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Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908;
Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, Westf. Wilhelms-Universität Münster, Münster, Germany; and
Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, and NK1.1 but did not
express the B cell and monocyte markers CD45RA, CD19, and CD11b. In
vitro stimulation assays revealed no difference in lymphocyte
proliferation and IL-2 secretion between EGFP+ and
EGFP- mice. Intravital microscopy of untreated or
TNF-
-treated cremaster muscle venules showed EGFP+ cells
in vivo, but these cells did not roll or adhere to the vessel wall. In
cremaster muscle venules treated with both TNF- and IFN-
,
EGFPhigh cells rolled, adhered, and transmigrated at a
rolling velocity slightly higher (11 µm/s) than that of neutrophils
(10 µm/s). Blocking 4 integrin with a mAb increased
rolling velocity to 24 µm/s. These findings show that
CD8+ T cells roll in TNF-/IFN--pretreated vessels in
vivo via an 4 integrin-dependent
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular and molecular mechanisms governing lymphocyte migration have been investigated in vitro for lymphocyte rolling and adhesion to plate-bound adhesion molecules or to tissue sections (5, 6). These assays have been extremely useful in defining adhesion molecules and their receptors. In vivo, cellular trafficking has been addressed using radiolabeled lymphocytes derived from the skin and the gut (7, 8).
Although the molecular mechanisms underlying the trafficking of naive lymphocytes to peripheral lymph nodes and Peyer’s patches has been studied in great detail (4, 9, 10), little is known about how CD8+ T cells get access to inflamed tissues. Based on evidence from homing of CD8+ T cell clones to the lung (11), islets of Langerhans (12), internal elastic lamina and lamina propria of the gut (13), and other inflamed tissues, it is clear that lymphocyte activation is required for recruitment of effector T cells. The sequence of events required is thought to encompass rolling, activation, arrest, and transmigration, akin to the paradigm known for neutrophil recruitment (10). However, it has not been possible to directly demonstrate the adhesion cascade for CD8+ effector T cells in vivo, nor is the adhesion molecule usage in different steps known.
In contrast to neutrophils, most lymphocytes express significant
amounts of 4 integrins, including
41 (very late Ag-4,
VLA-4) and 47
(14). VLA-4 has been shown to mediate lymphocyte rolling
(15, 16) and can be rapidly activated, which leads to
enhanced adhesiveness (17). Beyond its role in lymphocyte
trafficking, VLA-4 is important in the recruitment of monocytes
(18), eosinophils (19), and hemopoietic
progenitor cells (20). The avidity regulation of VLA-4 can
be triggered by tethered chemokines binding to their receptors
(17), but the molecular signaling mechanisms underlying
increased 4 integrin avidity are not
known.
Direct observation of cellular movement in the microvasculature in real time has become possible using intravital microscopy (21). This technique has permitted analysis of leukocyte-EC interactions that occur before a leukocyte can extravasate from the circulation into the tissue (22, 23). One limitation of this technique is its inability to identify individual cell types under observation, with the exception of one report identifying rolling neutrophils (24). This limits assessment of cellular interaction of a specific cell type with the endothelium. Recently, a CD4-green fluorescence protein (GFP)-transgenic mouse has been described to study CD8+ T cell differentiation in vivo (25). However, activated T cells lose GFP expression in the CD4-GFP mouse, making it less suitable for in vivo studies of effector T cell trafficking. Here, we describe the development of a transgenic mouse expressing enhanced GFP (EGFP) in T cells under a CD2 promoter with GFP expression even in activated T cells. We demonstrate that this mouse is a useful research tool for selective analysis of T cell rolling and adhesion during inflammation in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An EcoRI/NotI (blunted) fragment of
pEGFP-N1 (Clontech, Palo Alto, CA) was cloned into the
EcoRI/SmaI sites between the human CD2 promoter
and the CD2 locus control region of the pBS CD2 construct
(26). The construct (see Fig. 1
) was tested for GFP
expression in transient transfection of cultured Jurkat cells. The
construct was then prepared for pronuclear injection into fertilized
B6CBA/F1 eggs by removing the plasmid backbone by a
KpnI/NotI digestion and subsequent agarose gel
purification. Transgenic mice were generated using standard
microinjection techniques at the University of Virginia Transgenic
Mouse Core Facility (Charlottesville, VA). Tail DNA was analyzed for
the transgene by PCR, and tail bleeds from transgene positive mice were
analyzed for GFP expression by flow cytometry. Founder animals were
backcrossed into C57BL/6J wild-type mice for six generations and
maintained at the University of Virginia Center for Comparative
Medicine. Mice were used between 7 and 12 wk of age.
EGFP- littermates served as controls. All
experiments were conducted according to protocols approved by the
University of Virginia Institutional Animal Care and Use Committee.
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To characterize surface Ag expression of
EGFP+ cells, peripheral blood leukocytes, lymph
node cells, and splenocytes from EGFP+ mice were
labeled with PE-conjugated anti-mouse Abs (all obtained from BD
Phar Mingen, San Diego, CA): anti-CD2 (clone RM2-5), anti-CD4
(clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD8 (clone
53-5.8), anti-CD3 (clone 17A2), anti-TCR- (clone H57-597),
anti-NK1.1 (clone PK136), anti-CD45RA/B220 (clone RA3-6B2),
anti-CD19 (clone 1D3), anti-CD11b (clone M1/70), anti-Ly-6G
(clone RB6-8C5). After RBC lysis with a 1.5 M
NH4Cl solution, all samples were run on a FACScan
flow cytometer (BD Biosciences, San Jose, CA). Data analysis was
performed using CellQuest software (BD Biosciences). Activated
splenocytes were labeled with PE-conjugated anti-mouse Abs to
activation markers CD25 (clone M-A251), CD69 (clone FN50), and CD44
(clone C26) and analyzed as described above.
T cell proliferation and IL-2 assays
Splenocytes were plated at 4 x 105
cells per well in 96-well flat-bottom plates after RBC lysis and were
stimulated with graded doses of Con A (Sigma, St. Louis, MO),
anti-CD3
Ab clone 145-2C11 (BD PharMingen), or with 10 ng/ml PMA
(Sigma) plus 500 ng/ml ionomycin (Sigma) for 48 h. Controls were
run in parallel without any stimulation. Plates were pulsed with 1
µCi of 3H per well for the last 12 h of
the assay, and thymidine incorporation was analyzed by liquid
scintillation spectroscopy (LKB Pharmacia, Uppsala, Sweden). Data are
expressed as mean values of triplicate measurements ± SD. For the
IL-2 assay, culture supernatants were harvested 24 h
poststimulation, and IL-2 levels in the supernatant were estimated
using an IL-2 ELISA kit (BD PharMingen) as per manufacturer’s
instructions.
Intravital microscopy
Before surgery, mice were injected with an intrascrotal
injection of 250 ng murine rTNF- (Genzyme, Cambridge, MA) for 3–6
h, 2.5 µg murine rIFN- (Sigma) for 6 h, and 10 U hirudin
(Sigma) in 220 µl isotonic saline. Mice were anesthetized with i.p.
injections of ketamine (125 µg/g body weight; Ketalar; Parke-Davis,
Morris Plains, NJ), xylazine (12.5 µg/g body weight; Phoenix
Scientific, St. Joseph, MO), and atropine sulfate (0.025 µg/g body
weight; Elkins-Sinn, Cherry Hill, NJ), placed on a heating pad to
maintain body temperature, and prepared for intravital microscopy
(27). The cremaster muscle was prepared as described and
superfused with thermocontrolled (35°C)
HCO3-buffered saline (22).
Microscopy of the cremaster muscle was performed using an intravital
microscope (Axioskop; Carl Zeiss, Thornwood, NY) with a x40 saline
immersion objective (SW 40/0.75). Vessels to be analyzed were chosen on
the basis of video image clarity and resolution. Using stroboscopic (30
flashes/s) epifluorescence, intravital microscopy allowed for a clear
differentiation between EGFPhigh and
EGFPlow cells (see Fig. 2). Vessels were recorded
on videotape (S-VHS recorder; Panasonic, Osaka, Japan) with a SIT
camera (SIT 66; DAGE-MTI, Michigan City, IN). Mean blood flow velocity,
based on centerline RBC velocity (Circusoft; www.circusoft.com), and
wall shear rates were calculated as described elsewhere
(28). The flux fraction of EGFPhigh
cells was calculated by dividing the counted number of
EGFPhigh cells passing a line perpendicular to
the vessel by the total leukocyte (white blood cell) flux in the same
vessel.
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Rolling velocities of single leukocytes along a venule segment were determined for several leukocytes in postcapillary venules (diameter 30–90 µm). All leukocyte displacements and velocities were measured on a Macintosh computer (Apple Computer, Cupertino, CA) using the public domain NIH Image program (developed at the National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/). The rolling velocity was calculated as displacement in micrometers per second.
Adhesion molecule blocking experiments
Individual rolling EGFPhigh cells were
tracked from the time entering a large postcapillary venule (diameter
>50 µm) until the cells were lost. Displacements of tracked
EGFPhigh cells were measured offline every tenth
frame for the entire observation period. After tracking two to four
EGFPhigh cells per vessel observed, animals were
injected with 30 µg mAb PS/2 directed against murine
4 integrin intra-arterially; 10 min later, the
tracking experiments were continued studying one to four
EGFPhigh cells in the same vessel. In separate
experiments, P-selectin mAb RB40.34 (a gift from D. Vestweber,
Münster, Germany), E-selectin mAb 9A9 (a gift from B. Wolitzky,
Hoffman-La Roche, Nutley, NJ), L-selectin mAb MEL-14 (American Type
Culture Collection, Manassas, VA), or CD44 mAb KM81 (American Type
Culture Collection) were injected at 30 µg/mouse i.v. Blood samples
for white blood cell counts were taken immediately before and 5 min
after Ab injection.
Data analysis
All data are given as mean ± SEM. Statistical analysis was performed using Student’s t test, Mann-Whitney rank sum test, and ANOVA on ranks test. Differences were considered statistically significant if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate CD2-EGFP+-transgenic mice, the
EGFP coding sequence was placed under control of the human CD2 promoter
and locus control region (Fig. 1
). This
strategy has been shown to result in a position-independent, copy
number-dependent T cell-specific expression of the desired gene product
(26). Microinjection of the construct into
B6CBA/F1 fertilized eggs resulted in two positive founders,
identified by PCR, tail bleeding, and subsequent flow cytometry. These
mice were backcrossed into C57BL/6 wild-type mice. Whereas offspring
from one founder lost EGFP expression past N3, offspring from founder
two revealed a stable EGFP expression throughout (N7 today).
In vivo visualization of EGFP+ cells
Using intravital microscopy of the cremaster muscle, we sought to
visualize EGFP+ cell trafficking in vivo.
However, in two well-known models of inflammation in the cremaster
muscle, with or without TNF- pretreatment (22), no or
only short, transient interactions between EGFP+
cells and the endothelium could be observed. No stable rolling of
EGFP+ cells was seen in either model. Because
pretreatment of ECs with both TNF- and IFN- has been shown to
lead to firm adhesion of activated T lymphocytes in vitro
(29), cremaster muscles were pretreated with a combination
of TNF- for 3–6 h and IFN- for 6 h. This resulted in stable
of EGFP+ cells rolling at a median velocity
similar to that of neutrophils (11 µm/s), as well as occasional
adhesion and transmigration. Intravital microscopy was able to
distinguish between EGFPhigh and
EGFPlow cells (Fig. 2). EGFPhigh cells
accounted for 0.7% of all leukocytes.
Surface Ag expression of EGFP+ cells
Analysis of EGFP expression in splenocytes, peripheral lymph node
cells, and leukocytes in peripheral blood shows 33.4, 63.5, and 20.5%
of cells positive for EGFP in the spleen, lymph node, and peripheral
blood, respectively (Fig. 3).
Ninety-seven to 100% of all EGFP+ cells express
CD2. Both CD4 as well as CD8 T cells in all three tissues express EGFP.
CD4 T cells are predominantly EGFPlow, whereas
CD8 T cells are EGFPhigh. Between 0.9 and 15% of
EGFPlow cells express NK1.1 in these tissues
(Fig. 3). B cells, macrophages, and granulocytes do not express EGFP
(data not shown). Because intravital microscopy uses an epifluorescent
microscope to visualize cells, we looked at the intensity of EGFP in
these two T cell subsets under the epifluorescent microscope after
sorting the EGFPlow CD4 T cells and
EGFPhigh CD8 T cells. Clearly, CD4 T cells show
very poor fluorescence, whereas CD8 T cells appear to be very bright
(Fig. 4A).
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0.7% of all leukocytes as determined by intravital microscopy. The
brightest 1.0% of all analyzed cells were CD2+,
90% of the EGFPhigh cells were CD8 T cells,
2.5% were CD4 T cells, and 8% stained positive for the NK1.1 marker
(Fig. 4B). These findings identify peripheral blood
EGFPhigh cells in our EGFP-transgenic mouse as
predominantly CD8+ lymphocytes. In vitro stimulation and proliferation assays
Stimulation of splenocytes from EGFP+ and
EGFP- mice with anti-CD3 Ab, Con A, or
PMA showed similar rates of proliferation (Fig. 5, A, C, and
E) and IL-2 secretion (Fig. 5, B, D,
and F). These data suggest that the transgene did not impair
lymphocyte function.
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). In a previously
reported GFP-transgenic mouse (25), anti-CD3
Ab-activated CD8+ T cells showed loss of GFP
expression by day 7. In our mice, the activated
CD8+ T cells continued to express EGFP, albeit at
lower levels, even on day 7 (Fig. 6), thereby indicating that EGFP
expression is not lost postactivation.
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4 integrin in CD8+ T cell
rolling
To address the molecular mechanisms of rolling of
CD8+ T cells in inflamed venules in vivo, we
blocked several endothelial and leukocyte adhesion molecules using
mAbs. Injection of blocking concentrations of Abs to P-selectin,
E-selectin, or L-selectin failed to alter the rolling velocities of
EGFPhigh cells (Fig. 7A and data not shown) as well
as total leukocytes in the transgene-negative littermate controls (Fig. 8A). Although the rolling
velocities did not change after blocking P- or E-selectin, the rolling
flux fraction was altered significantly after blocking both P- and
E-selectin in the transgene-negative littermate controls (Fig. 8B). After injection of the 4
integrin mAb PS/2, the average rolling velocity of
EGFP+ cells was significantly elevated from 11 to
24 µm/s (Fig. 7B and Table I). This indicates that
4 integrin is required to stabilize rolling of
these CD8+ T cells in inflamed venules in vivo.
We also investigated the role of CD44 in CD8 T cell rolling as a
candidate other than 4 integrin. As shown in
Fig. 7C, CD44 does not contribute to CD8 T cell rolling in
our model, as blocking CD44 with an anti-CD44 Ab, KM81, did not
alter the rolling velocity. Unknown selectin- and CD44-independent
mechanisms prevent EGFPhigh cells from detaching
even when 4 integrins are blocked.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using intravital microscopy, it was possible to identify
EGFPhigh and EGFPlow
cells in the peripheral blood (Fig. 2) of untreated, EGFP-transgenic
mice. EGFPhigh cells are most readily detected by
intravital microscopy and accounted for 0.7% of peripheral blood
leukocytes. Ninety percent of these cells are comprised of CD8 T cells,
2% of CD4 T cells, and 8% of NK cells, with no B cells, monocytes, or
neutrophils. The fluorescence intensity of EGFP was at least one decade
higher in CD8+ T cells compared with
CD4+ T cells as analyzed by flow cytometry (Fig. 3) and by epifluorescent microscopy (Fig. 4A). Taken
together, these data show that almost all EGFP
high cells are CD8 T cells.
Introduction of the transgene did not perturb T cell function, because
the T cells from EGFP+ mice were comparable to
those from transgene-negative littermates, as assessed by T cell
proliferation and IL-2 production. In a previously reported
GFP-transgenic mouse (25), anti-CD3 Ab-activated
CD8+ T cells showed loss of EGFP expression by
day 7. In the transgenic mice reported here, although activation of T
cells using an anti-CD3 Ab led to a down-regulation of EGFP
expression in CD8+ T cells, the majority of these
activated cells (80%) continue to express EGFP even on day 7
postactivation (Fig. 6).
Recruitment of neutrophils is known to be achieved through a series of
well-defined interactions between the neutrophil and EC that encompass
rolling, activation, and arrest followed by transmigration
(10). Lymphocytes are thought to be recruited through a
similar mechanism (31), and a multistep process has
clearly been shown for homing of naive lymphocytes to secondary
lymphatic organs (4, 32), but not for effector T cells.
Selectin and integrin molecules appear to play an important role in
migration of T cells to sites of inflammation. E- and P-selectin are
involved in CD4+ T cell migration into inflamed
skin (33, 34), whereas migration of activated
CD8+ T cells to sites of viral infection appears
to be selectin independent (35). Integrins like VLA-4,
LFA-1, and Mac-1 and their ligands ICAM-1 and VCAM-1 appear to be
important for CD8+ T cell recruitment in a
lymphocyte choriomeningitis virus infection model
(36, 37, 38, 39). Another integrin,
47, plays an
important role in entry of T cells into the lamina propria and Peyer’s
patches (40). Despite this information, it has not been
possible to directly demonstrate the adhesion cascade for
CD8+ effector T cells in vivo, nor is the
adhesion molecule usage in different steps known.
Using the CD2-EGFP-transgenic mouse, we have begun to address the
adhesion cascade for CD8+ T cells in vivo. In the
present study, we analyzed the role for P-, E-, and L-selectin, CD44,
and VLA-4 in rolling and adhesion of EGFPhigh T
cells to inflamed endothelium in vivo. Blocking P-, E-, or L-selectin
alone or in combination did not change the rolling velocities of these
cells (Fig. 7A and data not shown). Although the CD44
ligand, hyaluronate, is known to be up-regulated by TNF- treatment
(41), blocking CD44 did not alter the rolling velocity
(Fig. 7C) indicating CD44-independent rolling of
EGFPhigh T cells on the inflamed endothelium in
our model. Injection of anti-4 Ab
increased the rolling velocity of EGFPhigh cells
significantly from 11 to 24 µm/s (Fig. 7B and Table I).
This 2-fold increase indicates a dependence on
4 integrin for slow rolling of T cells on
inflamed endothelium. Surface expression of 4
integrin appears to be important in T cell recruitment as the presence
of this molecule has been shown to enhance the pathogenicity of T cell
clones by virtue of their ability to transmigrate through the
endothelium in the experimental autoimmune encephalomyelitis model
(42), and the levels of VLA-4 on the cell surface have
been correlated to the degree of protection conferred by
CD8+ T cell clones in an animal model of malaria
(43).
Interestingly, pretreatment with either TNF- alone or IFN- alone
failed to induce any stable rolling of the
EGFPhigh cells. Pretreatment with TNF- alone
is sufficient for rolling and arrest of neutrophils (27).
In this model, the data suggests that IFN- plus TNF- produces
conditions not induced by TNF- alone. It is possible that
IFN--dependent chemokine up-regulation may contribute to CD8 T cell
rolling and/or adhesion. For example, IFN- alone or in combination
with TNF- is known to up-regulate the CXCR3 ligand IFN-inducible T
cell chemoattractant in human ECs (44). However, the
present study does not address the potential role of chemokines.
In conclusion, we use a novel CD2-EGFP-transgenic mouse to show that
CD8+ T cells use 4
integrins to roll on inflamed endothelium in vivo. This mouse will be
very useful in elucidating the adhesion cascade involved in recruitment
of CD8 T cells to sites of inflammation in vivo.
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Acknowledgments |
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Footnotes |
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2 K.S. and J.T. contributed equally to this work.
3 Address correspondence and reprint request to Dr. Klaus Ley, Department of Biomedical Engineering, Box 800709, University of Virginia, Charlottesville, VA 22908. E-mail address: klausley{at}virginia.edu
4 Abbreviations used in this paper: EC, endothelial cell; VLA-4, very late Ag-4; GFP, green fluorescence protein; EGFP, enhanced GFP.
Received for publication November 30, 2000. Accepted for publication April 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7 integrins in CD8 T cell trafficking during an antiviral immune response. J. Exp. Med. 189:1631.4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192:495.4 integrins in vivo. J. Clin. Invest. 94:1722.
7-integrins, and P-selectin in leukocyte rolling and adhesion in high endothelial venules of Peyer’s patches. J. Immunol. 161:2449.47 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multi-step model confirmed and refined. Immunity 3:99.[Medline]
4 integrin directs virus-activated CD T cells to sites of infection. J. Immunol. 154:5293.[Abstract]
2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice. J. Virol. 70:8997.[Abstract]
4 integrin and matrix metalloproteinase-2 in the pathogenesis of experimental autoimmune encephalomyelitis. Lab. Invest. 78:1445.[Medline]
chemoattractant by human endothelial cells is cyclosporin A-resistant and promotes T cell adhesion: implications for cyclosporin A-resistant immune inflammation. J. Immunol. 164:5383.This article has been cited by other articles:
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