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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1653-1661
By
From the Departments of Pathology and Immunology, University of
Toronto, Toronto; and Trauma Research Program, Sunnybrook Health
Science Centre, North York, Ontario, Canada.
Lymphocyte recirculation facilitates the detection and elimination
of pathogens and the dissemination of immunologic memory. It is
generally assumed that all small lymphocytes in the blood are actively
recirculating, yet there is little quantitative data directly comparing
the migration of this population with actively recirculating,
lymph-derived lymphocytes. In this study blood lymphocytes were labeled
with fluorescein isothiocyanate (FITC), and lymph lymphocytes were
labeled with CM-DiI, reinfused intravenously, and monitored in blood
and lymph. After equilibration the concentration of blood lymphocytes
was several times higher in blood than in lymph, whereas lymph
lymphocytes displayed the opposite behavior. This suggested that blood
lymphocytes did not recirculate as efficiently as lymph lymphocytes, so
we examined the following blood lymphocyte subsets in greater detail: B
cells, CD4+, CD8+, and
THE EXPERIMENTS of Gowans1,2
showed conclusively that lymphocytes recirculate from blood to lymph.
This landmark discovery triggered intense interest in the underlying
mechanisms and immunological relevance of this phenomenon. The majority
of lymphocyte recirculation experiments since performed have used rats
or mice, and these systems have provided the framework for our current
understanding of the molecular basis of lymphocyte-endothelial
interaction and lymphocyte homing. On the other hand, research using
larger species such as sheep has permitted the study of the flux of
lymphocytes through individual lymph nodes and discrete anatomic
compartments and has permitted a very detailed analysis of the
physiology of lymphocyte recirculation. The picture that has emerged
using all of these systems is that the migration of lymphocytes is not
entirely random but rather is regulated by the interaction of adhesion molecules on lymphocytes and vascular endothelium. Superimposed on
tissue-specific homing of lymphocyte pools is the phenomenon of
subset-specific homing; venous blood, afferent lymph, and efferent lymph all contain different proportions of lymphocyte subsets, such as
B cells and CD4+, CD8+, and The blood is a compartment that receives lymphocytes from the thoracic
duct (which receives lymphocytes from the lymph nodes and Peyer's
patches), other lymphatics from the head and neck, the spleen, bone
marrow, thymus, and other sources. Lymphocytes are exchanged between
the blood and these tissues many times per day, and the relationship
between blood lymphocytes and each of the different organs seems to be
quite unique. For example, different molecular mechanisms are involved
in the regulation of lymphocyte extravasation and migration into lymph
nodes versus the spleen. The exit routes are unique as well;
lymphocytes do not recirculate (travel from blood to lymph) through the
spleen as they do through lymph nodes, but rather they migrate directly
back to the blood.12 Also, the blood pool of lymphocytes is
in equilibrium with large reservoirs of lymphocytes that marginate in
the microvasculature of nonlymphoid organs, such as the
lung.13,14 It is likely that the cell adhesion mechanisms
in the microvessels of this tissue are unique.15 Because
the behavior of lymphocytes is different in each of these tissues, it
should not be assumed that the blood is a homogeneous source of
recirculating lymphocytes.
Lymphocyte recirculation experiments in the sheep have generally used
lymph-derived cells, because efferent lymph is a virtually pure source
of lymphocytes with a known capacity to recirculate.16,17 Fewer studies have examined the blood to lymph recirculation of blood
lymphocytes,18,19 particularly in a way that would reveal any differences in the recirculatory ability of lymphocyte subsets obtained from blood versus lymph. The most direct way to do this would
be to compare the recirculation of lymph lymphocytes from multiple
lymphoid compartments with that of blood lymphocytes obtained from the
same animal. If the latter population failed to recirculate as
efficiently as the former, this would provide direct evidence for a
unique blood lymphocyte population with distinctive recirculatory
patterns. In this report the two lymphocyte populations were labeled
with different fluorescent dyes, and their recirculation from blood to
lymph was monitored. Also, the concentration of directly labeled
lymphocytes was great enough that the recirculation of different
subsets of blood lymphocytes could also be examined. When the results
are considered in the context of the available literature on the
recirculation of lymph lymphocytes, it seems likely that blood Animals
Protocol 1. Comparison of the Recirculation of Blood
Lymphocytes and Lymph Lymphocytes
Surgery and lymphocyte labeling.
All surgical procedures were performed under sterile conditions.
Animals were anesthetized with pentothal sodium (15 to 25 mg/kg
intravenously; Boehringer Ingelheim, Burlington, Ontario, Canada). They
were intubated with an endotracheal tube, and anesthesia was maintained
with 2% halothane (Fluothane; Ayerst Laboratories, New York, NY) in
O2. A catheter with a three-way stopcock was surgically
positioned in the jugular vein. Also, surgical access was gained to an
efferent subcutaneous lymphatic vessel (two animals) or gut efferent
vessel (three animals), and a chronic fistula was established. Several
thorough descriptions are available of the precise anatomical location
of these lymph nodes and the surgical methods used.17,20
Essentially, 1 to 2 cm of the lymphatic vessel was exposed and ligated,
then a small incision was made in the wall. Polyvinyl chloride tubing
(SV45; Dural Plastics and Engineering, Dural, Australia) with an
outside diameter ranging from 0.6 to 1.2 mm was inserted into the lymph
vessel and sutured firmly in place. The wound was carefully closed, and
lymph was diverted into a sterile 250-mL or 500-mL bottle affixed to a
holder on the skin of the sheep. Heparin (300 to 500 IU; Hepalean;
Organon Teknika, Toronto, Ontario, Canada) and penicillin G potassium (7,000 to 10,000 IU; Marsam, Toronto, Ontario, Canada) were added to
the bottle every time a collection was made. Animals were allowed to
recover until at least the following day before experiments were
performed. All animals were given 0.005 mg/kg intramuscular buprenorphine HCl analgesic (Temgesic; Reckitt and Colman, Hull, UK)
immediately after waking from surgery and as needed thereafter.
Cell labeling and collection of blood and lymph samples.
The methodologies used for labeling and tracking blood lymphocytes with
fluorescein isothiocyanate (FITC) and lymph lymphocytes with CM-DiI
have been published elsewhere.21,22 These protocols were
shown to have minimal impact on the ability of the cells to
recirculate. Also, the viability of lymphocytes after labeling was
assessed by the exclusion of 2% trypan blue dye and found to be 80%
to 96% when labeling lymph lymphocytes with CM-DiI22 and
greater than 95% when labeling blood lymphocytes with
FITC.21 The labeling procedure for lymph lymphocytes was
similar to established protocols with other carbocyanine dyes, but the
blood lymphocyte labeling technique was unique in that all of the
cellular components in a sample of whole blood were labeled with FITC
and reinjected into the animal. The time required for blood labeling
was approximately 3 hours, and the temporary depletion was well
tolerated by the animals. The blood volume of the sheep is 75.1 ± 4.6 mL/kg,23 so the 300-mL sample represented about 11% to
16% of their blood volume. Other investigators have reported that
losses of 25% or more cause no apparent distress to
sheep.24 This blood labeling technique circumvented the use
of lymphocyte isolation procedures, which could potentially alter the
ability of the cells to recirculate. Dead, damaged, or overlabeled
cells are rapidly cleared following intravenous injection and never
reach lymph.21 To briefly describe the procedure, 300 mL
blood was obtained, washed two times with Hanks' Balanced Salt
Solution (HBSS) to remove free protein, and labeled with 600 mL FITC
for 30 minutes at 4°C. The cells were washed twice to remove free
FITC, resuspended in Ringer's lactate to a total volume of 300 mL,
then reinfused intravenously. Upon reinfusion, the FITC+
lymphocytes were distinguished from other labeled cells using flow
cytometry. Typically, 1 × 109 lymph
lymphocytes and 0.2 to 1.2 × 109 blood lymphocytes
were labeled and reinfused.
Control experiment.
These experiments showed clear differences in the recirculation of
blood lymphocytes and lymph lymphocytes, but it was necessary to rule
out the possibility that this was a consequence of the unique labeling
conditions used for blood lymphocytes. To determine whether the
recirculation of lymph lymphocytes would be hindered when subjected to
the blood-labeling protocol, the following experiment was performed. A
sample of blood was depleted of leukocytes by repeated centrifugation
and aspiration of the buffy coat. After the blood was over 90%
depleted of leukocytes as determined using a Coulter cell counter
(Coulter Immunology, Hialeah, FL), 1 × 109
lymph lymphocytes were added to the red blood cells
(RBCs), and this sample was labeled with FITC using the standard blood
lymphocyte labeling protocol. The cells were infused and their
recirculation monitored. If the labeling procedure damaged the cells or
altered their recirculation, it was expected that they would be
eliminated from the blood and their concentrations in lymph would not
approach the values predicted by previous investigations.10
Data analysis.
Lymph was obtained in sequential collections usually ranging from 6 to
12 hours, and a blood sample was taken every time the lymph collection
bottle was replaced. Erythrocytes were lysed using
Tris:NH4Cl solution, then the blood and lymph samples were washed 2 times in HBSS. After fixation in 1% paraformaldehyde for 1 hour the samples were washed in HBSS and stored for subsequent analysis. The concentration of labeled lymphocytes was determined using
a Coulter Epics Elite flow cytometer (Coulter Immunology) by gating on
small lymphocytes and further gating on FITC+ cells. At
least 1 × 105 lymphocytes were counted
per sample, and the number of FITC+ or CM-DiI+
cells detected varied from 100 to 1,000, depending on the nature of the
sample and the duration of the experiment (ie, the concentration of
labeled cells decreased over time). The sensitivity and alignment of
the instrument were monitored daily using Coulter Immunocheck fluorescent beads. For each time point the ratio of FITC+
and CM-DiI+ cells in blood and lymph was calculated. After
the initial equilibration period of approximately 1 day this ratio was
consistent throughout most experiments; therefore, the data from all
samples taken after 40 hours were pooled and expressed as an average
for each animal. Between three and eight paired blood and lymph samples
were obtained per animal over a period of 3 to10 days, depending on the
patency of the venous and lymphatic catheters.
Table 1 provides the duration of each
experiment (days) and the number of samples obtained (N).
Protocol 2. Analysis of the Recirculation of Blood Lymphocyte
Subsets
Animals and surgery.
Eight animals were used for this part of the study. As above, a venous
catheter was established in the jugular vein and one or more
subcutaneous (prescapular, prefemoral, or popliteal) efferent lymphatic
vessels was cannulated. Some experiments were incomplete because
lymphatic cannulae became dislodged or clotted, and in other
experiments it was not possible to phenotype the injected sample of
cells because the fluorescence intensity of the FITC+ cells
was too intense to permit immunophenotyping with a second color
antibody. Therefore, the tables and figures depicting the pooled data
for these experiments indicate in parentheses the number of samples
obtained.
Blood labeling, reinfusion, and sampling.
A 300-mL sample of blood was labeled with FITC and injected
intravenously, as described above. Following this, 60-mL samples of
blood were drawn 4 hours, 3 days, and 11 days later. These time points
were chosen so that the short-term distribution of blood lymphocytes
could be compared with that of more extended time points. The
erythrocytes were lysed using Tris:NH4Cl solution, then the
remaining leukocytes were washed 2 times in HBSS containing 1%
autologous lymph plasma by centrifugation at 600g for 10 minutes at 4°C.
Immunophenotypic analysis.
The cells were immunostained in microtitre wells using standard
procedures described elsewhere.25 They were phenotyped
using monoclonal antibodies (MoAbs) against the following surface
antigens: CD4,5 CD8,26 CD5,27
Analysis of samples.
A method similar to that used by other investigators7,25
was used to compare the migratory efficiency of the different lymphocyte subsets. The percentages of the various lymphocyte subsets
were determined and then used to calculate ratios. For example, in one
experiment the intravenously injected sample contained 18.94%
CD4+ and 30.24% B lymphocytes, so the ratio of CD4/B cells
was 18.94/30.24 = 0.63. The average CD4/B ratio of injected cells in
all experiments was 0.68 ± 0.18. The same ratio for labeled cells
that had actively recirculated to lymph 3 days later was 5.39 ± 1.74. The eightfold enrichment of CD4+ cells was
statistically significant, indicating that this subset was extracted
more efficiently from the blood than B cells.
Statistics
Kinetics of Recirculation and Concentration of Lymphocytes in Blood and Lymph Figure 1 shows the flow cytometric analysis of FITC+ (blood) and CM-DiI+ (lymph) lymphocytes in blood and efferent lymph following their intravenous infusion. The FITC+ cells remained higher in blood than in lymph at all times, whereas the CM-DiI+ lymphocytes disappeared rapidly from blood and reached higher concentrations in lymph. The difference in the distribution of the two lymphocyte populations was most striking in experiments where both populations were tracked in the same animal because it was obvious that each population was most concentrated in its respective compartment of origin (Fig 2). The pooled data for all seven experiments are shown in Table 1. After infusing FITC+ lymphocytes intravenously, the concentration of labeled blood lymphocytes averaged 3.50 ± 0.57 times higher in the blood than in the three lymph compartments examined: subcutaneous pseudoafferent and efferent lymph, and mesenteric efferent lymph. This difference was particularly large between the blood and mesenteric efferent lymph (Table 1). As expected, lymph lymphocytes were more concentrated in their respective lymph compartment than in blood; the blood/lymph ratio of CM-DiI+ lymphocytes was on average 0.51 ± 0.08. The ratio of FITC+ lymphocytes in blood/lymph was significantly different from that of CM-DiI+ lymphocytes (P < .05).
Effect of the Blood Labeling Protocol on Lymphocyte Recirculation In one experiment the leukocytes in a sample of blood were depleted and replaced with lymph lymphocytes before labeling with FITC (Fig 2, bottom panel). As would be expected of lymph-derived cells, the FITC+ lymphocytes achieved a higher concentration in lymph than in blood after about 1 day. The average blood/lymph ratio in this experiment was 0.54 ± 0.02 (as determined from six paired blood and lymph samples taken between 44.5 and 133.5 hours after the injection of labeled cells). This corresponded closely to the average blood/lymph ratio of 0.51 ± 0.08 for lymph lymphocytes labeled with CM-DiI (Table 1). Because lymph lymphocytes subjected to the blood lymphocyte labeling protocol recirculated normally, it seems highly unlikely that this methodology affected the migratory behavior of blood lymphocytes.The Redistribution of FITC+ Cells in the Blood The phenotypic profile of the labeled, injected lymphocytes was somewhat different from that of unlabeled blood lymphocytes (Fig 3). The percentage of CD5+ or CD4+ lymphocytes tended to be higher in the injected population, and B lymphocytes tended to be lower, but the only statistically significant difference was between injected CD4+ cells versus unlabeled CD4+ cells 4 hours postinjection. In some experiments it appears that some B cells were lost during labeling, perhaps because of adhesion to the polypropylene containers used for cell labeling. Because this depletion was not consistently observed, and by trypan blue exclusion the viability of labeled cells was greater than 95%, it was felt that the depletion was not caused by the selective damage of certain lymphocyte subsets. However, the changes in the distribution of each FITC+ subset were examined in two ways to determine whether similar conclusions were reached: (1) the concentration of labeled cells in the blood was compared with the original infused population and (2) in each sample the concentration of labeled and unlabeled cells of a given subset was compared.
The Recirculation of Blood B and T Cells
The Recirculation of Blood T-Cell Subsets
This study extends the observations of previous investigators by
directly comparing the recirculation of blood- and lymph-derived lymphocytes and showing that they do not recirculate to the same extent
through any of the lymph compartments examined. It might be argued that
this difference was caused solely by the differences in the phenotypic
profile of the starting populations. However, in sheep the migratory
kinetics of efferent subcutaneous lymph-derived T and B cells are
similar.30 The migratory kinetics of various lymph-born
T-cell subsets are also comparable; when CD4+,
CD8+, and
Submitted June 12, 1997;
accepted October 21, 1997.
The authors thank Cheryl Smith for her skilled analysis of samples using the flow cytometer. Monoclonal antibodies were generously provided by Dr Alan Young and Dr Wayne Hein.
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Abstract
Introduction
T cells.
Within 4 hours postinjection the percentage of FITC+
CD8+ and CD4+ lymphocytes fell in the blood
and remained significantly lower than the injected sample. In contrast,
the concentration of FITC+ 
Abstract
) than in lymph (
). Middle panel; in contrast, lymph
lymphocytes rapidly disappeared from the blood and reached a higher
concentration in lymph from about 1 day onward. Bottom panel; to rule
out the possibility that the blood labeling protocol hindered
lymphocyte recirculation, in one experiment the leukocytes were
depleted from a blood sample and replaced with lymph lymphocytes, then the blood was labeled with FITC and reinfused as usual. The lymph lymphocytes reached a higher concentration in lymph than in blood; therefore the blood labeling protocol does not appear to affect the
ability of lymphocytes to leave the blood and enter lymph.
) and
unlabeled lymphocytes (
) in the blood following the intravenous
injection of FITC+ blood lymphocytes. The number of
samples per time point is indicated in parentheses. *, indicates a
significant difference (P < .05) between the concentration of
labeled and unlabeled cells at the same time point; 
,
indicates a significant difference between the concentration of
FITC+ subsets in the original injected dose of labeled
cells (the first 
A disgraceful gap in medical knowledge.
Immunol Today
17:288,
1996