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Department of Respiratory, Allergy, Immunology, Inflammation, and Infectious Diseases, Pfizer Central Research, Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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levels
decreased. Among leukocytes, monocytes appeared to be the most affected
cellular targets of the ATP stimulus. These studies indicate that an
exogenous stimulus is required by blood for the efficient production of
IL-1ß and IL-18, and suggest that circulating blood monocytes
constitutively express a P2X7-like
receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activity attributed to IL-1 actually is derived from two separate but
related polypeptides, IL-1 and IL-1ß. Both of these cytokines are
produced as 31-kDa propolypeptides (8, 9). ProIL-1 is
fully competent to bind to IL-1 receptors on target cells and to elicit
a biologic response (10), but this polypeptide may be
cleaved to an equally active 17-kDa mature species by a calpain-type
protease (11). In contrast, proIL-1ß is incompetent as
an agonist of IL-1 receptors (11). Cleavage of this
propolypeptide to a 17-kDa species by caspase-1 is required to generate
an active signaling molecule (12, 13). Caspase-1 also
functions in the activation of the proinflammatory cytokine IL-18
(14). ProIL-1, proIL-1ß, and proIL-18 all are
synthesized without signal peptides (9, 15); these
signature peptides normally direct a polypeptide that is destined to be
secreted to the secretory apparatus of the cell (16). In
the absence of the signal peptide, the procytokines accumulate within
the cytosolic compartment of activated monocytes/macrophages
(17). Release of IL-1 to the medium from
monocytes/macrophages that are actively engaged in cytokine synthesis
is an inefficient process (18, 19, 20, 21). However, the
cell-associated molecules can be proteolytically processed and
externalized rapidly when the producing cells encounter an
extracellular initiator of post-translational processing such as ATP,
cytolytic T-cells, bacterial toxins, and potassium ionophores
(18, 20, 21, 22, 23). These agents ultimately compromise the
integrity of the cytokine-producing cell’s plasma membrane, suggesting
that cellular necrosis and/or apoptosis may accompany IL-1 release
(18, 20). However, not all agents that promote cell death
cause proteolytic processing of proIL-1ß (20), and the
possibility exists that IL-1 release in vivo may be achieved in the
absence of overt cell lysis.
ATP acts as an agonist of IL-1 post-translational processing by binding to the P2X7 receptor (24, 25, 26). Like other members of the P2X family of ligand-gated ion channels, the P2X7 receptor forms a nonselective channel in the presence of ATP (27, 28, 29, 30). However, unlike other members of the family, the channel formed by the P2X7 receptor rapidly transitions to a pore-like structure that allows passage of molecules as large as 900 Da (29, 31, 32). Prolonged ligation of the P2X7 receptor (>15 min) commits the cell to death (33, 34). By activity, the P2X7 receptor (formerly called P2Z) is restricted to a limited number of cell types including monocytes, macrophages, microglial cells, and some lymphocytes and cancer cells (33, 35). The physiological function of this unusual receptor remains unclear.
Studies that have demonstrated the need for a secondary stimulus to promote efficient IL-1 post-translational processing have employed purified preparations of LPS-activated monocytes, macrophages, and microglial cells (18, 24, 25, 34, 36). One could argue that the isolated cells lacked important extracellular components and/or growth factors that were necessary for efficient cytokine processing, and that the requirement for a secretion stimulus (in addition to LPS) was an artifact of the experimental system. However, an in vivo study demonstrated that LPS-activated murine peritoneal macrophages also require a secretion stimulus to achieve maximum IL-1 production capacity (37). To further address this issue, the current study investigates production of IL-1ß by human blood. A cohort of 12 individuals was bled twice daily for 4 consecutive days, after which the blood samples were analyzed in a two-step cytokine release assay. Results indicate that LPS-activated human blood samples generate greater levels of IL-1ß in the presence of a secretory stimulus such as ATP or nigericin. The magnitude of the increase was consistent over the course of analysis. The ATP stimulus also increased extracellular levels of IL-18, and caused a high percentage of monocytes to demonstrate altered physical properties. These changes in monocytes did not require pretreatment with LPS, suggesting that a high percentage of these cells constitutively express a P2X7-like receptor. Therefore, efficient release of IL-1 from LPS-activated human blood is dependent on extracellular effectors such as ATP. The need for separate stimuli to promote synthesis and release of IL-1ß punctuates the importance served by post-translational processing in the regulation of this cytokine’s activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood was collected from normal volunteers and RA patients in heparin-containing vacutainer tubes; these samples could be stored on ice for up to 4 h with no adverse effect on assay performance. A total of 75 µl of blood was placed into an individual well of a 96-well plate and diluted with 75 µl RPMI 1640 medium containing 20 mM HEPES (pH 7.3). The diluted blood samples then were incubated for 2 h in the absence or presence of 100–200 ng/ml LPS (Escherichia coli serotype 055:B5; Sigma, St. Louis, MO) at 37°C in a 5% CO2 environment. After this incubation, ATP was introduced as a secretion stimulus (by addition of the appropriate volume of a solution of 100 mM ATP in 20 mM HEPES (pH 7)) where indicated, and the mixtures were incubated at 37°C for an additional 2 h. The 96-well plates then were centrifuged at 700 x g for 10 min, and the resulting plasma samples were harvested; these samples were stored at -20°C. In some experiments, ADP, UTP, or GTP was substituted for ATP as the secretion stimulus (all were obtained from Sigma). When nigericin was employed as the secretion stimulus, this potassium ionophore (Sigma) was prepared as a 10-mM stock solution in ethanol. Each condition was assayed in a minimum of triplicate wells. None of the agonists or antagonists appeared to cause lysis of RBC as evidenced by the absence of visible hemoglobin within the plasma supernatants.
ELISA measurements
Plasma supernantants were analyzed in the following ELISAs:
IL-1ß (R&D Systems, Minneapolis, MN), IL-18 (Medical and Biological
Laboratories, Nagoya, Japan), and TNF- (R&D Systems). The assays
were performed following the manufacturer’s specifications, and
absolute cytokine levels were calculated based on comparison to assay
performance in the presence of known quantities of recombinant cytokine
standards.
Laser flow analysis
Blood was collected in heparin-containing vacutainer tubes. A total of 0.25 ml of each sample was diluted with 0.25 ml of RPMI 1640 medium containing 20 mM HEPES (pH 7.3) in a 1.5 ml Eppendorf tube (Eppendorf Scientific, Westbury, NY). The diluted samples were incubated for 2 h in the absence or presence of 100 ng/ml LPS at 37°C. ATP then was introduced (by addition of 0.03 ml of a 100 mM stock solution) where indicated, and the samples were incubated for an additional 2 h at 37°C. These incubation mixtures then were analyzed on a Cell Dyne (Abbott Laboratories, Abbott Park, IL; model 3500) for leukocyte content.
FACS analysis
Blood samples (collected in heparin) were diluted with an equal volume of RPMI 1640 medium containing 20 mM HEPES (pH 7.3) in polypropylene tubes. Where indicated, ATP was added to achieve a final concentration of 6 mM, and the samples were incubated at 37°C for 2 h. The 0.1 ml blood samples then were centrifuged, the plasma supernatants were discarded, and the cells were suspended in 1 ml of PBS and again were subjected to centrifugation. The resulting cell pellets were suspended in 10 µl of heat-aggregated human IgG and incubated at 4°C for 10 min, after which 10 µl FITC-conjugated anti-CD33 and 10 µl PE-conjugated anti-CD14 (both mAbs were obtained from PharMingen, San Diego, CA) were added and the samples were incubated for an additional 30 min at 4°C. At this point, 1 ml of PBS containing 2% FCS and 0.2% sodium azide (PBS+) was added to each sample and the cells were collected by centrifugation. These cell pellets were suspended in 1 ml of FACS lysing solution (Becton Dickinson, Franklin Lakes, NJ) and the samples were incubated for 10 min at room temperature to lyse RBC. Intact leukocytes then were collected by centrifugation, washed three times with PBS+ by repeated centrifugation, and resuspended in 0.5 ml of PBS+. These samples were analyzed by FACScan; a total of 20,000 ungated events were collected per analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly isolated human monocytes maintained in culture
produce large quantities of proIL-1ß in response to LPS activation,
but release little of the cytokine product to the medium as the mature
polypeptide species (21, 24, 26, 38, 39). However, the
subsequent addition of ATP to the LPS-activated cells causes a high
percentage of the cytokine to be externalized as the mature polypeptide
species (21, 24, 26, 39). Monocytes are considered to be
the major cell-type in blood responsible for IL-1 production
(40). To determine whether the blood environment alters
cytokine production requirements, individual blood samples from 12
normal volunteers were stimulated with LPS for 2 h and then
treated with or without ATP for an additional 2 h. IL-1ß
released to the medium subsequently was determined by ELISA. In the
absence of LPS and ATP, no significant IL-1 was produced by any of the
blood samples (Fig. 1
). Addition of LPS
in the absence of ATP caused production of measurable quantities of
IL-1 in 10 of the 12 blood samples (Fig. 1). Addition of ATP to the
LPS-activated samples greatly enhanced the quantity of IL-1 generated
by all 12 blood samples (Fig. 1). The relative increase in cytokine
levels produced by the LPS-treated samples in the presence and absence
of ATP, referred to as the stimulus-induced secretion (SIS) ratio,
ranged from a low of 3.9 for subject III to an incalculable value for
subjects X and XI (Fig. 1). In the absence of prior LPS stimulation,
ATP did not cause production of IL-1 by blood samples (see below).
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). LPS-activated blood
treated with concentrations of ATP 
0.5 mM produced quantities of IL-1
comparable to those generated in the absence of the nucleotide
triphosphate (Fig. 2). In contrast, concentrations of ATP 
1 mM
increased levels of extracellular cytokine well above those observed in
the absence of the nucleotide triphosphate; maximum production was
observed at 6 mM ATP, the highest concentration tested (Fig. 2). In
contrast to ATP, the nucleotide triphosphates UTP and GTP and the
nucleotide diphosphate ADP were inactive as secretion stimuli (Fig. 2
inset).
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). Addition of nigericin to
the LPS-activated samples stimulated cytokine release; a final
concentration of 100 µM nigericin appeared to be optimal (Fig. 3). Quantities of IL-1ß produced in the presence of nigericin were
comparable to those produced by 6 mM ATP.
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Blood IL-1 production is constant and not subject to diurnal variance
To determine whether the observed characteristics of the IL-1ß
production assay were consistently demonstrated, the same 12 subjects
who donated blood for the assay shown in Fig. 1
were bled twice daily
for 4 consecutive days. After each bleed, the blood samples were
subjected to the two-step IL-1 release assay. Although differences were
observed in the total quantity of IL-1ß released in response to the
+LPS/+ATP combination between individual subjects, the quantities
produced by any one individual were remarkably consistent throughout
the 78-h time frame encompassed by the eight assays (Table I and Fig. 4A). For example, subjects XII
and VIII generated higher amounts of IL-1ß in response to LPS/ATP on
average than did the other 10 subjects. Total IL-1ß produced over the
4-day period for these two individuals ranged from a low of 13 ng/ml to
a high of 19 ng/ml (Table I). In contrast, subjects II and V
consistently generated less IL-1ß; in response to LPS/ATP, blood
samples from these subjects generated cytokine levels ranging between
5.8 and 8.9 ng/ml (Table I).
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B); the mean and SD over the eight bleeds for this
individual were 5.5 ± 1.2. The SIS response ratio for subject VI
was intermediate to that observed for subjects II and III, and ranged
between 6.8 and 16 (Table I and Fig. 4B); the mean and SD
over the eight bleeds were 11 ± 3.9. Therefore, quantities of
IL-1 released in response to the LPS-only and the LPS/ATP stimuli are
consistent traits of an individual’s blood. Blood samples from RA patients also require a secretion stimulus for efficient IL-1ß release
Literature reports suggest that blood-borne
cells obtained from RA patients may be in an activated state and
circulate with IL-1 on board (41, 42). To explore the
possibility that blood samples obtained from RA patients may perform
differently in the IL-1 production assay, several samples of blood from
affected individuals were analyzed; no consideration of current therapy
was taken into account in the selection of these individuals.
Preliminary experiments indicated that blood samples derived from
normal volunteers could sit on ice for up to 4 h with no change in
their cytokine production properties (data not shown). RA patient blood
samples obtained at a local rheumatologist’s office were transported
to the lab on ice and then subjected to the two-step IL-1ß production
assay. In the absence of LPS stimulation, no significant IL-1ß was
detected in three separate samples (Table II); this was true for blood samples
treated with (-LPS/+ATP) or without (-LPS/-ATP) ATP. Addition of LPS
(+LPS/-ATP) led to generation of IL-1ß by all three blood samples;
IL-1ß levels ranged between 0.023 to 0.2 ng/ml (Table II). Treatment
of the same blood samples with the combination of LPS and ATP
(+LPS/+ATP) generated quantities of IL-1ß ranging from 1.7 to 10.4
ng/ml (Table II). The lower quantities of IL-1ß produced by RA
patient A correlated with a reduced number of blood monocytes (Table II). Therefore, performance of RA patient blood samples in the IL-1ß
production assay is similar to that observed with samples obtained from
normal healthy subjects.
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IL-18, like IL-1ß, is produced as a leaderless propolypeptide
that requires cleavage by caspase-1 to generate an active mature
cytokine (14). To determine whether ATP enhanced IL-18
production, blood samples were subjected to the two-step activation
protocol and cytokine levels were assessed by ELISA. IL-1ß production
by these blood samples behaved as expected. In the absence of an
activator (-LPS/-ATP) no IL-1ß was detected, and treatment with ATP
alone (-LPS/+ATP) did not enhance cytokine production (Table III). In contrast, blood samples treated
with LPS individually (+LPS/-ATP) or in combination with ATP
(+LPS/+ATP) generated 1.9 and 16.1 ng/ml, respectively, of IL-1 (Table III). In these same blood samples, IL-18 production demonstrated a
different response pattern. In the absence of a stimulus (-LPS/-ATP),
0.04 ng/ml IL-18 were detected. Treatment with LPS only (+LPS/-ATP)
did not increase levels of IL-18 significantly, but samples treated
with ATP only (-LPS/+ATP) yielded 0.29 ng/ml, or a 7-fold increase
above the inactivated comparator. The combination of LPS and ATP
(+LPS/+ATP) promoted a slightly higher yield of IL-18 corresponding to
0.38 ng/ml (Table III).
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is secreted from cells via a
process involving the normal cellular secretory apparatus consisting of
the endoplasmic reticulum and Golgi apparatus (43). To
assess whether this type of secretory product was affected by ATP, the
same blood samples used above were analyzed for their content of
TNF-. As expected, TNF- was produced in samples stimulated with
LPS but not in samples stimulated with ATP only (Table III). The
combination of LPS and ATP decreased TNF- production relative to
blood samples stimulated with LPS only (Table III). ATP alters specific leukocyte populations
ATP binding to the P2X7 receptor can promote
cell volume changes and death if the receptor remains ligated for >15
min (33, 34). To identify leukocyte populations responsive
to ATP, blood samples from three separate donors were subjected to the
two-step IL-1ß production assay after which they were characterized
by Cell Dyne laser flow analysis; this instrument distinguishes
individual leukocytes based on their size and granularity
(44). For donor no. 1, the blood sample treated with ATP
(-LPS/+ATP) yielded 12% fewer total leukocytes than the sample
exposed to no stimulus (-LPS/-ATP; Table IV). Neutrophil and eosinophil numbers
were essentially unchanged between these two blood samples, but
lymphocyte and monocyte numbers decreased by 39 and 93%, respectively
(Table IV). Basophil numbers also decreased after ATP treatment;
however, based on their limited number it is not clear that this change
reflects an actual decline in the number of basophils or of a
contaminating cell-type. Treatment with LPS alone (+LPS/-ATP) reduced
total leukocyte counts by 10% vs the untreated blood sample, with a
reduction in the number of monocytes accounting for the bulk of this
change; loss of monocytes may occur as a result of the activated cells
adhering to the plastic culture tubes and/or changing shape such that
they no longer appear in the gated window. Relative to the LPS-only
treatment, blood treated with the combination of LPS and ATP
(+LPS/+ATP) contained a reduced number of total leukocytes, and this
loss again reflected reductions in the monocyte and lymphocyte
populations (Table IV). Similar trends were observed with blood
obtained from two other donors (Table IV). ATP treatment of each of the
three blood samples, either alone or in combination with LPS, reduced
lymphocyte and monocyte numbers without significantly affecting the
numbers of neutrophils and eosinophils (Table IV). This effect of ATP
was not shared with UTP. Addition of 6 mM ATP reduced the percentage of
monocytes and lymphocytes within non-LPS treated blood samples (Table V). In contrast, the same concentration
of UTP produced no change in leukocyte composition (Table V).
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). In contrast, the
percentage of leukocytes that stained positive for CD-33 following ATP
treatment was reduced to 2.9%, and the positive cells were distributed
between two distinct populations. Some of the CD-33-positive cells
persisted within the original monocyte position, but a new population
possessing reduced forward and side scatter signals was apparent within
the ATP treated samples (Fig. 5). Thus, a volume and/or shape change
occurs to a high percentage of monocytes following ATP treatment,
resulting in their failure to gate in the expected region of monocytes.
In the same experiment, monocytes were also traced using PE-labeled
anti-CD14. Following ATP treatment, a lower percentage of
CD-14-positive cells was detected, and the fluorescence intensity of
the positive cells was reduced. This reduction in fluorescence
intensity suggests that the CD-14 Ag is cleaved and released post-ATP
activation. It should be noted that blood samples subjected to the FACS
analysis were not pretreated with LPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of human blood with LPS caused IL-1 immunoreactivity to be
released extracellularly. However, like the behavior of isolated human
monocytes, the quantity of IL-1 released in response to LPS represented
only a fraction of the total cytokine generated in response to the
inflammatory stimulus. The subsequent addition of an exogenous
secretion stimulus consisting of ATP or nigericin increased
extracellular IL-1ß levels dramatically. Similarly, ATP treatment
enhanced release of IL-18 from blood, but the nucleotide triphosphate
did not elevate extracellular levels of TNF-. Synthesis of IL-18 was
not as dependent on LPS as was IL-1ß. This observation is consistent
with findings of a previous study that found IL-18 to be constitutively
produced by peripheral blood monocytes, whereas IL-1ß was synthesized
only after LPS activation (50). Despite differences in
their expression requirements, both cytokine products require a
secretion stimulus such as ATP to achieve efficient release. ATP
promotes release of mature IL-1ß from monocytes and macrophages
(18, 20, 24, 26, 39), and we assume that the cytokine
products released in the blood assay also correspond to mature
polypeptides; the ELISA kit employed recognizes mature IL-1ß and, to
a lesser degree, the prospecies (51). In contrast to its
ability to enhance production of IL-1ß and IL-18, ATP decreased
production of TNF-; death of the cytokine producing monocytes may
account for this reduction.
A number of studies with isolated human monocytes provide compelling
evidence that ligation of the P2X7 receptor can
activate proIL-1ß post-translational processing
(24, 25, 26), and a human monocyte cDNA library was employed
as a source for cloning the human receptor (28). Agonist
requirements of the blood response are consistent with a
P2X7 receptor involvement. ATP, but not UTP, GTP,
or ADP, stimulated IL-1 release, and the concentrations of ATP required
to promote the response (1 mM) are in line with requirements of the
P2X7 receptor (28, 29). The
effectiveness of ATP is somewhat surprising based on conclusions of
several previous studies noting that only a small percentage of
isolated blood monocytes express P2X7 receptor
activity (52, 53). These studies demonstrated an inability
of ATP to induce accumulation of a fluorescent dye (52) or
to promote sustained depolarization (53) of freshly
isolated monocytes. However, following in vitro differentiation of the
monocytes to macrophages a higher percentage of cells demonstrated
these P2X7 receptor-associated activities
(52, 53). In contrast, our findings demonstrate that ATP
is an effective stimulus of IL-1 release in blood and this effect
appears to be a monocyte P2X7-mediated response.
To explain this discrepancy, we considered the possibility that only a
small percentage of monocytes actually expressed the
P2X7 receptor, and this subpopulation responded
to ATP and released its cytokine load. This possibility seems unlikely
because the total quantities of IL-1 produced in response to nigericin
and ATP were comparable, and nigericin is expected to act independently
of the P2X7 receptor. Moreover, a high percentage
of monocytes (>90%) in blood samples treated with ATP responded and
changed their light scatter properties, suggesting that a majority of
blood-borne monocytes possess the P2X7 receptor.
The blood-borne monocyte responded to ATP even without prior LPS
activation, suggesting that unstimulated monocytes express the ATP
receptor. Differences in protocols between the current study and the
two previous studies may account for the dissimilar findings. In the
IL-1 production assay, blood cells are treated with ATP for longer
times than employed in the earlier studies. In addition, plasma
components were present in the blood-based IL-1 assay and these
components were not present in the previous cultured monocyte
experiments. External factors are known to influence
P2X7 receptor activity; for example, THP-1 cells
do not constitutively express the P2X7 receptor,
but after treatment with a combination of LPS and IFN-
, both
P2X7 receptor message and functional protein are
expressed (54).
Alternatively, perhaps the monocyte P2X7 receptor demonstrates only a subset of activities that have been attributed to this receptor in other cell types. For example, BW5147 mouse lymphoma cells express the message for the P2X7 receptor and respond to high ATP concentrations by activating a calcium conductance (55); this conductance was inhibited by KN-62, an antagonist of the P2X7 receptor. However, BW5147 cells do not accumulate ethidium bromide in response to ATP, suggesting that their receptor does not form a pore-like structure. Interestingly, a recent report presented evidence suggesting that the P2X7 receptor and the pore represent distinct functional units (56). If true, then perhaps monocytes lack the essential pore component and/or the P2X7 receptor fails to recruit it appropriately.
Laser flow analysis of ATP-treated blood samples indicated that some lymphocytes responded to the nucleotide triphosphate in addition to monocytes; the most abundant white cell population, neutrophils, in contrast, showed no response to the ATP stimulus. To our knowledge, neutrophils and eosinophils have not been reported to express this receptor. At present, it is not clear whether the responding lymphocytes represent a previously characterized subset of these cells. Isolated murine (57) and human (58, 59, 60) lymphocytes are reported to demonstrate P2X7 receptor activity, but properties of the lymphocyte-associated pore appear distinct from those typically attributed to the P2X7 receptor (61). Additional work is needed to characterize the T cell response. Therefore, leukocyte populations that we observed to respond in ATP-treated blood samples correspond to cell types known from in vitro studies to express P2X7 receptor activity.
The total amount of IL-1ß produced by an individual donor’s blood in response to the combination of LPS and ATP was consistent throughout eight different assays over a 4-day period. This indicates a lack of a significant diurnal variation in cytokine production capacity. Surprisingly, a significant donor dependence was observed with respect to the SIS ratio. This ratio reflects the amount of IL-1 released in response to the combination of LPS/ATP relative to amount released with LPS only. Over the 4-day period, blood cells from each individual released a remarkably consistent SIS ratio. This consistency suggests that the inherent ability of a monocyte to release IL-1 in response to LPS is influenced by genetic factors and/or physiological state. Both the total amount of IL-1ß released in response to LPS and ATP and the SIS ratio appear to be gender-independent traits. Peripheral blood mononuclear cell preparations isolated from men and women previously were shown to differ with respect to the basal unstimulated secretion of IL-1ß, but not with respect to the LPS-inducible component (62). The efficiency at which purified LPS-activated monocytes release IL-1ß also is known to be affected by the absolute LPS concentration (63); in the current study, a limited range of LPS concentrations was employed (100–200 ng/ml), and we have not investigated whether changes in LPS concentration affect the SIS ratio.
Blood samples obtained from RA patients behaved comparably to samples obtained from normal volunteers with respect to the two step IL-1ß production assay. These samples required both LPS and ATP for maximal production of cell-free IL-1ß. In the presence of only ATP, quantities of IL-1ß detected extracellularly were minimal and not different from untreated blood samples. Previous studies suggested that circulating blood cells in RA patients are in an activated state and express cell-associated IL-1 (41, 42). Because ATP alone did not promote release of IL-1 from the RA patient blood samples, we conclude that the level of cell-associated cytokine was minimal relative to their LPS-induced production capacity, or that the cells producing IL-1 did not express the P2X7 receptor.
Demonstration that blood-borne monocytes require an exogenous stimulus to efficiently generate cell-dissociated IL-1ß and IL-18 provides additional evidence that the process of stimulus-induced secretion is important and physiologically relevant. The requirement for a separate stimulus to promote post-translational processing may provide a final checkpoint to ensure that a monocyte and/or macrophage does not inadvertently release multipotential inflammatory mediators such as IL-1 and IL-18 in the absence of a genuine need. Identification of this post-translational processing checkpoint offers the possibility of identifying novel therapeutic strategies aimed at controlling inflammatory mediators whose export is dependent on this unusual cellular process.
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Footnotes |
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2 Abbreviations used in this paper: RA, rheumatoid arthritis; SIS, stimulus-induced secretion.
Received for publication May 17, 2000. Accepted for publication July 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in rheumatoid arthritis. Arthritis Rheum. 38:151.[Medline]
precursor but not the interleukin-1ß precursor. J. Biol. Chem. 262:2941. precursor. J. Biol. Chem. 266:12162. inducing factor mediated by interleukin-1ß converting enzyme. Science 275:206.-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J. Immunol. 156:4274.[Abstract]
and IL-1ß occurs via a similar mechanism. J. Immunol. 160:2469. and interleukin 1ß detected with three-color immunofluorescence technique. Histochem. Cell Biol. 106:593.[Medline]
staining circulate in the blood of patients with rheumatoid arthritis. Clin. Exp. Immunol. 80:25.[Medline]
in the human THP-1 monocytic cell line. J. Immunol. 157:5627.[Abstract]
, IL-1ß, and IL-1 receptor antagonist secretion from mononuclear cells and uninary excretion. J. Immunol. 153:300.[Abstract]
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 L. Raffaghello, P. Chiozzi, S. Falzoni, F. Di Virgilio, and V. Pistoia The P2X7 Receptor Sustains the Growth of Human Neuroblastoma Cells through a Substance P-Dependent Mechanism Cancer Res., January 15, 2006; 66(2): 907 - 914. [Abstract] [Full Text] [PDF]
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J. M. Kahlenberg, K. C. Lundberg, S. B. Kertesy, Y. Qu, and G. R. Dubyak Potentiation of Caspase-1 Activation by the P2X7 Receptor Is Dependent on TLR Signals and Requires NF-{kappa}B-Driven Protein Synthesis J. Immunol., December 1, 2005; 175(11): 7611 - 7622. [Abstract] [Full Text] [PDF]
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E. Bulanova, V. Budagian, Z. Orinska, M. Hein, F. Petersen, L. Thon, D. Adam, and S. Bulfone-Paus Extracellular ATP Induces Cytokine Expression and Apoptosis through P2X7 Receptor in Murine Mast Cells J. Immunol., April 1, 2005; 174(7): 3880 - 3890. [Abstract] [Full Text] [PDF]
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L. C. Denlinger, G. Angelini, K. Schell, D. N. Green, A. G. Guadarrama, U. Prabhu, D. B. Coursin, P. J. Bertics, and K. Hogan Detection of Human P2X7 Nucleotide Receptor Polymorphisms by a Novel Monocyte Pore Assay Predictive of Alterations in Lipopolysaccharide-Induced Cytokine Production J. Immunol., April 1, 2005; 174(7): 4424 - 4431. [Abstract] [Full Text] [PDF]
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 J. M. Kahlenberg and G. R. Dubyak Differing caspase-1 activation states in monocyte versus macrophage models of IL-1{beta} processing and release J. Leukoc. Biol., September 1, 2004; 76(3): 676 - 684. [Abstract] [Full Text] [PDF]
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H. L. Wilson, S. E. Francis, S. K. Dower, and D. C. Crossman Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation J. Immunol., July 15, 2004; 173(2): 1202 - 1208. [Abstract] [Full Text] [PDF]
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 C. Andrei, P. Margiocco, A. Poggi, L. V. Lotti, M. R. Torrisi, and A. Rubartelli From The Cover: Phospholipases C and A2 control lysosome-mediated IL-1{beta} secretion: Implications for inflammatory processes PNAS, June 29, 2004; 101(26): 9745 - 9750. [Abstract] [Full Text] [PDF]
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 M. Aga, J. J. Watters, Z. A. Pfeiffer, G. J. Wiepz, J. A. Sommer, and P. J. Bertics Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-{kappa}B signaling pathways in murine RAW 264.7 macrophages Am J Physiol Cell Physiol, April 1, 2004; 286(4): C923 - C930. [Abstract] [Full Text] [PDF]
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R. Sluyter, A. N. Shemon, and J. S. Wiley Glu496 to Ala Polymorphism in the P2X7 Receptor Impairs ATP-Induced IL-1{beta} Release from Human Monocytes J. Immunol., March 15, 2004; 172(6): 3399 - 3405. [Abstract] [Full Text] [PDF]
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A. Colomar, V. Marty, C. Medina, C. Combe, P. Parnet, and T. Amedee Maturation and Release of Interleukin-1{beta} by Lipopolysaccharide-primed Mouse Schwann Cells Require the Stimulation of P2X7 Receptors J. Biol. Chem., August 15, 2003; 278(33): 30732 - 30740. [Abstract] [Full Text] [PDF]
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 A. N. Guerra, P. L. Fisette, Z. A. Pfeiffer, B. H. Quinchia-Rios, U. Prabhu, M. Aga, L. C. Denlinger, A. G. Guadarrama, S. Abozeid, J. A. Sommer, et al. Purinergic receptor regulation of LPS-induced signaling and pathophysiology Innate Immunity, August 1, 2003; 9(4): 256 - 263. [Abstract] [PDF]
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L. Gudipaty, J. Munetz, P. A. Verhoef, and G. R. Dubyak Essential role for Ca2+ in regulation of IL-1{beta} secretion by P2X7 nucleotide receptor in monocytes, macrophages, and HEK-293 cells Am J Physiol Cell Physiol, August 1, 2003; 285(2): C286 - C299. [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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V. Budagian, E. Bulanova, L. Brovko, Z. Orinska, R. Fayad, R. Paus, and S. Bulfone-Paus Signaling through P2X7 Receptor in Human T Cells Involves p56lck, MAP Kinases, and Transcription Factors AP-1 and NF-kappa B J. Biol. Chem., January 10, 2003; 278(3): 1549 - 1560. [Abstract] [Full Text] [PDF]
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 T. Into, M. Fujita, T. Okusawa, A. Hasebe, M. Morita, and K.-I. Shibata Synergic Effects of Mycoplasmal Lipopeptides and Extracellular ATP on Activation of Macrophages Infect. Immun., July 1, 2002; 70(7): 3586 - 3591. [Abstract] [Full Text] [PDF]
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M. Aga, C. J. Johnson, A. P. Hart, A. G. Guadarrama, M. Suresh, J. Svaren, P. J. Bertics, and B. J. Darien Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X7 J. Leukoc. Biol., July 1, 2002; 72(1): 222 - 232. [Abstract] [Full Text] [PDF]
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J. M. Labasi, N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M. M. Payette, W. Brissette, J. R. Wicks, L. Audoly, and C. A. Gabel Absence of the P2X7 Receptor Alters Leukocyte Function and Attenuates an Inflammatory Response J. Immunol., June 15, 2002; 168(12): 6436 - 6445. [Abstract] [Full Text] [PDF]
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D. G. Perregaux, K. Bhavsar, L. Contillo, J. Shi, and C. A. Gabel Antimicrobial Peptides Initiate IL-1{beta} Posttranslational Processing: A Novel Role Beyond Innate Immunity J. Immunol., March 15, 2002; 168(6): 3024 - 3032. [Abstract] [Full Text] [PDF]
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E. Adinolfi, L. Melchiorri, S. Falzoni, P. Chiozzi, A. Morelli, A. Tieghi, A. Cuneo, G. Castoldi, F. Di Virgilio, and O. R. Baricordi P2X7 receptor expression in evolutive and indolent forms of chronic B lymphocytic leukemia Blood, January 15, 2002; 99(2): 706 - 708. [Abstract] [Full Text] [PDF]
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L. C. Denlinger, P. L. Fisette, J. A. Sommer, J. J. Watters, U. Prabhu, G. R. Dubyak, R. A. Proctor, and P. J. Bertics Cutting Edge: The Nucleotide Receptor P2X7 Contains Multiple Protein- and Lipid-Interaction Motifs Including a Potential Binding Site for Bacterial Lipopolysaccharide J. Immunol., August 15, 2001; 167(4): 1871 - 1876. [Abstract] [Full Text] [PDF]
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F. Di Virgilio, P. Chiozzi, D. Ferrari, S. Falzoni, J. M. Sanz, A. Morelli, M. Torboli, G. Bolognesi, and O. R. Baricordi Nucleotide receptors: an emerging family of regulatory molecules in blood cells Blood, February 1, 2001; 97(3): 587 - 600. [Abstract] [Full Text] [PDF]
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