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Departments of
* Antibacterials, Immunology, and Inflammation and
Drug Safety Evaluation, Pfizer Global Research and Development, Pfizer Inc., Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but
sequential treatment of wild-type, but not P2X7R-deficient,
blood with LPS and ATP yielded large amounts of cell-free cytokine.
Based on these differences, wild-type and P2X7R-deficient
animals were compared following induction of monoclonal
anti-collagen-induced arthritis. Ab-treated wild-type animals
subsequently challenged with LPS developed inflamed, swollen paws;
their joint cartilage demonstrated lesions, loss of proteoglycan
content, and the presence of collagen degradation products.
P2X7R-deficient animals subjected to the same challenge
were markedly less affected; both the incidence and severity of disease
were reduced. These data indicate that ATP does act via the
P2X7R to affect leukocyte function and that the
P2X7R can serve as an important component of an in vivo
inflammatory response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P2X7R activity is reported to exist in a limited
number of cell types but is readily detectable in cells of hemopoietic
lineage including monocytes, macrophages, and lymphocytes (15, 16). The physiological function of this receptor remains a
subject of investigation, and a number of diverse activities have been
proposed, including activation and maturation of T cells (17, 18), formation of giant cells (19, 20), killing of
invading microorganisms in macrophages (21, 22), and
activation of various signaling cascades (23, 24, 25). The
receptor also has been proposed to serve as a regulator of
inflammation, based on its ability to initiate posttranslational
processing of leaderless cytokines such as IL-1
(26, 27, 28). When stimulated by an inflammatory insult such
as LPS, monocytes, macrophages, and microglial cells generate large
quantities of proIL-1 but release very little of the mature
biologically active cytokine to the extracellular environment. However,
efficient posttranslational processing of the procytokine molecules is
rapidly engaged by treating the LPS-activated cells with ATP
(26, 27, 28, 29). Nucleoside triphosphate-induced cytokine
processing is accompanied by a necessary K+
efflux, activation of caspase-1 (the protease responsible for cleavage
of proIL-1 to its mature form), and cell death (27, 28, 30). LPS-activated peritoneal macrophages isolated from mice
engineered to lack the P2X7R fail to generate
mature IL-1 in response to ATP, confirming the role of
P2X7R in this unusual biologic response
(31).
ATP is considered to be a physiological ligand of the
P2X7R (4, 16), but concentration
requirements for this nucleoside triphosphate in normal tissue culture
media can exceed millimolar values (1, 5). These
high concentrations are reduced by removal of divalent cations from the
medium, suggesting that the relevant ligand is ATP4-
(5). Although the source of the ATP that engages the
P2X7R in vivo remains to be established, in vitro
studies have demonstrated that ATP can be released to the medium from
degranulating platelets, dying cells, or viable cells via various
transport processes (32, 33, 34). Nonetheless, the high ATP
concentration requirements of the P2X7R remain
problematic, and it is difficult to envision how millimolar levels of
extracellular nucleotide triphosphate are achieved by any of the known
ATP export mechanisms. A recent study using cocultures of astrocytes
and microglia suggested that astrocytes released ATP in response to
mechanical or bradykinin activation in quantities sufficient to
activate P2X7Rs on neighboring microglial cells
(35). Thus, the context in which cells are exposed to ATP
may influence ligand concentration requirements. In the present study
we compare the ATP responsiveness of blood-derived leukocytes from
wild-type and P2X7R-deficient mice, and
demonstrate that absence of the receptor leads to loss of ATP-dependent
leukocyte functions, including IL-1 production and L-selectin
shedding. Moreover, wild-type and P2X7R-deficient
mice are compared with respect to their susceptibility to monoclonal
anti-collagen-induced arthritis, a model of inflammatory joint
disease. Absence of the P2X7R is associated with
less severe disease outcomes, indicating that the
P2X7R can function as an integral component of an
in vivo proinflammatory mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of the P2X7R mice was described previously (31). These animals were maintained on a mixed genetic background (129/Ola x C57BL/6 x DBA/2). Breeding P2X7R-/- males with P2X7R-/- females was used to maintain the colony of receptor-deficient animals. Likewise, genetically comparable wild-type animals were maintained by crossing homozygous animals. Mice used in the current studies generally were 12 wk of age.
Cell Dyne assay
Wild-type and P2X7R-deficient mice were euthanized by CO2 fixation and blood was collected into 1-ml syringes (containing 10 U/ml heparin) by cardiac puncture. Blood from individual animals was diluted 1/1 with RPMI 1640 medium containing 20 mM HEPES (pH 7.5), 100 U/ml penicillin, and 100 µg/ml streptomycin. A total of 0.28 ml of each diluted blood sample was placed into a 1.5-ml capped tube and LPS (Escherichia coli serotype 055:B5; Sigma-Aldrich, St. Louis, MO) was added to some tubes to achieve a final concentration of 1 µg/ml. These blood samples were incubated for 3 h at 37°C in a 5% CO2 environment after which 5 mM effector (ATP, ADP, or UTP) was introduced to some tubes, and the incubation continued for an additional 2 h. At this point, samples were analyzed for leukocyte content by laser flow analysis using a Cell Dyne 3700 instrument (Abbott Laboratories, Abbott Park, IL) (36).
FACS analysis for leukocyte L-selectin expression
Heparinized blood was isolated from wild-type and P2X7R-deficient animals as described above, samples from several individual animals of each genotype were combined, and 0.5 ml of the resulting pools was placed in 1.5-ml capped tubes. ATP was added to achieve a final concentration of 5 mM, and the samples were incubated at 37°C in a 5% CO2 environment for 15 min. The nucleoside triphosphate (disodium salt from Sigma-Aldrich) was added from a 100 mM concentrate preneutralized to pH 7 by NaOH addition. MgCl2 subsequently was added (final concentration of 20 mM) to quench the ATP-dependent response. At this point, the blood samples were split into two 0.2-ml aliquots and each was diluted with 1 ml of heat-inactivated fetal serum with azide (HiFAZ) reagent (PBS containing 0.02% sodium azide, 2% heat-inactivated FBS). These samples were subjected to centrifugation, and the resulting cell pellets were resuspended in 0.1 ml of HiFAZ reagent. Each of the duplicate samples received 5 µl of allophycocyanin-labeled anti-CD62, after which one received 5 µl of FITC-labeled anti-CD3 and the other received 5 µl of PE-labeled anti-CD45; all Abs were obtained from BD PharMingen (San Diego, CA). Ab complexes were allowed to form at room temperature for 30 min, then the samples were diluted with 1 ml of HiFAZ reagent and cells was collected by centrifugation. Cell pellets were suspended in 2 ml of FACS lysing solution (BD Biosciences, San Jose, CA) and incubated for 10 min at room temperature to lyse RBCs. Leukocytes were recovered by centrifugation and washed once with 1 ml of HiFAZ reagent and suspended in 0.5 ml of this buffer. Samples were analyzed on a FACSCalibur instrument (BD Biosciences).
Blood-based IL-1 assay
Heparinized blood (pooled samples from multiple animals of the
same genotype) was dispensed into 96-well plates (0.12 ml/well) and
diluted with an equal volume of RPMI 1640 medium containing 25 mM HEPES
(pH 7.5), 1% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.
LPS was introduced to some wells (final concentration of 1 µg/ml),
and the plates were incubated for 3 h at 37°C in a 5%
CO2 environment to promote proIL-1 synthesis.
ATP subsequently was introduced to some wells to achieve a final
concentration of 5 mM, and the plates were incubated for an additional
2 h at 37°C. The plates then were subjected to centrifugation
(700 x g for 10 min), the resulting plasma
supernatants were harvested, and their content of murine IL-1
subsequently was determined by ELISA (Amersham Pharmacia Biotech,
Piscataway, NJ).
Peritoneal macrophage ATP response
Murine peritoneal macrophages were recovered as described
previously (30). Isolated cells were washed with RPMI 1640
medium containing 5% heat-inactivated FBS, 100 U/ml penicillin, and
100 µg/ml streptomycin, and then seeded into Natrix-coated six-well
plates (BD Biosciences) at a density of 2 x
106 cells/well. After an overnight incubation,
media were removed, 1 ml of fresh RPMI 1640 medium containing 5%
heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 1 µg/ml LPS was added to each well, and the cultures were
incubated for 2 h at 37°C. ATP (5 mM) then was introduced, after
which individual cultures were incubated for different periods of time
before being photographed (x40 objective) by light microscopy or
harvested for analysis of the distribution of -hexosaminidase
between the cell-associated and media fractions (27).
mAb-induced arthritis
Arthritis was induced in mice using mAbs directed against type II collagen (37, 38). All procedures involving animals were approved by the Institutional Animal Care and Use Committee. The treatment consisted of i.p. injection (400 µl) of a 10 mg/ml mAb mixture (Chemicon International, Temecula, CA) on day 0. Twenty-four hours later the mice were injected i.p. with LPS (100 µl of a 0.25 mg/ml solution). During the subsequent 15 days, arthritis was assessed by the degree of swelling, redness, and ankylosis of the joints. All visual scores were combined into a score of 0–3 per paw and summed for a total score of 0–12 per animal. On day 15, hind paws were removed in some experiments for histological processing.
Both hind limbs (distal to the mid-femur) were collected and placed in 10% neutral buffered formalin for 24–48 h. The specimens were then decalcified in Immunocal (Decal Chemical, Congers, NY) for 48 h. The femorotibial (knee) and tibiotarsal (ankle) joints were cut in frontal and sagittal planes, respectively, dehydrated through graded alcohols, embedded in paraffin wax, and sectioned at 5 µm. Sections were stained with safranin-O for acid mucopolysaccharides. For 9A4 immunohistochemistry (39), sections from the stifle joint were stained using DAKO ARK kit (DAKO, Carpinteria, CA) according to the manufacturer’s directions. Ag retrieval was performed with Decal Solution (Biogenex, San Ramon, CA) at room temperature for 30 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide followed by rinsing with distilled water. Ab 9A4 (2.3 µg/ml) or a mouse isotype control IgG1 were biotinylated (DAKO biotinylation reagent) and incubated with tissue samples at room temperature for 45 min. Slides subsequently were washed and then incubated with streptavidin-HRP (DAKO) for 30 min. Binding of the primary Ab was detected by incubating the slides for 5 min in chromagen DAB+ substrate-chromagen (DAKO), followed by counterstaining with hematoxylin.
Histologic evaluation on safranin-O sections was performed by a single blinded observer using a modified Mankin scale (40) as described below. This scale was developed to quantify changes in articular cartilage in humans with osteoarthritis, and was modified to reflect rodent size and to include synovial inflammation. This scale grades on a total composite scale of 0–17 and evaluates the severity of arthritis lesions based upon the following criteria: cartilage structural changes (0–6), cartilage cellularity changes (0–3), loss of safranin-O staining within the articular cartilage (0–4), and synovial inflammation and hyperplasia (0–4). 9A4 immunohistochemical staining was graded in reverse of the scale used for safranin-O (on a scale of 0–4); immunostaining was not included in the modified Mankin score but paralleled the loss of staining observed for glycosaminoglycans.
Tetanus toxin challenge
Ten wild-type and 10 P2X7 knockout (KO) mice were immunized with tetanus toxoid (NDC 49281-800-83; Aventis Pasteur, Swiftwater, PA); 0.1 ml was injected i.m. per mouse. On day 14, serum samples were collected by cardiac puncture and the mice were euthanized. These serum samples were tested for the presence of tetanus toxoid-specific IgG by ELISA as follows. Tetanus toxoid (10 µl) was added to each well of 96-well Maxisorp plates (Nalge Nunc International, Naperville, IL) and incubated overnight in Dulbecco’s PBS (D-PBS). The wells subsequently were washed three times with D-PBS containing 0.05% Tween 20. A solution of 5% BSA in D-PBS then was introduced to block remaining reactive sites, the plates were incubated for 2 h, and the wells again were washed three times. The collected serum samples were diluted in D-PBS, and 100-µl aliquots were added to individual wells in triplicate of the tetanus toxin-coated plates. Following a 2-h incubation, the serum-containing samples were removed, the wells were washed, and a solution of peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was added; the plates again were incubated for 2 h and then washed. Turbo TMB (Pierce, Rockford, IL) was added and the reaction was allowed to proceed for 20 min, after which sulfuric acid was added as a stop reagent. Reaction product was assessed with a ThermoMax plate reader (450 nm; Molecular Devices, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte populations reported to express the
P2X7R include monocytes, T cells, and B cells
(41, 42, 43), and ATP addition to human blood is known to
promote physical alterations in specific leukocyte populations,
possibly as a result of a P2X7R-mediated response
(44). To determine whether murine blood leukocytes respond
to extracellular ATP, a laser flow assay was formatted to allow
simultaneous assessment of multiple cell types. When wild-type blood
samples were treated with ATP, a decrease in the total number of
leukocytes was observed (Fig. 1
A). Treatment with LPS alone
did not alter leukocyte numbers, nor did it augment the ATP-dependent
response (Fig. 1A). Blood samples from
P2X7R-deficient animals, in contrast, showed no
decline in leukocyte numbers following ATP treatment (Fig. 1B); pretreatment with LPS did not uncover an ATP response.
To identify cell types that responded to ATP, individual leukocyte
populations were analyzed based on their gating properties.
Approximately 3.5% of total leukocytes associated with blood samples
from wild-type animals displayed gating properties expected of
monocytes. When treated with ATP (with or without prior LPS treatment),
a near complete loss of monocytes was observed in wild-type blood
samples (Fig. 1B). In this type of analysis a decline within
a given population may result from an actual reduction in cell number
(i.e., cell death) or from a change in cell morphology resulting in the
affected cell demonstrating altered gating properties; in either case,
a reduction in the number of cells gating as monocytes indicates that
this population was affected by the extracellular stimulus. Loss of
wild-type monocytes was not observed when ADP or UTP were substituted
for ATP (Fig. 1E). Monocytes associated with blood samples
derived from P2X7R-deficient animals comprised a
similar overall percentage of the leukocyte population (3.5%) as found
in wild-type blood samples. However, following treatment with ATP,
monocyte numbers within the P2X7R-deficient
samples remained constant (Fig. 1B).
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C). In contrast, lymphocytes within blood
samples derived from P2X7R KO animals were not
affected by the ATP stimulus (Fig. 1C).
Thus, like monocytes, lymphocytes from wild-type mice experience a
morphology change and/or die in response to ATP via a
P2X7R-mediated process. In contrast, no change in
the number of neutrophils following ATP challenge was observed in blood
samples from either wild-type or P2X7R-deficient
animals (Fig. 1D). Therefore, neutrophils either lack
surface expression of the P2X7R and/or fail to
respond to extracellular ATP in a detectable manner. Blood-based IL-1 response
Following LPS treatment of human blood, extracellular IL-1 can
be detected but levels of the cell-dissociated cytokine increase
markedly following subsequent treatment with exogenous ATP
(44). To determine whether murine blood samples
demonstrate a similar requirement for a secretory stimulus and to
assess how the absence of the P2X7R affects IL-1
production capacity, blood samples from wild-type and
P2X7R-deficient animals were subjected to LPS/ATP
challenge. These blood samples were stimulated with LPS for 3 h to
allow synthesis of proIL-1 and then treated for an additional 2
h with or without 5 mM ATP. In the presence of LPS only, no significant
IL-1 was detected in plasma harvested from wild-type or KO blood
samples (Fig. 2A). Likewise,
blood samples treated with ATP without prior LPS activation generated
minimal levels of extracellular IL-1 (Fig. 2A). However,
wild-type blood samples yielded large quantities of
plasma-associated IL-1 following serial treatment with LPS and ATP
(Fig. 2A). In contrast, blood samples from
P2X7R-deficient animals yielded no significant
extracellular IL-1 following LPS and/or ATP challenge (Fig. 2A).
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from LPS-activated wild-type
blood (Fig. 2B). However, increasing the ATP concentration
to 0.5 mM generated extracellular cytokine. Increasing the ATP
concentration further to 2 mM led to additional extracellular cytokine,
and higher ATP concentrations produced a comparable response (Fig. 2B). All tested ATP concentrations were unable to promote
IL-1 externalization from LPS-activated blood derived from
P2X7R-deficient animals (Fig. 2B).
Benzoylbenzoyl-ATP, an alternate ligand for the
P2X7R (1), also promoted
externalization of IL-1 from LPS-activated blood samples derived
from wild-type animals (Fig. 2B). Although
benzoylbenzoyl-ATP often acts as a more potent agonist of the
P2X7R than ATP (45), in the
blood-based assay this enhanced potency was not observed. The free
concentration of benzoylbenzoyl-ATP is likely reduced by binding to
serum proteins, and this may affect potency. Importantly, LPS-activated
blood from P2X7R-deficient animals did not
externalize IL-1 in response to benzoylbenzoyl-ATP challenge (Fig. 2B). L-Selectin shedding in response to ATP is absent in P2X7R-deficient animals
Extracellular ATP is known to promote L-selectin shedding from
human lymphocytes (41, 46, 47). To demonstrate that this
process is P2X7R dependent, blood samples from
wild-type and KO animals were treated with ATP, after which they were
analyzed by FACs for leukocyte surface L-selectin expression. In this
analysis, enriched T and B cell populations were distinguished based on
costaining with anti-CD3 and anti-CD45, respectively, whereas
monocytes and neutrophils were identified based on their distinct
forward scatter properties. Monocytes, T cells, B cells, and
neutrophils all stained positive for L-selectin, and no marked
difference was observed in the intensity of staining between matched
cell types derived from wild-type and KO animals (Table I).
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A and Table I). T cells that
responded to ATP and shed L-selectin, for the most part, were
completely devoid of surface L-selectin Ag (evidenced by their low
fluorescence intensity; Fig. 3A and Table I). In contrast,
those T cells that did not respond retained their pre-ATP level of
L-selectin staining. The percentage of T cells recovered from
P2X7R-deficient animals staining positive for
L-selectin was comparable to that seen in wild-type animals (>90%).
However, after ATP treatment there was no significant decrease in the
percentage of L-selectin-positive cells (Fig. 3A and
Table I).
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B and Table I). In
this case, 84% of the CD45+ cells initially
stained positive for L-selectin, and the 15-min treatment with ATP
caused >50% of these cells to shed L-selectin (evidenced by the shift
in fluorescence intensity). However, the affected cells retained some
L-selectin staining, suggesting incomplete cleavage of
surface-associated lectin. Whether complete L-selectin shedding could
be achieved by increasing the length of ATP exposure was not addressed.
CD45+ cells recovered from the
P2X7R-deficient animals showed no significant
L-selectin shedding (Fig. 3B and Table I). Following the
15-min treatment with ATP, blood samples from wild-type animals also
demonstrated a 66% reduction in the number of L-selectin-positive
monocytes, and the responding cells appeared to lose all surface Ag
(Table I). Blood samples from the P2X7R-deficient
animals, in contrast, demonstrated no decrease in monocyte-associated
L-selectin staining following ATP treatment (Table I). Finally,
neutrophils recovered from both wild-type and
P2X7R-deficient animals showed no loss of
L-selectin when challenged with ATP (Table I). Morphology changes associated with P2X7R activation
Prolonged (>15 min) ligation of the P2X7R
is reported to cause cell death and dramatic morphology changes in
monocytes/macrophages (5, 26, 27, 48). To confirm
the role of the P2X7R in mediating these effects,
peritoneal macrophages from wild-type and receptor-deficient animals
were isolated and treated sequentially with LPS and ATP. Wild-type
macrophages treated with LPS were heterogeneous in appearance; many
were firmly attached to the plastic tissue culture dishes, allowing
their nuclei and intracellular architecture to be readily visualized
while others were rounded and nonadherent (Fig. 4). LPS-treated macrophages isolated from
P2X7R-deficient animals demonstrated similar
morphological attributes (Fig. 4). However, following treatment with
ATP, dramatic differences in appearance were observed between the
P2X7R+/+ and
P2X7R-/-
macrophages. After a brief 5-min exposure to ATP no significant changes
were evident in either culture, but following 15 min of treatment many
wild-type macrophages demonstrated loss of cytoplasmic density and
their nuclei became very pronounced (Fig. 4). Increasing the ATP
treatment to 30 min caused the majority of wild-type macrophages to
demonstrate loss of cytoplasmic density and to appear swollen; most of
these cellular corpses detached from the plastic surface. In sharp
contrast, ATP-treated
P2X7R-/-
macrophages retained their normal morphological appearance (Fig. 4).
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-hexosaminidase. An increase in extracellular
-hexosaminidase already was detected after 5 min of ATP treatment in
wild-type cultures, and extracellular levels of this enzyme continued
to increase throughout the initial 45-min treatment period (Fig. 5). At this time, 64% of the total
culture-associated enzyme activity was present in the medium (Fig. 5).
Importantly, cultures of LPS-treated macrophages from
P2X7R-/- animals
showed no time-dependent release of -hexosaminidase to the medium
during a 60-min incubation with ATP (Fig. 5).
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Collagen-induced arthritis in mice is a commonly used inflammatory
disease model. However, the P2X7R deficiency is
established on a mixed genetic background, and such a strain is not
likely to be susceptible to collagen-induced arthritis
(49). Therefore, an alternate method was used to induce an
arthritic type of disease involving injection of animals with a panel
of four different mAbs generated against type II collagen (37, 38). One day later, the Ab-treated mice were injected with LPS
and subsequently assessed for arthritis outcome measures. Gross
assessment of paw swelling and inflammation indicated that the
wild-type animals developed a severe arthritic phenotype within 7 days
of the LPS injection (Fig. 6A). In the course of four
independent experiments, the mean maximal arthritis score in the
wild-type animals achieved a value near 7 (Fig. 6A). The
severity of arthritis gradually declined during an additional 8 days of
observation (Fig. 6A). By comparison, arthritis severity in
the P2X7R-/-
animals was greatly attenuated; the maximum mean score in the
receptor-deficient animals was <3 (Fig. 6A). Overall, the
time course of the disease appeared comparable in the receptor-positive
and -deficient animals (Fig. 6A). In wild-type animals,
nearly 100% of the mice developed disease (Fig. 6B,
Incidence), and >40% of their limbs showed visual signs of
inflammation on day 15 (Fig. 6B, arthritic limbs). In
contrast, 
30% of the receptor-deficient animals developed disease,
and the number of affected limbs in the individual mice was reduced
(Fig. 6B). Viewed in another way, the receptor-deficient
population contained many animals on day 15 with few or no inflamed
limbs, but the majority of the wild-type population contained two or
more affected limbs (Fig. 6C).
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). Whereas all joints in wild-type
animals subjected to the mAb-induced arthritis were affected,
receptor-deficient animals showed a much reduced incidence of disease
(Table II). A modified Mankin score (40) was calculated
based on cartilage structure, cellularity, safranin-O staining of
mucopolysaccharides, and synovial inflammation/hyperplasia. In
addition, to assess collagen degradation, sections were stained with a
mAb (9A4) that specifically recognizes the neoepitope generated by
collagenase digestion (39). Lesions observed in the
articular cartilage of the stifle and hock joints recovered from
wild-type animals included slight to moderate reductions in
glycosaminoglycans (extending to the tidemark of the articular
cartilage), which correlated inversely with increased 9A4
immunohistochemical staining (Fig. 7).
Changes in cartilage cellularity, ranging from slight diffuse increases
in cellularity to multifocal areas of necrosis and loss of
chondrocytes, and structural changes ranging from slight surface
irregularities to clefts in articular cartilage extending to the
tidemark, also were observed. In severely affected joints there was
occasional slight periosteal new bone formation (data not shown). The
average total modified Mankin score (and SD) in all joints from
wild-type animals was calculated to be 10.5 ± 1.7 (Table III). Joints from the
P2X7R-/- animals
displayed an overall reduction in all affected parameters, resulting in
a reduced total modified Mankin score of 5.3 ± 4.7 (Table III).
The mean score (and SD) for 9A4 staining in stifle joints was 2 ±
0 and 0.8 ± 1 for wild-type and
P2X7R-/-
animals, respectively (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
response to ATP challenge (31). The current study extends
our initial findings by showing that blood leukocytes isolated from
wild-type animals, but not P2X7R-deficient
animals, adopt altered morphological and/or viability states, shed
L-selectin, and generate extracellular IL-1 in response to ATP
challenge. Moreover, we demonstrate that the severity of mAb-induced
arthritis is attenuated in the receptor-deficient animals relative to
that achieved in their wild-type counterparts. These findings provide
additional evidence that the P2X7R is an
important regulator of inflammatory cell function, and suggest that
levels of ATP generated endogenously as a result of an inflammatory
response are sufficient to engage this unusual ligand-gated ion
channel. Human blood monocytes and lymphocytes are reported to express cell surface-associated P2X7Rs, and these receptors appear functionally competent as judged by the ability of the cells to alter their shape and/or volume and to accumulate fluorescent tracer molecules following ATP challenge (6, 41, 42, 43, 44). Human neutrophils also are reported to possess the P2X7R, but the majority of their receptors appear to reside intracellularly (41). However, a recent study reported that neutrophils do respond to extracellular ATP via the P2X7R (50). Murine blood leukocytes demonstrate ATP-induced changes in cell size/shape that are consistent with previously reported patterns of P2X7R expression observed in human blood leukocytes. When blood samples from wild-type mice were treated with ATP, both lymphocytes and monocytes responded, as evidenced by the reduction in the numbers of these cells detected by laser flow analysis. Importantly, the ATP response was observed in the absence and presence of LPS prestimulation, indicating that the receptor is present and functional on resting blood-borne leukocytes. Murine blood-borne neutrophils demonstrated no discernable change in volume/shape in response to ATP challenge.
The leukocyte composition of blood samples derived from
P2X7R-deficient mice was not dissimilar to that
found in wild-type animals with respect to the presence of the four
major cell types; however, a detailed analysis of individual T cell
populations has not yet been performed. Despite this similarity in
steady-state leukocyte populations, ATP-challenged
P2X7R-deficient leukocytes demonstrated complete
tolerance to the nucleoside triphosphate; therefore, the observed
decrease in the number of wild-type leukocytes following ATP treatment
is attributed to the presence of the P2X7R.
Whether the decline in leukocyte numbers detected by laser flow
analysis is due to an actual loss of cells or to a shape/volume change
that causes the affected cell to move out of a predetermined gating
window is presently unknown. However, ATP treatment of isolated mouse
lymphocytes is known to cause cell death (51), and
P2X7R-mediated apoptosis is observed in other
cell systems (26, 52). Moreover, analysis of isolated
peritoneal macrophages provided a dramatic example of the ability of
ATP to mediate morphology changes and cell death. Wild-type peritoneal
macrophages, but not those from
P2X7R-/-
animals, responded to extracellular ATP and underwent a rapid
morphological change marked by loss of substratum adherence, clearing
of the cytoplasm, and extensive swelling. This dramatic transition in
cell morphology was complete within 30 min of ATP addition and
coincided temporally with loss of plasma membrane latency as measured
by the release of the lysosomal enzyme -hexosaminidase. Therefore,
ATP-induced macrophage death is accompanied by changes (e.g., swelling
and release of cytosolic elements) not typically associated with an
apoptotic process; this type of cell death demonstrates attributes of
oncosis (53).
In response to LPS, murine blood samples failed to generate significant
levels of extracellular IL-1. However, following a sequential
two-step activation process with LPS and ATP, large quantities of
IL-1 were generated by wild-type, but not
P2X7R-/-, blood
samples. Thus, mouse blood is even more dependent on the presence of a
secretory stimulus such as ATP than is human blood. With the latter,
LPS alone is sufficient to promote synthesis and externalization of
some IL-1, but the subsequent addition of ATP to the LPS-treated
samples increases the amount of externalized cytokine 10- to 30-fold
(44). Murine blood samples, in contrast, generated no
significant extracellular IL-1 in the absence of the ATP stimulus.
Like the human system, ATP treatment without prior LPS exposure
generated no cell-dissociated IL-1; LPS is needed to initiate
proIL-1 synthesis. LPS-activated blood samples derived from
P2X7R-/- animals
generated no cell-dissociated IL-1 in the presence of ATP,
confirming that the export process is dependent on the
P2X7R. We previously demonstrated that peritoneal
macrophages isolated from wild-type and
P2X7R-deficient animals respond to LPS to
generate similar quantities of cell-associated proIL-1. However, in
response to exogenous ATP challenge, LPS-treated macrophages from
wild-type animals, but not from receptor-deficient animals, released
mature IL-1 to the medium (31). Therefore, even within
the context of a complex mixture of blood proteins and blood cells,
efficient generation of cell-dissociated IL-1 requires a secretory
stimulus, and ATP working via the P2X7R can serve
in this capacity.
Shedding of L-selectin from the surface of ATP-treated human lymphocytes is reported to be a P2X7R-dependent response based on the required ATP concentrations, the ability of benzoylbenzoyl-ATP to substitute as the agonist, and the effect of receptor antagonists (46). Our findings demonstrating that ATP-induced L-selectin shedding is absent in leukocytes isolated from P2X7R-deficient animals confirms that the P2X7R is a necessary component of this response. Within the CD3+ T cell population, the majority of cells expressed L-selectin and responded to a 15-min treatment of ATP by shedding essentially all of their surface Ag. The majority of CD45+ cells also expressed L-selectin. However, following a 15-min treatment with ATP, a subset of these cells (32%) retained their full complement of L-selectin and those that did shed Ag did so incompletely. Therefore, the B cell-enriched fraction appears to be less responsive than T cells to ATP-induced L-selectin shedding. Comparison of matched leukocyte populations isolated from wild-type and receptor-deficient animals showed no significant differences in the resting levels of L-selectin surface expression, suggesting that ATP-induced L-selectin shedding does not take place during the normal circulation of leukocytes.
ATP-induced responses demonstrated by wild-type leukocytes (i.e., cell
activation/death, IL-1 production, and L-selectin shedding) are
expected to impact the outcome of an inflammatory response. Indeed, in
response to mAb-induced arthritis,
P2X7R-deficient animals demonstrated reduced
susceptibility and severity of disease relative to their wild-type
counterparts. This resistance manifested as less swelling and redness
of affected joints and less destruction of cartilage. In wild-type
animals, the mAb stimulus led to a phenotype not dissimilar to that
observed in collagen-induced arthritis in terms of both quantitative
and qualitative changes (49, 54). This latter model is
dependent on IL-1, and inhibitors of the production and/or activity of
this cytokine suppress disease outcomes (55, 56). At the
histological level, mAb-induced arthritis led to a disruption of the
normal cartilage architecture, loss of proteoglycan content, synovial
inflammation, and the appearance of collagen cleavage products as
detected with the mAb 9A4 (39). The time required to
achieve disease in the monoclonal arthritis model is reduced relative
to that required in the collagen-induced model, but the severity of the
disease appears comparable in the two formats. All assessed
histological changes were less severe in the
P2X7R-deficient animals relative to those
observed in matched wild-type controls. This suggests that the absence
of the P2X7R leads to a general suppression of
the inflammatory response, perhaps reflecting a diminished output of
inflammatory mediators such as IL-1. Importantly, wild-type and
receptor-deficient animals were equally competent in their response to
tetanus-toxoid Ag; this type of an immune response requires Ag
presentation, Th cell function, and B cell Ab production. Thus,
P2X7R-deficient animals are not generally
immunocompromised.
To date, no activity has been reported for the P2X7R that does not involve ligand-induced activation of the channel/pore. Therefore, demonstration that absence of the P2X7R alters disease outcome in an in vivo model of inflammation suggests that this receptor encounters sufficient levels of an endogenous ligand to promote its activation in wild-type animals. This is an important finding, as previous in vivo studies investigating this receptor have used exogenous ATP as the activating ligand (31). In the mAb-induced arthritis model no exogenous ATP was introduced, so receptor activation must result from the presence of an appropriate endogenous ligand. Whether ATP is released to local environments where its concentration can achieve levels sufficient to activate the receptor, or whether other endogenous effectors can lower ATP concentration requirements remains to be established.
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Footnotes |
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2 Abbreviations used in this paper: P2X7R, P2X7 receptor; KO, knockout; HiFAZ, heat-inactivated fetal serum with azide.
Received for publication February 5, 2002. Accepted for publication April 10, 2002.
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 H. Z. Ke, H. Qi, A. F. Weidema, Q. Zhang, N. Panupinthu, D. T. Crawford, W. A. Grasser, V. M. Paralkar, M. Li, L. P. Audoly, et al. Deletion of the P2X7 Nucleotide Receptor Reveals Its Regulatory Roles in Bone Formation and Resorption Mol. Endocrinol., July 1, 2003; 17(7): 1356 - 1367. [Abstract] [Full Text] [PDF]
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S. M. Joseph, M. R. Buchakjian, and G. R. Dubyak Colocalization of ATP Release Sites and Ecto-ATPase Activity at the Extracellular Surface of Human Astrocytes J. Biol. Chem., June 20, 2003; 278(26): 23331 - 23342. [Abstract] [Full Text] [PDF]
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P. A. Verhoef, M. Estacion, W. Schilling, and G. R. Dubyak P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1{beta} Release J. Immunol., June 1, 2003; 170(11): 5728 - 5738. [Abstract] [Full Text] [PDF]
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R. E. Laliberte, D. G. Perregaux, L. R. Hoth, P. J. Rosner, C. K. Jordan, K. M. Peese, James. F. Eggler, M. A. Dombroski, K. F. Geoghegan, and C. A. Gabel Glutathione S-Transferase Omega 1-1 Is a Target of Cytokine Release Inhibitory Drugs and May Be Responsible for Their Effect on Interleukin-1beta Posttranslational Processing J. Biol. Chem., May 2, 2003; 278(19): 16567 - 16578. [Abstract] [Full Text] [PDF]
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J. S. Wiley, L.-P. Dao-Ung, C. Li, A. N. Shemon, B. J. Gu, M. L. Smart, S. J. Fuller, J. A. Barden, S. Petrou, and R. Sluyter An Ile-568 to Asn Polymorphism Prevents Normal Trafficking and Function of the Human P2X7 Receptor J. Biol. Chem., May 2, 2003; 278(19): 17108 - 17113. [Abstract] [Full Text] [PDF]
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G. R. Dubyak Knock-Out Mice Reveal Tissue-Specific Roles of P2Y Receptor Subtypes in Different Epithelia Mol. Pharmacol., April 1, 2003; 63(4): 773 - 776. [Full Text] [PDF]
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S. Duan, C. M. Anderson, E. C. Keung, Y. Chen, Y. Chen, and R. A. Swanson P2X7 Receptor-Mediated Release of Excitatory Amino Acids from Astrocytes J. Neurosci., February 15, 2003; 23(4): 1320 - 1328. [Abstract] [Full Text] [PDF]
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R. Sluyter and J. S. Wiley Extracellular adenosine 5'-triphosphate induces a loss of CD23 from human dendritic cells via activation of P2X7 receptors Int. Immunol., December 1, 2002; 14(12): 1415 - 1421. [Abstract] [Full Text] [PDF]
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S. Adriouch, C. Dox, V. Welge, M. Seman, F. Koch-Nolte, and F. Haag Cutting Edge: A Natural P451L Mutation in the Cytoplasmic Domain Impairs the Function of the Mouse P2X7 Receptor J. Immunol., October 15, 2002; 169(8): 4108 - 4112. [Abstract] [Full Text] [PDF]
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