where they
undergo maturation into potentially immunocompetent
cells (Moore and Owen, 1967a), which enter the circulation and localize, at least transiently, throughout the
0 1988 ALAN R. LISS, INC.
lymphoid system (Le Gross et al., 1983; Rocha et al.,
1983).
It is uncertain whether cells of the T-cell lineage must
first enter the thymus before acquiring the ability to
home to lymphoid tissues, although experiments that
might favor such a conclusion are reported (Rouse et
al., 1984; Carrol and de Sousa, 1983; Le Gross et al.,
1983; Van Ewijk et al., 1982). Ideally, prethymic stem
cells would be isolated, labeled, and injected into recipients where their localization could be observed. However, the complete isolation of prethymic BM cells, or
prothymocytes, defined by their ability to repopulate
the T-cell compartments of irradiated recipients, has
not yet been accomplished (reviewed in Silverstone and
Cayre, 1985).
In an effort to determine what signals prethymic cells
to enter the thymus, several studies utilizing in vitro
migration assays in mice (Haar and Loor, 1981; Haar
et al., 1987; Taubenberger and Haar, 1987a)b) and in
birds (Champion et al., 1986) have demonstrated the
ability of factors derived from the thymus to act as
chemoattractive agents to subpopulations of BM cells.
Received September 21, 1987; accepted December 9, 1987.
Address reprint requests to Dr. Jack L. Haar, Department of Anatomy, P.O. Box 709, MCV Station, Richmond, VA 23298.
HOMING OF THYMUS ENRICHED BONE MARROW CELLS
715
Such cells appear to be the progenitors of mature T
cells; they home to the thymus in vivo and they lack
surface antigens characteristic of mature T cells (but
have been shown to develop mature T-cell markers after
arriving in the thymus in the avian system). From those
studies we conclude that thymic epithelium produces
chemoattractive peptides that may specifically recruit
BM precursors destined to develop into mature T cells,
which in turn localize according to their role in lymphoid tissues. Moreover, analogous or homologous mechanisms are working in birds and mice.
The present study demonstrates utilization of the in
vitro migration assay of Haar and Loor (1981) to collect
migration-enriched BM (MEBM)cells. The in vivo homing of injected MEBM cells labeled with H33342 (according to Brenan et al., 1985) in lymphoid organs of
irradiated recipients is compared with the homing of
nonenriched BM (NEBM) cells. A characteristic difference in homing by MEBM cells is seen in the spleen:
MEBM cells home to T-dependent areas. The results
are discussed in terms of the homing and distribution
of mature lymphocytes.
lowing modifications: Chemotactic chambers (Nuclepore 440900) with upper and lower well volumes of 0.2
ml separated by a 13-mm diameter, 5-pm pore size filter
(Nucleopore 110413)were used. The bottom (blind)wells
were filled with 0.2 ml TS (experimental) or 0.2 ml
IMDM alone (control). To the upper wells, 0.2 ml of the
BM suspension (containing 1 x lo6 cells) was added.
Chambers were incubated for 90 min at 37°C in a humidified atmosphere of 5% CO, in air. After incubation
the cells that migrated to the bottom wells were collected, washed in PBSBSA, and counted in a hemacytometer, and their viability was ascertained by trypan
blue exclusion. A migration index for each group of
chambers was calculated as the percentage of the cells
applied to the top well that were collected in the bottom
well. The pooled MEBM cells from wells containing TS
were pelleted in preparation for labeling. In order to
determine what direct influenceTS might have on NEBM
cells, which were not subjected to the migration assay,
some NEBM cells were incubated in TS (1 x lo6 cells
per 1 ml) for 90 min at 37°C prior to labeling.
MATERIALS AND METHODS
Animals
Two populations of MB cells were labeled with H33342
by the method outlined by Brenan and Parish (1984):
1) MEBM cells and 2) NEBM cells-BM cells that were
not subjected to the enrichment migration. Prior to labeling, all cells had been washed twice in PBSBSA and
pelleted. In all experiments (1-4) x 106 pelleted MEBM
cells and an equal number of NEBM cells were resuspended in a 6-pglml solution of H33342 in phosphatebuffered saline (PBS) to a concentration of 1.5 x lo6
cells per 1 ml. The suspensions were incubated for 15
min at 37°C. Labeling was stopped by adding cold PBS.
The cells were washed twice in PBS and resuspended
at a concentration of 1 x 108 cells per 1ml for injection.
Age- and sex-matched syngeneic 6 to 8-week-old adult
CBA/J mice of both sexes were used as BM cell donors
and recipients. Recipients were irradiated (900 rads)
with a Picker teletherapy radiation unit equipped with
a cobalt 60 gamma ray source (Neutron Products, Inc.)
24 hr prior to injection. Neonatal (4- to 6-day-old)CBA/
J mice were used to prepare thymus supernatant (TS).
Thymus Supernatant Preparation
The procedures followed to make TS as an attractant
for BM cells were those previously reported by Haar
and Loor (1981),with the following modifications: The
thymus was removed from decapitated neonatal mice
and cut into fragments (8-10 equal pieces per thymic
lobe) under a dissecting microscope. Fragments from
3-4 neonates were placed in 35-mm petri dishes containing 3 ml Iscove's modified Dulbecco's Medium
(IMDM) (Gibco 430-2200) and incubated at 37" for 48
hr. After incubation, the culture medium was centrifuged to remove cells and debris, sterilized by filtration
through a 0.22-pm disposable filter assembly (Gelman,
41921, and stored a t - 70°C until used.
Bone Marrow Cell Preparation
Femora and tibiae were dissected from adult mice
sacrificed by cervical dislocation. Cells were obtained
by flushing the marrow cavity with 5 ml phosphatebuffered saline with 0.5% added bovine serum albumin
(PBSBSA). Following sedimentation for 5 rnin at lg,
erythrocytes were lysed with 0.05 M Tris-NH,C1 buffer,
washed in PBSBSA, and counted in a hemacytometer;
viability was determined by trypan blue exclusion. Cells
were resuspended in IMDM at a concentration of 5 x
106 viable cells per 1 ml in preparation for migration
to TS.
Migration of Bone Marrow Cells to Thymus Supernatant
BM cells were collected after migration to TS as previously described (Haar and Loor, 1981) with the fol-
Labeling With H33342
Injection of Cells and Tissue Preparation
Into the tail vein of each animal (1-4) x lo6H33342labeled MEBM or NEBM cells (the same number for
each pair of animals) were injected in a volume of 0.1
-0.4 ml PBS into each of three to six mice for each time
period specified. At 2, 6, and 16 hr after injection, recipient spleen, pelvic lymph nodes, distal ileum, liver,
lung, and thymus were dissected free. The spleen was
weighed immediately upon removal and half (weighed
separately) was placed in 2 ml PBSBSA to prepare a
cell suspension. The remaining spleen and other tissues
were placed into specimen molds (Miles 4565) and COVered with embedding medium (Miles 4583) for 30 rnin
in the dark a t 4°C prior to freezing. The tissues were
snap-frozen for 2 min in isopentane cooled with liquid
nitrogen and transferred to a cryostat microtome (Damon/IEC 3398), where they equilobrated to - 18°C for
1 hr in the dark. Tissue sections were cut at 16-pm
thickness, mounted onto acid-cleaned glass slides, and
stored in the dark a t 4°C. Tissues obtained 16 hr after
injection of NEBM cells, incubated with TS prior to
labeling, were prepared from two animals. Tissues from
animals injected with unlabeled MEBM and NEBM cells
were also prepared to serve as negative controls. In
order t o verify the splenic architecture seen in unstained tissue sections, several adjacent sections were
prepared for light microscopy with 0.1% methylene blue/
7 16
K.G. KLUSSMANN AND J.L. HAAR
toluidine bluelsodium borate after fixing with a 4% formaldehydelglutaraldehyde for 2 min.
labeled cells were also observed t o verify the absence
of autofluorescence by injected or resident cells.
Counting and Antibody Labeling of Cells Localized in the
Spleen
RESULTS
The percentage injected cells localized in the spleen
was determined from cell suspensions of weighed spleen.
Spleen halves were pressed gently through a fine-wire
mesh washed with 5 ml PBS, leaving the capsule behind. Suspensions of the total cells obtained were prepared and counted exactly as were the BM cells. The
number of H33342-labeled cells in each suspension was
determined with a hemacytometer under epifluorescent
illumination. The total number of cells that had homed
to the spleen was calculated by multiplying the number
of H33342-labeled cells counted in suspension by the
ratio: weight of whole spleedweight of splenic half used
for suspension; thus, the calculations are based on the
assumption that injected cells are distributed evenly
throughout both halves of the spleen.
The total cells collected from the spleen 16 hr after
injection were labeled with rhodamine-conjugatedmonoclonal anti-mouse Thy-1.2 antibody (a-Thy-1; Miles 63351). The collected cells were resuspended in Dulbecco's
modified Eagle's medium with 5%added fetal calf serum
(DMEM/FCS)to a concentration of 1 x lo6 cells per 1
ml. Equal volumes of cell suspensions and a 1:lOO dilution of a-Thy-1 in DMEMIFCS were incubated for 15
min at 4°C in the dark. Cells were washed twice with
PBS and were counted in a hemacytometer. A total of
400 of each of H33342-positive MEBM and NEBM cells
were examined for Thy-1. Positive controls were H33342labeled BM donor thymocytes treated identically to the
splenic cells. Unlabeled thymocytes were observed for
autofluorescence.
Fluorescent Microscopy
A Nikon microscope with a mercury vapor lamp and
exciter and barrier filters for epi-illumination of H33342
(365-nm excitation and >420-nm barrier filters) and
rhodamine (550-nm excitation and >580-nm barrier filters) was used throughout. Tissue sections were observed and photographed within 1hr of sectioning, and
cell suspensions were counted immediately after preparation. Tissue from control animals injected with un-
Epifluorescent microscopic examination of tissue sections revealed injected H33342-labeled MEBM and
NEBM cells in the spleen, lymph nodes, liver, lung,
Peyer's patches, and thymus at 2, 6, and 16 hr after
injection. The H33342-labeled cells were clearly seen
in each section as brightly fluorescent individual cells,
which were best visualized in spleen tissue sections
(Fig. 1 a-f) where characteristic differences between
MEBM and NEBM cell localization was observed.
In the spleen, red and white pulp areas were easily
distinguished as light and dark areas, respectively, in
unstained tissues under epifluorescent illumination
(Fig. 1). At 2 hr after injection both MEBM and NEBM
cells were located in the marginal zone and in the red
pulp (Fig. l a , b). At 6 hr the MEBM cells were distributed in the marginal zone and the white pulp (Fig. lc),
whereas the NEBM cells remained predominately in
the marginal zone or red pulp (Fig. Id). By 16 hr after
injection, MEBM cells were located within the white
pulp (Fig. le), whereas NEBM cells were seen primarily
in the marginal zone (Fig. 10. NEBM cells incubated
with TS prior to injection were distributed identically
to the NEBM cells at 16 hr. In order to confirm the
localization described, adjacent splenic tissue sections
stained for light microscopy (not shown) were examined, verifying that MEBM cells were located in the
white pulp, frequently surrounding small central arterioles. Germinal centers contained few of either injected cell types.
The distribution of the injected cells in other tissues
(not shown) was generally sparse at all times examined,
and no obvious differences in distribution patterns was
noted. The greatest number of injected cells of either
type per tissue section were observed, in decreasing
order, in spleen, lung, liver, lymph nodes, Peyer's patches,
and thymus. The total number of injected MEBM and
NEBM cells was calculated for the spleen.
As shown in Table 1,the greatest average number of
injected cells was counted in the spleen a t 16 hr, although there was no statistical difference (based on a
Student t test, P < 0.05)between the number of MEBM
TABLE 1. Percentage of injected cells recovered from recipient spleen and
migration indices' of MEBM cells injected'
Time
after
injection
2hr
6hr
16 hr
Cells
recovered
Cell
type
injected
6%* S.D.)
MEBM
NEBM
MEBM
NEBM
MEBM
NEBM
2.6 f 0.4
1.7 0.1
7.0 2 1.0
5.5 2 0.2
9.0 2 2.5
8.4
2.0
*
*
Migration index (5% f S.D.)
of BM cells migrated to:
TS
IMDM
17.1 2 5.3"
3.7
2
1.6
13.7 2 5.8"
3.2
&
0.1
15.2
4.8 f 2.0
2 4.9"
'Migration index defined as percentage of cells applied to top well that are collected in bottom well of
migration chamber.
'Abbreviations: MEBM, migration-enriched bone marrow; BM, bone marrow; TS, thymus supernatant;
IMDM, Iscove's modified Dulbecco's medium (control); NEBM, nonenriched bone marrow.
*Statistically significant difference (P< 0.05) between in vitro migration to TS vs. IMDM (Student t
test).
HOMING OF THYMUS ENRICHED BONE MARROW CELLS
Fig. 1, Sections of spleen viewed under epi-illumination demonstrating
localization differences between MEBM cells (a,c,e)and NEBM cell contmls
at 2 hr (a,b), 6 hr (c,d), and 16 hr (e,D after injection into
jrradiated recipient mice. Individual cells are brightly fluorescent. Note
that MEBM cells are located predominately in the marginal zones at 2
hr,that MEBM cells have distributed into the RP and particularly the
717
marginal zones and white pulp at 6 hr; and that MEBM cells home
almost exclusively within the white pulp at 16 hr. In contrast, NEBM
cells remain predominately in the marginal zones or red pulp for the
duration of the experiment. MZ, marginal zones; RP, red pulp; WP, white
pulp. a-d, X 180 e,f, X 118.
K.G. KLUSSMANN AND J.L. HAAR
718
and NEBM cells at any one time studied. Furthermore,
there was no statistically significant difference in the
number of either MEBM or NEBM cells at 6 or 16 hr
but there were significantly more cells at those two
times than at 2 hr. Based on these data (Table l),an
average of 1.7-2.6% of injected NEBM or MEBM cells
appeared in the spleen by 2 hr after intravenous injection, and the number of cells of either type increased
to an average of 5.5-9.0% from 6 to 16 hr. Thus, by 2
hr there was neither a difference in the number of localized MEBM or NEBM cells nor a difference in their
apparent distribution within the spleen (Fig. la,b). In
contrast, there was was a distinct differencein the splenic
localization between MEBM and NEBM cells by 6 hr
(Fig. lc,d) and 16 hr (Fig. le,Q after injection although
there was still no significant difference in the number
of cells that had homed.
Table 1also shows the migration indices of the MEBM
cells injected for each time period evaluated. The migration index is a measure of the selectivity with which
BM cells migrate in vitro, as previously reported (Haar
and Loor, 1981). Overall, the number of BM cells migrated to TS was significantly more than those that
migrated to media alone. The net difference between
the percentage of cells that migrated t o TS and those
that migrated to media alone is on the order of 10%
and is assumed to reflect the actual percentage of BM
cells that respond to TS. Their morphology has previously been described (Haar and Loor, 1981).
Antibody labeling of splenic cell suspensions with aThy-1 a t 16 hr after injection demonstrated that all of
the MEBM cells were negative for Thy-1, compared to
4.0 & 1.0% (mean percentage ? S.D.) NEBM cells that
were positive. Control H33342-labeled Thy-1.2 mouse
thymocytes were clearly double-labeled with a-Thy-1.
The morphology of individual MEBM and NEBM cells
was observed and showed the majority of cells to be
mononuclear lymphoid cells.
DISCUSSION
The localization of H33342-labeledMEBM and NEBM
cells in the spleen a t 2 hr after injection (Fig. la,b)
indicates that both cell populations enter the spleen in
the marginal zones, as suggested for mature B and T
cells seen at a similar time after injection of H33342labeled lymphocytes (Brenan et al., 1985). The difference in distribution betwen the MEBM (Fig. lc) and
NEBM (Fig. Id) cells at 6 hr after injection can be explained by a tendency of the MEBM cells to home towards T-cell-dependent regions of the spleen, in the
splenic white pulp, whereas the NEBM cells tend to
remain in the marginal zone or move into the red pulp,
in a direction outward from the PALS. The selective
homing behavior of MEBM cells is further evidenced at
16 hr aRer injection when the majority of cells are within
the PALS (Fig. le). This same tendency to home selectively to and localize in the white pulp has been demonstrated for mature T cells at similar times by Brenan
et al. (1985),although the percentage of injected mature
T cells collected from spleen (23.2%) in their study is
greater, and is maximal at 2 hr after injection, compared with an average of 9.0% MEBM cells collected
here at 16 hr (Table 1). In contrast, the NEBM cells
remain in the marginal zone or red pulp, although a
few can be seen in the white pulp a t 16 hr. Since the
MEBM cells are a subpopulation of NEBM cells and
because NEBM cells are made up, in part, of mature
(Thy-l-positive) lymphocytes, it might be expected that
some NEBM cells appeared in the white pulp.
A question that arises is whether the TS migration
assay simply selects for a population of MEBM with an
intrinsic homing capability or whether it also modifies
the homing properties of the MEBM cells during their
90 min incubation with TS. It appears that TS alone is
not sufficient to confer the homing ability observed for
MEBM cells; NEBM cells incubated with TS prior to
injection were distributed in the same manner as NEBM
cells unexposed to TS. However, the potential direct
influence of TS on the homing of MEBM cells remains
uncertain. It is still possible that TS not only selects
MEBM cells but also modifies their homing properties.
’ The homing ability of MEBM cells does not appear
to correlate with the expression of Thy-1 antigen. The
MEBM cells do not express it before (Haar and Loor,
1981) or up to 16 hr after injection. It may be that
MEBM cells possess the marker in quantities insufficient to be demonstrated here. Thy-1 has been detected
on up to 25-30% of murine BM cells (Berman and Basch,
1985) including prothymocytes. Basch and Berman
(1982) showed that pretreatment of injected BM cells
with monoclonal a-Thy-1 reduced the thymic repopulation in irradiated recipients, compared with no effect
on repopulation with conventional a-Thy-1 antiserum
(see also Boersma et al., 1981).
The in vivo mechanisms by which MEBM cells home
to T-cell areas in the spleen are presumed to be analogous to the incompletely understood mechanisms governing mature T cells. In the case of mature T cells,
migration to lymph node and Peyer’s patch (reviewed
in Rouse et al., 1984) occurs through high endothelial
venules (HEV).But the spleen does not possess classical
HEV (Kraal et al., 1983). Futhermore, the ability to
to migrate through specialized vessels does not in itself
account for T-cell and B-cell localization differences once
the cells have entered lymphoid tissues. A more likely
explanation (Rouse et al., 1984; Witmer and Steinman,
1984; Veerman and van Ewijk, 1975) involves direct
cell contact between lymphocytes and interdigitating or
follicular dendritic cells, respectively, in T-cell and Bcell areas once the cells enter lymphoid tissues.
Another unresolved issue is the developmental age
at which lymphocyte progenitors and subsets possess
the ability to home. It has been shown (reviewed in
Rouse et al., 1984) that immature BM precursors do
not bind nearly as well to HEV as mature B and T cells,
indicating that this ability is acquired through cell maturation. Furthermore, the homing abilities of mature
T cells differ depending on the organ source and type
of antigenic determinants expressed (Jonjic et al., 1987;
Kraal et al., 1983; Rocha et al., 1983; Carroll and de
Sousa, 1983; Kumararatne and MacLennon, 1981). In
this study we demonstrate that MEBM cells, presumed
to represent immature lymphoid cells, possess the ability to home to T-cell-dependent splenic areas.
The observed migration of MEBM cells is an important finding; it shows that there is a Thy-l-negative
BM cell population capable of selective homing to T-cell
areas, at least in the spleen. And though there is evidence that functional T cells must first mature in the
thymic microenvironment in order to repopulate lym-
HOMING OF THYMUS ENRICHED BONE MARROW CELLS
phocyte-depleted tissues (Le Gross et al.,1983; Boersma
et al., 198l),our data suggest that specific localization
to T-cell areas may not require physical interaction between thymic stromal and BM cells. Although we cannot rule out the possibility that MEBM cells have passed
through the donor thymus prior to isolation from the
BM, the absence of surface Thy-1 on MEBM cells, even
after localizing in the spleen at 16 h r argues against
such a conclusion.
Our results add credence to the suggestion that MEBM
cells represent prethymic cells resident in the BM. The
migration indices of MEBM cells (Table 1)are in good
agreement with previous work (Haar and Loor, 1981;
Taubenberger and Haar, 1987a) that further characterizes the in vitro migration of MEBM cells. Since the
original characterization of the in vitro migration assay,
considerable work has demonstrated a selective migration of BM cells to thymic epithelial cell culture medium
(Taubenberger and Haar, 1987a) which contains peptides (Taubenberger and Haar, 1987b) responsible for
the chemoattraction. Other work (Haar et al., 1987)
employing flow cytometry analysis has shown significantly more MEBM cells homing to the thymus in vivo
although in the present study we were unable to observe a morphological difference in the distribution of
MEBM and NEBM cells in thymic sections (not shown).
The results presented here are the first description
of H33342-labeled BM cells localized in irradiated recipients. The methods used were particularly useful for
describing the profound morphological differences in in
vivo homing observed between MEBM and NEBM cell
populations, differences that were not reflected in the
absolute numbers of homing cells. The work required
to more fully characterize MEBM cells includes the
purification of peptideb) responsible for the in vitro
migrations and the development of monoclonal antibodies against the cells selected.
ACKNOWLEDGMENTS
The authors wish to kindly thank Dr. Robert F. Spencer for his expertise in preparing frozen tissue sections
and Dr. Jeffery K. Taubenberger for helpful discussion.
This work was supported by a grant from the National
Institutes of Health, AG 04384 (J.L.H.).
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