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*
Department of Immunology and
Human Genome Research, Schering-Plough Research Institute, Kenilworth, NJ 07033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase gene
in multiple tissues. In these transgenic mice, doxycycline treatment
induced a strong up-regulation in the expression of KC in several
tissues, including heart, liver, kidney, skin, and skeletal muscle.
Expression of KC within these tissues led to a rapid and substantial
increase in the serum levels of KC (serum KC levels were higher than
200 ng/ml 24 h after treatment). Accordingly, -galactosidase
expression was also detected after injection of doxycycline and was
highest in skeletal muscle, pancreas, and liver. Surprisingly, despite
expression of KC in multiple tissues, no neutrophil infiltration was
observed in any of the tissues examined, including skin. Doxycycline
treatment of nontransgenic mice grafted with transgenic skin caused
dense neutrophilic infiltration of the grafts, but not the surrounding
host skin, indicating that the KC produced in transgenic tissues was
biologically active. In separate experiments, neutrophil migration
toward a localized source of recombinant KC was impaired in animals
overexpressing KC but was normal in response to other neutrophil
chemoattractants. Analysis of transgenic neutrophils revealed that high
concentrations of KC in transgenic blood had no influence on L-selectin
cell surface expression but caused desensitization of the receptor for
KC, CXCR2. These results confirm the neutrophil chemoattractant
properties of KC and provide a mechanistic explanation for the
paradoxical lack of leukocyte infiltration observed in the presence of
elevated concentrations of this chemokine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence supporting an important role for chemokines in guiding leukocyte migration and homing in vivo has been provided by analysis of a variety of genetic models (reviewed in Ref. 6). Loss-of-function experiments have shown that deletion of chemokine receptors significantly disrupts homing and inflammatory trafficking of specific leukocyte subsets (7, 8, 9, 10, 11). In contrast, gain-of-function transgenic studies have shown that tissue-specific expression of chemokines is associated with recruitment of specific leukocyte subsets into the expressing tissues. For instance, tissue-specific expression of the murine chemokines KC and JE (murine monocyte chemoattractant protein-1 (MCP-1)2), induces recruitment of neutrophils and monocytes, respectively, to the expressing tissues (12, 13, 14, 15, 16, 17). The ability of JE and other chemokines to recruit leukocytes in transgenic mice seems to depend on the mode of expression of the transgenes. Widespread rather than localized overexpression of MCP-1 or IL-8 prevents or inhibits leukocyte recruitment (18, 19), but the mechanistic basis for these paradoxical findings is unclear at present (20, 21, 22).
To better understand the mechanisms regulating leukocyte recruitment in vivo, we have applied transgenesis to study the function of the murine CXC chemokine KC. KC, also known as N51, is a murine immediate early gene (23, 24) with a low or undetectable expression in most tissues (23, 25). KC expression is highly up-regulated in vitro and in vivo by a variety of factors, such as LPS, platelet-derived growth factor, and bombesin (23, 25, 26, 27, 28, 29). Similar to its human relatives growth-related oncogene/melanoma growth-stimulating activity (CXCL1) and IL-8 (CXCL8), KC is a potent inducer of neutrophil migration in vitro and in vivo (25). In humans, these molecules promote chemotaxis via their interaction with the receptors CXCR1 and CXCR2, but only one such receptor, CXCR2, has been identified thus far in the mouse (30, 31, 32, 33). In previous transgenic experiments, tissue-specific expression of KC in thymus, skin, heart, lung, and brain was associated with recruitment of neutrophils into these organs (6, 12, 14, 34). The recruited neutrophils accumulated in the vicinity of cells expressing the transgene and did not appear to be activated. Interestingly, neutrophil infiltration was attenuated over time in some of these models, which was attributed to the constitutive, unphysiological expression of KC (12, 34). Thus, although genetic studies have clearly demonstrated that expression of KC is sufficient to promote neutrophil infiltration into tissues, they have also suggested that an experimental system mimicking the physiological, inducible pattern of expression of KC may be required to better understand its function.
Here we describe the generation of transgenic mice conditionally expressing the chemokine KC. To this end, we used the tetracycline-dependent gene expression system developed by Gossen and Bujard (35). In the transgenic model described here, expression of KC was induced by doxycycline, a tetracycline analog, in a wide variety of tissues. In agreement with previous reports, we found that localized, conditional expression of KC was associated with significant recruitment of neutrophils, but that its widespread expression inhibited neutrophil recruitment. The predominant mechanism underlying the failure of neutrophils to migrate under these conditions was the desensitization of the CXCR2 in response to high circulating levels of KC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A bidirectional reporter transgene was constructed by cloning
the cDNA for KC (+2 to +140) into pBI-G (Clontech, Palo Alto, CA). The
activator transgene was constructed by cloning the cDNA for
rtTA obtained from the pTet-On vector (Clontech) into
the EcoRI site of an expression vector containing the CMV
enhancer/chicken -actin promoter and the rabbit -globin
polyadenylation signal (36). Separation of the transgenes
from vector sequences after digestion with restriction enzymes was
accomplished by zonal sucrose gradient centrifugation as described
(37). Fractions containing the transgene were pooled,
microcentrifuged through Microcon-100 filters (Amicon, MA), and washed
five times with microinjection buffer (5 mM Tris-HCl (pH 7.4), 5 mM
NaCl, 0.1 mM EDTA).
Generation of transgenic mice
DNA containing the transgene was resuspended in microinjection
buffer to a final concentration of 1–5 ng/µl, microinjected into
mouse eggs (C57BL/6J x DBA/2 F2; Charles
River Laboratories, Wilmington, MA), which were then transferred into
oviducts of CD1 (Charles River Laboratories) foster mothers, according
to published procedures (38). By 10 days of life, a piece
of tail from the resulting animals was clipped for DNA analysis.
Identification of transgenic founders was conducted by PCR analysis, as
previously described (39). Identification of transgenic
mice was accomplished by amplification of a segment of the
rtTA transgene using primers
(5'-CGGGTCTACCATCGAGGGCCTGCT-3') and
(5'-CCCGGGGAATCCCCGTCCCCCAAC-3') and amplification of a
segment of the-galactosidase (-gal) gene
(5'-ACCAGCGAATACCTGTTCCGTCATGC-3' and
5'-AGTAAGGCGGTCGGGATAGTTTTCTTGC-3'). As an internal control for the
amplification reaction, primers for the endogenous ZP3 gene
were used (5'-CAGCTCTACATCACCTGCCA-3'; 5'-CACTGGGAAGAGACACTCAG-3').
These primers amplify a 242-bp segment of the rtTA
transgene, a 791-bp segment of the -gal gene, and a
511-bp segment of the ZP3 gene. PCR conditions were: 95°C
for 30 s; 60°C for 30 s; 72°C for 60 s for 30
cycles. The resulting transgenic mice were kept under specific
pathogen-free conditions. Experiments were performed following the
guidelines of the Schering-Plough Animal Care and Use Committee.
RNA analysis
RNA was extracted from tissues using RNA STAT-60, following specifications from the manufacturer (TEL-TEST, Friendswood, TX). Total RNA (20 µg) was denatured and blotted onto Biotrans membrane (ICN Biomedicals, Costa Mesa, CA). Transgene expression was assessed by hybridization to randomly primed 32P-labeled rtTA cDNA (Stratagene, La Jolla, CA).
Quantitative PCR analysis
cDNA was generated by reverse transcription using random hexamers (Promega, Madison, WI) and oligo(dT) primers (Life Technologies, Gaithersburg, MD) from RNA. Quantitative PCR analysis was performed on an ABI 7700 sequence detection instrument (TaqMan) following manufacturer’s instructions. For TaqMan analysis, 25 ng cDNA were amplified with primers at 0.9 µM final concentration, and FAM-labeled diagnostic probe at a final concentration of 0.25 µM. Primers/probe sequences for KC were as follows: (forward 5'-TGCACCCAAACCGAAGTCA-3', reverse 5'-AGCTTCAGGGTCAAGGCAAG-3', probe 5'-6FAM-AGCCACACTCAAGAATGGTCGCGAG-TAMRA-3'). rRNA primers/probe (PE Applied Biosystems, Foster City, CA) were used as an internal control. Quantitative PCR conditions were as follows: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s, 60°C for 1 min. A plasmid containing the KC gene was used as standard, ranging from 200 pg to 20 fg. Data were analyzed using Sequence Detection Systems software version 1.7.
ELISA
Sera were stored at -80°C until assayed for KC concentrations using a KC-specific ELISA kit (R&D Systems, Minneapolis, MN).
Air pouch model
The mouse air-pouch model for in vivo chemotaxis has been described in detail elsewhere (40). On experimental day 0, 5 ml sterile air was injected under the dorsal skin; the resultant space was reinjected with 3 ml sterile air on day 3. On day 5, 1 µg recombinant KC (R&D Systems) or 0.2 µg LPS (Sigma, St. Louis, MO) in 1 ml 0.5% carboxymethylcellulose (CMC; Fluka, Buchs, Switzerland) in saline was injected into the pouches. The animals were sacrificed 4 h later, and the air pouches were lavaged with 2 ml sterile PBS. The resulting cell suspensions were pelleted, resuspended in PBS, and counted under a hemocytometer. Cytospin slides were prepared, stained (Diff-Quik Stain Set; Dade Behring, Newark, DE), and differential cell counts were determined. The absolute number of neutrophils in each sample was determined by multiplying the percentage of this cell type by the total number of leukocytes per sample.
Flow cytometry
Blood was collected from the retro-orbital sinus into tubes containing EDTA (Terumo Medical, Elkton, MD) to prevent clotting. After RBC had been lysed, 106 cells were incubated with 5 µg/ml Fc block (BD Biosciences, San Diego, CA) and 300 µg/ml mouse IgG (Pierce, Rockford, IL). Cells were then stained with the directly conjugated primary mAbs Gr-1 (RB6–8C5) and L-selectin (CD62L; MEL-14; BD Biosciences) in PBS, 1% BSA, 0.1% sodium azide for 20 min at 4°C in the dark. To determine viability, samples were subsequently stained with 20 µl 5 µg/ml propidium iodide (Calbiochem, San Diego, CA). Events were acquired on a BD Biosciences FACScan and analyzed using the CellQuest software.
Skin grafts
A patch of skin (5 x 7 mm) was removed from the back of donor mice and kept in PBS until host animals were ready for grafting. Host Rag1-/- (The Jackson Laboratory, Bar Harbor, ME) were anesthetized and shaved on the back. A graft bed was prepared on the back by carefully removing a rectangular piece of skin (5 x 7 mm) without disturbing the vascular layer underneath. The donor skin was then placed onto the graft bed with the direction of hair growth opposite to that of the host. A wound dressing (Spenco Medical, Waco, TX) and a petroleum gauze (Johnson & Johnson Medical, Arlington, TX) were then placed over the graft. Finally, a Band-Aid was wrapped around the graft and thorax to prevent dislodgment of the graft. The mice were monitored during recovery, and the bandage was removed 7 to 10 days later.
Histology and -gal histochemistry
After euthanasia, tissues were either fresh frozen with OTC
medium for cryosection or fixed by immersion in 10% phosphate-buffered
formalin for processing as paraffin sections. Tissues for light
microscopic examination were routinely processed, sectioned at 5 µm,
and stained with H&E. Fresh frozen sections (10 µm) were fixed with
2% paraformaldehyde in PBS and then used for -gal histochemistry
following a procedure described previously (41). Briefly,
the slides were incubated overnight or for a specific length of time in
the presence of
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (Roche
Molecular Biochemicals, Indianapolis, IN) and counterstained with
neutral red.
Measurement of neutrophil intracellular calcium response
Neutrophils were isolated from the peripheral blood of wild-type animals or transgenic mice 24 h after injection of 500 µg doxycycline i.p. To prevent receptor recycling, all steps were performed at 4°C within 1 h after blood collection. RBC were lysed, and white blood cells (WBC) were resuspended in PBS and suspended for analysis. Cells were loaded with fluo-3-acetoxymethyl ester (Sigma) in culture medium (DMEM containing 10% serum) for 1 h at 37°C after which cells were washed three times in HBSS containing 20 mM HEPES and 0.1% BSA. Cells were then aliquoted into 96-well black-wall plates at a density of 1 x 106 cells/well and centrifuged briefly (300 x g, 2 min). All plates were precoated with poly-L-lysine. Intracellular calcium flux was measured in all 96 wells simultaneously and in real time using a Fluorescent Imaging Plate Reader (Molecular Devices, Sunnyvale, CA), and data were expressed as maximum fluorescence vs concentration of agonist. Recombinant KC was purchased from R&D Systems.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate transgenic mice in which expression of the chemokine
KC could be induced conditionally, we used the tetracycline-dependent
gene expression system originally described by Gossen and Bujard
(35). In this bigenic system the tet-activator protein
(rtTA) is expressed constitutively from the "activator" transgene
(Fig. 1
A). Then, in the
presence of the tetracycline analog doxycycline, the rtTA protein binds
to a tetracycline-responsive promoter element (TRE) present on a
"reporter" transgene and induces expression of the transgenes of
choice (Fig. 1C). The activator transgene used here was
driven by the CMV enhancer/-actin promoter, which promotes
expression of transgenes in multiple tissues (42, 43). We
generated 15 founder mice carrying the activator transgene and derived
8 independent transgenic lines from them. Activator transgene
expression was detected in four of these lines by analysis of skeletal
muscle RNA. Further analysis of one of these lines (line 72) showed
high levels of transgene expression in heart; moderate expression
levels in skin, kidney, thymus, and lung; and low expression levels in
spleen and liver (Fig. 1B). This line was selected as the
activator line.
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-gal (Fig. 1
C). Because this transgene caries only
minimal promoter sequences, it should remain silent or nonexpressed in
the absence of the transcriptional factor encoded by rtTa. To test
whether reporter transgene expression was indeed silent in the absence
of rtTA, we analyzed a large variety of tissues (bladder, bone, brain,
esophagus, heart, kidney, liver, lung, muscle, pancreas, skin, spleen,
stomach thymus, trachea) from all four transgenic lines for -gal
activity. With the exception the brain, where a small number of cells
stained blue, -gal activity was not detected in any other organs
examined, indicating that reporter transgene expression was silent in
these transgenic mice. Animals from reporter line 19 were crossed to
animals from activator line 72 to generate double-transgenic animals. Induction of transgene expression is dependent on doxycycline and occurs in multiple tissues of double-transgenic mice
To test whether doxycycline treatment induced KC expression and to
determine whether this effect was dose dependent, we injected
double-transgenic mice with a single i.p. dose of 20, 100, or 500 µg
doxycycline. Blood was collected 24 h after injection, and KC
serum levels were determined using a KC-specific ELISA. As shown in
Fig. 2A, the levels of KC in
serum increased proportionally to the amount of doxycycline injected,
reaching concentrations of 224 ± 112 ng/ml in response to the
highest dose of doxycycline.
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2000-fold over baseline; Fig. 2B). After this
time, the concentration of KC declined steadily reaching baseline
levels 7 days after treatment. Thus, injection of a single dose of
doxycycline resulted in a rapid, strong, and reversible induction of KC
gene expression.
The pattern of expression of the activator transgene (Fig. 1B) suggested that the elevated levels of KC in circulation
could be caused by doxycycline-induced expression of KC in multiple
tissues. To more accurately define the source of KC in circulation, we
examined KC mRNA expression in various tissues by real time PCR.
Tissues (thymus, kidney, spleen, lung, heart, liver, skeletal muscle,
tongue, and skin) were harvested from untreated or doxycycline-treated
transgenic mice (24 h after injection of doxycycline 500 µg i.p.).
RNA was prepared and translated into cDNA, which was amplified using
primers and probe specific for KC. As shown in Fig. 3, most tissues of untreated transgenic
mice expressed very low levels of KC (<1 fg KC/20 ng total cDNA). Low
levels of KC expression (1–7 fg KC/20 ng total cDNA) were detected in
spleen, lung, liver, and skeletal muscle. This low basal level of KC
expression in mouse tissues is in agreement with results published by
Bozic et al. (25), who have used a sensitive RNase
protection assay to examine expression of KC. In contrast, levels of KC
in tissues of treated animals were 4- to 500-fold higher than those of
untreated animals, except in spleen, where there was no induction of KC
over baseline levels (Fig. 3). KC expression was highest in skeletal
muscle, followed by kidney, liver, tongue, and skin.
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To map the expression of the reporter transgene at the tissue and
cellular level, we examined tissues from doxycycline-treated and
untreated transgenic mice for evidence of -gal activity. No -gal
activity was detected in rtTa-expressing tissues in the
absence of doxycycline (Fig. 4A and data not shown). In
contrast, -gal activity was easily detected in multiple organs
including skeletal muscle, pancreas, liver, kidney, stomach, small and
large intestine, tongue, and heart 24 h after doxycycline
treatment (500 µg i.p) (Fig. 4, B–L). In some organs,
transgene expression was restricted to morphologically defined areas
such as the cortex of the kidney (Fig. 4E), the exocrine
pancreas (Fig. 4C), the epithelial lining of the stomach
(Fig. 4G), or the epithelium of the small (Fig. 4H) and large intestine (Fig. 4I). In other
tissues, such as skeletal muscle (Fig. 4B), tongue (Fig. 4K), and heart (Fig. 4L), a mosaic pattern of
transgene expression could be clearly appreciated. In thymus (Fig. 4F), spleen (Fig. 4J), bone marrow, and brain
(not shown), -gal activity was low or absent, despite expression of
the activator transgene (Fig. 1C). In summary, -gal
activity was detected in most of the transgenic tissues expressing the
activator transgene after, but not before, doxycycline treatment.
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Expression of KC in a tissue-specific fashion induces neutrophil
recruitment (12, 14, 34). To determine whether neutrophils
would infiltrate tissues of animals expressing KC ubiquitously, we
collected tissues from animals treated with increasing doses of
doxycycline (20, 100, and 500 µg) at different time points (6–96 h
postinjection) and examined them microscopically. Surprisingly, we did
not detect neutrophil infiltration in the parenchyma of any of the
tissues examined, regardless of the dose or duration of doxycycline
treatment (Fig. 5). Neutrophils were seen
only within vascular structures of skeletal muscle between 24 and
96 h after administration of the highest dose of doxycycline (Fig. 5F, inset).
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The simplest explanation for the paradoxical lack of neutrophil
infiltration observed in the previous experiment would be that the KC
produced was biologically inactive. To test whether KC produced by the
double-transgenic mice was biologically active, we performed skin
grafting experiments. If biologically active KC were produced by the
grafts, we would expect to observe a pattern of neutrophil infiltration
similar to that observed in transgenic mice expressing KC in the skin
(12). We grafted skin from untreated double-transgenic
mice onto Rag1-/- mice and used as control the
skin from ROSA mice. ROSA mice express -gal constitutively in the
majority of tissues (44) including skin (Fig. 6A). Four weeks after the
procedure, when the grafts had healed, we injected the host mice with a
single dose of doxycycline (500 µg i.p.); 48 h after injection
of doxycycline, we collected grafted and host skin and examined -gal
expression. As expected, grafted ROSA skin stained positive for -gal
expression whereas surrounding host tissue did not stain (Fig. 6A). Similarly, doxycycline treatment of host mice resulted
in the induction of transgene expression in grafted transgenic skin,
but not in the surrounding host skin (Fig. 6B). To test
whether neutrophils had infiltrated the grafts, we stained adjacent
sections to those used for detection of -gal activity with an Ab
specific to Gr-1, a neutrophil marker. No Gr-1-positive staining was
detected in the grafted skin from ROSA mice or in the adjacent host
tissue (Fig. 6C), but a significant accumulation of
Gr-1+ cells was detected in tissue grafts derived
from double-transgenic mice (Fig. 6D). Accumulation of
neutrophils occurred specifically in the grafted tissue, and no
infiltration was observed in the adjacent host tissue (Fig. 6, E and F). Thus, the localized induction of
transgene expression in our conditional system resulted in infiltration
of neutrophils, indicating that the KC produced in the transgenic
tissues during doxycycline treatment is biologically active.
Infiltration of neutrophils after induction of KC expression is also
associated with a thickening of the epidermis in the grafted transgenic
skin. Because such thickening of the epidermis is absent in the grafted
control skin as well in the skin of doxycycline-treated transgenic
donor mice (Fig. 5D), it remains to be seen whether these
changes are an immediate response to the influx of neutrophils, the
induction of KC expression, or a combination of these factors.
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The absence of neutrophil infiltration into tissues expressing
high levels of KC could be caused by a number of factors including
decreased mobilization of neutrophils from the bone marrow or
marginating pool, or reduced expression of adhesion molecules important
for the transmigration process, such as L-selectin. To test these
hypotheses, we analyzed the number of total neutrophils and
L-selectin-positive neutrophils in blood after injection of doxycycline
(500 µg i.p.). As shown in Fig. 7, induction of KC expression led to a 2- to 3-fold increase in
circulating neutrophils at 4 and 24 h, suggesting that the
mobilization responses were preserved or even elevated
(p = 0.02). In addition, the expression of
L-selectin on the cell surface of neutrophils from doxycycline-treated
transgenic animals was equivalent to that of nontreated transgenic
controls, demonstrating that shedding of L-selectin did not occur in
response to high levels of circulating KC.
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Next we investigated whether neutrophils could migrate toward a
localized source of KC or other chemoattractants in the presence of
high systemic levels of KC. To this end, we generated a s.c. air pouch
on the back of transgenic animals and injected it with vehicle, LPS, or
KC. As shown in Fig. 8, large numbers of
neutrophils infiltrated the air pouch of untreated transgenic animals
in response to KC. In contrast, significantly fewer neutrophils
migrated into the air pouch in response to KC, when the transgenic mice
had received doxycycline treatment 24 h earlier
(p = 0.008). The number of neutrophils
recovered from the air pouch of these animals was equivalent to that
obtained by injection of vehicle alone. The number of neutrophils
migrating toward KC under these conditions did not change even when 10
µg recombinant KC were injected into the air pouch (data not shown).
Interestingly, neutrophils of transgenic animals treated with
doxycycline were still able to migrate toward other
neutrophil-attracting stimuli such as those induced by LPS (Fig. 8).
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The previous results indicated a selective deficit of neutrophil
migration toward KC in transgenic animals overexpressing this
chemokine. This defect could have resulted from down-regulation or
desensitization of the receptor for KC, CXCR2. At present, we are not
aware of any Ab suitable to detect surface expression of murine CXCR2.
We therefore tested whether transgenic blood neutrophils could respond
to KC stimulation with an intracellular calcium flux. In peripheral
blood, CXCR2 is primarily expressed by neutrophils (our unpublished
observation). As shown in Fig. 9, WBC from wild-type mice demonstrated a dose-dependent increase in
intracellular calcium when stimulated with recombinant KC. In contrast,
the KC-dependent maximum calcium response in WBC from transgenic mice
was reduced by 4-fold. Interestingly, the KC concentration inducing
a half-maximal response (EC50) was similar (12
nM) for both control and transgenic WBC. These results suggest that the
defect in neutrophil response in induced transgenic animals is indeed
caused by receptor desensitization, down-regulation, or a combination
of both.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bigenic conditional system described here consists of two
transgenes. The first transgene (the activator transgene) encodes a
transcriptional factor (rtTa), which can complex with doxycycline to
bind a tetracycline response DNA element present in the second
transgene (reporter transgene). In agreement with previous transgenic
studies using the human CMV/-actin promoter (42, 43),
high expression of the activator transgene were found in skeletal
muscle, heart, tongue, and skin. Crossing animals carrying the
activator transgene with animals carrying the reporter transgene
yielded double-transgenic mice that did not express KC or -gal in
the absence of doxycycline. These animals, however, expressed high
levels of KC and -gal in multiple tissues after doxycycline
treatment. In general, the inducibility of KC correlated with the
levels of expression of the activator transgene in most tissues
(skeletal muscle, tongue, skin, liver, and spleen). However, high
levels of activator transgene expression were detected in the lung, and
yet there was a relatively low induction of KC expression (4-fold). The
converse was true for kidney, where low levels of activator transgene
expression were detected, but where there was a robust KC induction
(680-fold over background). Coexpression of -gal (encoded
alongside KC in the reporter transgene) allowed precise mapping of the
reporter transgene expression. Upon administration of doxycycline,
-gal expression was detected in several tissues and correlated with
the expression of the activator transgene. The highest expression of
-gal was detected in exocrine pancreas and skeletal muscle. We
suggest that the high expression of -gal in pancreas was the direct
result of activator transgene expression, because we and others have
shown that transgenes under control of the CMV/-actin promoter are
highly expressed in the exocrine pancreas (43, 45).
-Gal was not expressed in all tissues in which the activator
transgene was expressed. For instance, -gal expression was minimal
or not detected in lymphoid tissues, in lung, or in the brain, tissues
in which low but detectable expression of the activator transgene was
demonstrated. The lack of -gal expression in these settings could
have been caused by low levels of rtTa protein or by other
tissue-specific transcriptional requirements. Finally, we should point
out that there was a very good concordance between -gal and KC
expression. These genes were found to be expressed at high levels in
skeletal muscle, liver, heart, kidney, and skin. There was low
expression of both genes in thymus and lung and no expression of either
gene in spleen.
Arguably, the most interesting finding of our studies was the observation that despite high transgene expression in many tissues and increased numbers of neutrophils in circulation, no neutrophil infiltration could be detected in any of the expressing tissues. This finding suggested that neutrophil migration from the blood stream into tissue parenchyma was impaired. At least two general hypotheses could be formulated to explain this impairment in neutrophil migration: one would suggest that the transgenic mice would express a biologically inactive KC; the other would suggest a defect in neutrophil function.
To investigate whether the KC produced in the transgenic tissues was
biologically active, we performed skin grafting experiments. Here we
took advantage of the fact that the reporter transgene encoded both KC
and -gal, and we used -gal expression to mark the cells
expressing the transgene. Transgene expression in the grafted
transgenic skin was induced by administration of doxycycline to the
host animals. As expected, -gal expression was visualized in the
grafted transgenic skin, but not in the surrounding host skin. Again,
as previously reported (12), expression of KC in the skin
promoted neutrophil recruitment. This process was specific to the
grafted skin from double-transgenic mice and was not observed in
grafted control skin (derived from ROSA mice) expressing -gal. These
results indicated that the KC produced by the transgenic tissues was
biologically active. Thus, besides validating the original observations
regarding KC, these findings suggest that skin and other tissues from
these transgenic animals can now be used to investigate the role of KC
and neutrophils in transplantation biology. The ability to regulate
gene expression in multiple, transplantable tissues will likely
facilitate the analysis of the biological role of other chemokines.
As discussed above, the paradoxical absence of neutrophil infiltration into tissues expressing KC could also be the result of a general defect in the ability of the transgenic neutrophils to migrate. To test this hypothesis, we promoted expression of neutrophil chemoattractants by injection of LPS in a s.c. air pouch in transgenic mice with or without doxycycline treatment. LPS is by itself a potent inducer of neutrophil migration and also induces other neutrophil chemoattractants such as C5a (46). There was no difference in the number of neutrophils recruited in the pouch, suggesting that the overall capacity of neutrophils to respond to chemoattractants had been preserved, even in the presence of high systemic levels of KC. There was, however, a significant impairment in the response of neutrophils to KC after but not before doxycycline treatment. These results indicated again that the transgenic neutrophils were essentially normal before treatment and suggested that they became less capable to migrate toward a localized source of KC after doxycycline treatment. We hypothesized that the high systemic levels of KC were directly causative of the abnormal neutrophil response. A number of in vivo and in vitro studies in the literature would point in this direction. In vivo, high levels of circulating chemokines inhibit leukocyte infiltration into tissues. High systemic levels of IL-8 attenuate proinflammatory effects of locally administered IL-8 (21, 22) or neutrophilia induced by LPS in lung (20). Furthermore, high constitutive expression of MCP-1 or IL-8 in transgenic mice does not result in leukocyte infiltration into the expressing organ or any other organ (18, 19). Moreover, tissue-specific constitutive transgenic expression of KC results over time in attenuation of neutrophil recruitment (14, 34), and monocyte recruitment induced by tissue-specific expression of MCP-1 can be abrogated in the presence of high systemic levels of MCP-1 (15). The reduced ability of leukocytes to migrate from the blood into tissue in these circumstances has been attributed to a number of factors such as shedding of L-selectin, the absence of a chemoattractant gradient, or desensitization of the cognate chemokine receptor (15, 18, 19).
Leukocyte extravasation from the blood stream into tissue is dependent on P- and possibly L- and E-selectins that mediate a low affinity adhesive rolling interaction of leukocytes on activated endothelium (47). Neutrophil emigration into tissues is dependent on shape change and shedding of L-selectin (reviewed in Ref. 48), but this effect is not dominant, because it has been demonstrated that neutrophils lacking L-selectin are still capable of infiltrating tissues (49, 50). Simonet et al. (19) found reduced L-selectin expression on the surface of blood neutrophils of mice overexpressing IL-8 and argued that this reduction could explain lack of neutrophil infiltration observed in their model. Here we found that surface expression of L-selectin on blood neutrophils remained constant even after prolonged exposure of neutrophils to high concentrations of KC. We therefore ruled out shedding of L-selectin as a factor contributing to the lack of neutrophil migration in our system.
Neutrophil migration to KC in mice is mediated through the CXCR2
receptor, a G protein-coupled seven-transmembrane receptor (33, 51). Like other G-protein-coupled seven-transmembrane receptors,
the murine CXCR2 induces calcium mobilization and cell migration in
response to agonist binding (52). Thus, freshly isolated
transgenic WBC were tested for calcium mobilization in response to
recombinant KC by calcium fluorometry. The response was greatly reduced
in WBC derived from doxycycline-treated mice, indicating that the
functionality of the CXCR2 receptor was affected by high concentrations
of KC. Indeed, induction of transgene expression in multiple tissues
resulted in a rapid increase in the levels of circulating KC reaching
maximal levels of 224 ng/ml 24 h after doxycycline injection.
In vitro, similar concentrations of neutrophil chemoattracting
molecules arrest neutrophil movement (53). Thus, the
neutrophil migration deficit seen in our model could be due to an
uncoupling of the receptor in response to KC binding, leading to
desensitization or to increased internalization of the receptor.
Although we have clearly demonstrated a deficit in the response of WBC
to KC, we cannot rule out at this point that additional factors such as
the absence of a chemotactic gradient may have prevented cell
infiltration into tissues.
In summary, the development of the ubiquitous conditional system described here provided us with a tool to investigate the mechanisms underlying inhibition of neutrophil migration associated with high systemic expression of KC. Multiple tissues were shown to express KC, which resulted in high serum levels of this chemokine. Grafting of tissues conditionally expressing KC into Rag1-/- mice established that the KC was biologically active to promote neutrophil recruitment into the grafts, confirming previous studies (54). In the presence of high systemic levels of KC, we found an impaired neutrophil response to KC and identified desensitization of CXCR2 as a mechanism to explain the migration defect. These findings suggest that the ability of KC to promote neutrophil recruitment is dependent on how it is expressed. On the basis of these results, we hypothesize that elevated levels of KC or other CXCR2 ligands may attenuate or reduce neutrophil infiltration during pathological situations and profoundly impact host responses. We anticipate that the conditional system described here will prove to be a powerful experimental tool to test this and other hypotheses related to the biological function of chemokines.
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Acknowledgments |
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-actin promoter cassette. |
Footnotes |
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2 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; rtTA, tet-activator protein; CMC, carboxymethylcellulose; TRE, tetracycline-responsive promoter element; WBC, white blood cells; -gal, -galactosidase.
Received for publication August 1, 2001. Accepted for publication October 22, 2001.
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)
or treated mice 24 h after injection of doxycycline (500 µg
i.p.; 
). Values are expressed as femtograms KC amplicon per 20 ng
total cDNA. Results are representative of three experiments. sk,
skeletal.
) and 24 h (
). Cells were analyzed
for their ability to flux calcium in response to varying concentrations
of recombinant KC. The response to KC is displayed as relative
fluorescence change. Data are representative of two independent
experiments.