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División Inmunología and
División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of CD11b/CD18 on neutrophil
surface. Although several reports have shown that CD11b engagement
modulates neutrophil responses, the effect of human Fbg (hFbg), one of
CD11b physiologic ligands, has not been exhaustively investigated. We
have now shown that incubation of purified neutrophils with hFbg
induces a transient and rapid elevation of free intracellular
Ca2+. This early intracellular signal is accompanied by
changes in the expression of neutrophil activation markers, including
enhancement of CD11b and CD66b, and down-regulation of Fc
RIII. In
addition, we have evaluated the effect of hFbg on two functional events
related to expression and resolution of inflammation: cytotoxic
capacity and rate of neutrophil apoptosis. We have found that
activation of neutrophils by hFbg resulted in both enhancement of
phagocytosis and Ab-dependent cellular cytotoxicity, and delay of
apoptosis. We conclude that during inflammatory processes, soluble Fbg
could influence neutrophil responses, increasing and prolonging their
functional capacity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, and ). It has been demonstrated that
Fbg binds to 2 integrin CD11b/CD18 on human
neutrophils at a single saturable binding site with an apparent
Kd of 0.17 µM and 140,000 sites/cell
(1, 2). An internal peptide sequence in the fibrinogen
chain has been shown to function as the high affinity binding site for
CD11b/CD18 on human neutrophils (3, 4). This sequence
binds to peripheral human neutrophils (PMN) when Fbg is in the soluble
or surface-bound form and is preserved even after extensive plasmin
digestion of Fbg (5). Recent structural studies have
identified two conformational states of the ligand binding site of
CD11b/CD18 that may correspond to two functional states, one active
(which binds to activation-dependent ligands such as iC3b and
fibrinogen) and one inactive (which binds only to
activation-independent ligands such as the neutrophil adhesion
inhibitor) (6). There is considerable evidence for the
role of CD11b/CD18 as the receptor for C3bi during host defense by
facilitating phagocytosis of opsonized particles (7). In
contrast, the exact physiological role of the fibrinogen binding
capacity in CD11b/CD18 has not been precisely defined
(8).
Leukocyte interaction with fibrinogen or its degradation products has
special importance at sites of inflammation, since fibrinogen may gain
access to the extravascular compartment by exudation, where it
encounters migrating leukocytes (9). It is well known that
both the extent of leukocyte recruitment and the proinflammatory action
of the migrating leukocytes determine the intensity of an inflammatory
reaction. Moreover, neutrophil recruitment is intimately linked to
neutrophil activation and cell survival (10). In this
context, it has been recently established that integrin aggregation on
PMN is able to initiate intracellular signaling events that lead to
changes in gene expression (8) and promotes phagocytosis,
spreading, and chemotaxis (11, 12, 13). However, the
modulatory effects of soluble fibrinogen on the process of sequential
neutrophil activation have been poorly investigated. In this context,
we examined the potential contributions of soluble human Fbg (hFbg) to
the regulation of neutrophil functionality and survival. We
demonstrated that hFbg triggered the activation of purified neutrophils
through a CD11b-dependent mechanism. This activation resulted in an
increase in intracellular free calcium
([Ca2+]i), up-regulation
of CD11b, and neutrophil degranulation. We also observed the loss of
surface type III receptors for the Fc region of IgG (FcRIII) (CD16).
Moreover, we found that purified neutrophils have an increased
cytotoxic capacity and a delayed apoptosis in response to hFbg
challenge.
We conclude that during an inflammatory process, soluble fibrinogen could contribute to the sequential activation of neutrophil functionality and may influence other aspects of neutrophil behavior, such as gene expression, which would control apoptotic mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following reagents were used: acridine orange, ethidium
bromide, propidium iodide (PI), ionomycin, fluo-3/AM, and FMLP (Sigma,
St. Louis, MO); human albumin (Baxter Inmuno, Buenos Aires, Argentina);
bovine thrombin-TT (Diagnostic Grifols, Buenos Aires, Argentina);
and FITC-conjugated mouse mAb C1KM5 (IgG1) anti-human FcRII
(CD32), R-PE-conjugated mouse mAb 3G8 (IgG1) anti-human FcRIII
(CD16), FITC-conjugated mouse mAb Bear-1 (IgG1) against human
CD11b/CD18 (Mac-1), FITC-conjugated mouse mAb 80H3 (IgG1) against
human CD66b, FITC-conjugated isotype control of mouse IgG1,
and R-PE-conjugated isotype control of mouse IgG1 (Caltag, Burlingame,
CA). Azida-free mAb Bear-1 against CD11b (IgG1) and isotype control of
mouse IgG1 were obtained from Immunotech (Marseilles, France). Mouse
anti-human CD11c clone 3.9 was purchased from Caltag. The purity of
pyrogen-free hFbg (Baxter Inmuno) used in the treatment of patients
with hypo/afibrinogenemia was confirmed electrophoretically.
Blood samples
Blood samples were obtained from healthy volunteer donors who had taken no medication for at least 10 days before the day of sampling. Blood was drawn from the forearm vein and was discharged directly into citrated plastic tubes.
Neutrophil isolation
Neutrophils were isolated by Ficoll-Hypaque gradient centrifugation (Ficoll Pharmacia, Uppsala, Sweden; Hypaque, Wintthrop Products, Buenos Aires, Argentina) and dextran sedimentation as previously described (14). Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (>96% neutrophils on May-Grünwald-Giemsa-stainedCyto-preps) were suspended in RPMI 1640 supplemented with 1% heat-inactivated FCS.
Ex vivo experiments
Care was taken to handle all samples quickly and identically.
Samples were injected into heparinized plastic tubes, inverted once to
mix. To asses the degree of responsiveness of PMN to hFbg, aliquots of
fresh blood, each containing 2.5 x 105
leukocytes, were placed into tubes containing medium or 50 ng/ml of
TNF- at 37°C. After 60 min of incubation, medium or different
concentrations of hFbg were added. After 1 h, cells were stained
with specific mAb for 30 min at 4°C. Then RBC were lysed using FACS
lysing solution (Becton Dickinson, San Jose, CA), and cells were washed
with cool PBS and suspended in 0.4 µl of ISOFLOW (International Link,
Buenos Aires, Argentina). The fluorescence was measured with a Becton
Dickinson FACScan. The analysis was made on 10,000 events on each
sample using the CellQuest program.
Quantification of cellular apoptosis and viability by fluorescence microscopy
Quantification was performed as previously described (15), using the fluorescent DNA-binding dyes acridine orange (100 µg/ml) to determine the percentage of cells that had undergone apoptosis and ethidium bromide (100 µg/ml) to differentiate between viable and nonviable cells. With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment.
Quantification of neutrophil apoptosis by propidium iodide staining and flow cytometry
The proportion of neutrophils that displayed a hypodiploid DNA peak, i.e., apoptotic cells, was determined using a modification of Nicoletti’s protocol (16). Briefly, cell pellets containing 2.5 x 106 neutrophils were suspended in 400 µl of hypotonic fluorochrome solution (50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100) and incubated for 2 h at 4°C. The red fluorescence of PI of individual nuclei was measured using a FACScan flow cytometer (Becton Dickinson). The forward scatter and side scatter of particles were simultaneously measured. Cell debris were excluded from analysis by appropriately raising the forward-scattered threshold. The red fluorescence peak of neutrophils with normal (diploid) DNA content was set at channel 250 in the logarithmic mode. Apoptotic cell nuclei emitted fluorescence in channels 4–200.
Measurement of fluctuations in [Ca2+]i
Changes in [Ca2+]i were monitored using fluo-3/AM, as previously described (17). Briefly, neutrophils suspended at a concentration of 5 x 106 cells/ml in RPMI 1640 were incubated with 4 mM fluo-3/AM for 30 min at 30°C. Then loaded cells were washed twice and suspended at 5 x 106 cells/ml in RPMI 1640 supplemented with 5% heat-inactivated FCS. Aliquots of 50 µl of this cell suspension were added to 450 µl of RPMI 1640 medium containing 5% FCS and 1 mM CaCl2 and warmed at 37°C. The samples were immediately loaded onto the flow cytometer, and the basal fluorescence (FL1) was recorded during 30 s. Then cells were stimulated with precipitating immune complexes, FMLP, or purified hFbg, and the fluorescence was recorded during an additional 400 s. Acquisition of samples was performed at 37°C. Fluctuations in cytoplasmatic free calcium concentrations were recognized as alterations in fluo-3 fluorescence intensity over time. The adequacy of cellular loading and instrumental alignment was determined by treating a sample of cells with 1 µg/ml of ionomycin. We found that 98% of neutrophils respond to this calcium ionophore. Data were analyzed using CellQuest software (Becton Dickinson). A gate based on forward and side scatters was used to exclude debris. Cells responding to immunocomplexes, FMLP or hFbg, show a 3- to 5-fold increase in mean fluorescence intensity (MFI) over that shown by 97% of resting cells.
Flow cytometric analysis
Measurement of the expression of FcR, FcRII, and FcRIII
(CD32 and CD16, respectively) and CD11b (Mac-1) on neutrophils was
performed by direct immunofluorescence flow cytometry. For analysis of
FcRII and FcRIII expression, PMN were treated with mouse IgG in
excess to avoid nonspecific binding of whole mAb molecules. After
preincubation at 37°C with or without Fbg, neutrophils were washed
with cool PBS supplemented with 1% FCS and incubated with mAbs against
FcRs and CD11b for 30 min at 4°C. The control of isotype-matched
Ab was assayed in parallel. Then cells were washed with cool PBS
supplemented with 1% FCS and suspended in 0.3 µl of ISOFLOW.
Fluorescence was measured with a Becton Dickinson FACScan. The analysis
was made on 10,000 events on each sample using the CellQuest
program.
Degranulation of neutrophils
The expression of the surface marker CD66b on the neutrophil surface was used as an indicator of degranulation (18). After preincubation at 37°C with or without hFbg, neutrophils were washed with cool PBS supplemented with 1% FCS and incubated with mAb against CD66b. The control of isotype-matched Ab was assayed in parallel. Then cells were washed with cool PBS supplemented with 1% FCS and suspended in 0.3 µl of ISOFLOW. Fluorescence was measured with a Becton Dickinson FACScan. The analysis was made on 10,000 events on each sample using the CellQuest program.
Ab-dependent cellular cytotoxicity (ADCC)
ADCC was assayed by the chicken erythrocyte (E) IgG anti-E system as previously described (19). Briefly, neutrophils were suspended at a concentration of 2.5 x 106/ml in culture medium supplemented with 1% heat-inactivated FCS. One hundred microliters of this suspension was preincubated with or without hFbg for 1 h at 37°C. Then 40 µl of 51Cr-labeled E (2.5 x 105) sensitized with different amounts of subagglutinating concentrations of rabbit IgG anti-E (E-IgG) were added. After incubation for 18 h at 37°C in 5% CO2-95% humidified air, the culture plate was centrifuged, and the radioactivity of supernatants and pellets was measured in a gamma counter. The mean release of 51Cr in triplicate samples was expressed as a percentage of the total radioactivity. Spontaneous release was always <3%.
Ab-mediated erythrophagocytosis
Erythrophagocytosis was performed as previously described (20). Briefly, after preincubation with or without hFbg for 1 h at 37°C, human neutrophils (50 µl, 7 x 106/ml) were mixed with sheep erythrocytes (50 µl; 3%, v/v) sensitized with subagglutinating amounts of rabbit IgG anti-sheep erythrocytes (Sigma). After incubation for 30 min at 37°C in 5% CO2-95% humidified air, the noningested erythrocytes were lysed by hypotonic shock. The percentage of phagocytic neutrophils was evaluated by microscopic examination. At least 100 cells were scored in each sample. No phagocytosis was detected when neutrophils were incubated with unsensitized erythrocytes.
Statistical analysis
Results are expressed as the mean ± SEM. Statistical analysis of the data was performed using a nonparametric paired Mann-Whitney test. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-linking of CD11b by mAbs has been described to induce an
increase in [Ca2+]i in
PMN (21, 22); therefore, we first investigated whether
hFbg was able to trigger this signaling mechanism. Free
[Ca2+]i was measured
using the Ca2+-sensitive dye fluo-3/AM in
neutrophils exposed to different concentrations of hFbg. We observed
dose-dependent elevation of
[Ca2+]i when hFbg was
added to purified PMN. The higher concentration of hFbg used (2 mg/ml),
which is within the normal range of Fbg concentration in plasma
(23), caused a rapid and transient rise in cytosolic
calcium, similar to that induced by FMLP (10-7
M; Fig. 1
) or 20 µg of immune complexes
(not shown), two well-known Ca2+-mobilizing
stimuli. This increase in free
[Ca2+]i by hFbg was CD11b
dependent, since it could be blocked by clone Bear-1 anti-CD11b
mAb, but not by an irrelevant isotype-matched mAb (IgG1) or by
anti-CD11c (data not shown). Since Ca2+
mobilization triggered by hFbg is an instantaneous reaction, we were
able to exclude that hFbg adherence mediates this response.
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Since neutrophil degranulation is an important
inflammatory event secondary to PMN activation, we quantified
degranulation upon hFbg incubation. Degranulation of PMN was studied
phenotypically by measuring the up-regulation of membrane marker CD66b.
This molecule resides in the specific granules of resting PMN and
appears in the membrane upon degranulation (18, 24).
Considering the dose-response pattern induced by hFbg upon
Ca2+ mobilization, in the following experiments
we used the lower effective concentration within the physiologic range.
Incubation with hFbg (2 mg/ml) resulted in a time-dependent
up-regulation of CD66b expression, which was maximal at 60 min
(
100%; Fig. 2). Control of PMN
incubated with human albumin (20 mg/ml) or mAb anti-CD11b did not
show differences with the basal expression of CD66b. Cross-linking of
CD11b with mAb anti-CD11b and a second
F(ab')2 Ab anti-mouse IgG increased CD66b
expression to the same levels as those reached after hFbg incubation.
Finally, preincubation of the cells with anti-CD11b abolished the
hFbg effect on CD66b expression, confirming that the hFbg effect was
CD11b dependent.
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Rs
and CD11b on purified neutrophils
Considering that FcRs and 2 integrins
are required for several inflammatory responses mediated by PMN
(25) and that changes in their expression have been
associated with neutrophil activation (26), we
investigated the effect of hFbg on surface expression of FcRII,
FcRIII, and CD11b. Isolated neutrophils were preincubated with
purified hFbg (2 mg/ml) for 1 h at 37°C or with medium alone
(control). It is important to note that this incubation time in general
represents the optimal time to observe a decrease in FcRIII,
although some individuals showed a maximal decrease at earlier times
(data not shown). Subsequently, aliquots of cells (5 x
105/50 µl) were washed and incubated with
labeled mAbs or isotype controls. The cells were then analyzed by flow
cytometry as described in Materials and Methods. The results
shown in Fig. 3 indicate that hFbg
induced the loss of FcRIII compared with controls. Cross-linking of
CD11b with anti-CD11b and a second Ab gave similar results (Fig. 3). Preincubation of the cells with anti-CD11b alone abolished the
hFbg effect on FcRIII expression. Meanwhile, FcRII expression was
not significantly modified (MFI (mean ± SEM): control, 624
± 49; hFbg, 629 ± 47; human albumin, 682 ± 15;
anti-CD11b, 536 ± 56; anti-CD11b and
F(ab')2 anti-mouse IgG, 600 ± 35;
n = 6).
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Rs or
CD11b surface expression. Human Fbg enhances neutrophil functional activity
Since hFbg incubation in vitro modified the surface expression of
FcRIII and CD11b, two receptors directly or indirectly involved in
FcR-dependent functions, we next determined Fc-dependent neutrophil
cytotoxicity and phagocytosis. Both assays were measured after 1 h
of PMN incubation with hFbg, at the time at which membrane receptor
variations were maximal. As depicted in Fig. 5, erythrophagocytosis and ADCC conducted
by PMN against optimally sensitized target cells, were significantly
increased by hFbg incubation (2 mg/ml). Similar results were obtained
with CD11b cross-linking (data not shown). In both reactions,
hFbg-dependent enhancement was abolished by previous incubation with
mAb anti-CD11b (Fig. 5b). However, at suboptimal Ab
concentrations for target sensitization (3 ng), ADCC was not modified
upon hFbg incubation.
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Elevation of free [Ca2+]i (27) and different activating stimuli (28, 29, 30) exert an inhibitory effect on PMN apoptosis. On the other hand, other mediators accelerate the apoptotic cell death of human PMN (31, 32, 33). Taking these data into account, we examined whether the incubation with hFbg induced the modulation of neutrophil apoptosis.
Apoptosis was detected by the loss of cellular DNA content by flow
cytometry using PI-permeabilized and stained PMN after 24 h of
culture. As shown in Fig. 6, preincubation for 1 h with 2 mg/ml of hFbg reduced the neutrophil
apoptotic population from 40% (control) to 12%.
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). Human Fbg induces full activation of neutrophils after priming
Considering the above-described results, circulating neutrophils
would continuously be exposed to an activating concentration of
fibrinogen. To understand the biological meaning of such findings and
taking into account that standard preparative techniques for PMN
purification using a Ficoll-Hypaque gradient may result in neutrophil
priming (34), we hypothesized that hFbg would not be able
to mediate neutrophil activation until priming occurs. To test this
possibility we performed ex vivo experiments conducted on whole blood
as previously described (35). The primed status of
purified neutrophils was confirmed by CD66b up-regulation (MFI
(mean ± SEM): PMN in whole blood, 147 ± 12; after PMN
purification, 284 ± 45 (p < 0.04); whole
blood plus TNF-, 340 ± 37 (not significant vs after PMN
purification); n = 6). Then hFbg was assayed in both
conditions, resting and TNF--primed PMN. As shown in Fig. 7, exogenously added hFbg was able to
enhance in a dose-dependent fashion CD66b surface expression only in
PMN preincubated with TNF-. Since these experiments were conducted
on whole blood, the concentration of hFbg indicated in the figure
corresponds to the fibrinogen added to the basal plasma concentration.
Taking this observation into account, it was not surprising that 0.25
mg/ml of hFbg was able to increase CD66b expression on primed
neutrophils. Similar results were obtained when CD11b expression was
measured (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrins on myeloid
cells were initially identified as cell surface structures mediating
the traffic and localization of circulating immune cells
(37), they are also able to regulate multiple cellular
responses to environmental stimuli, leading to growth, differentiation,
gene expression, and apoptosis (38). We now demonstrate
that soluble hFbg is capable of triggering neutrophil activation
through a CD11b-dependent mechanism. It is well known that several
products of coagulation and fibrinolysis profoundly affect many facets
of inflammatory reactions, such as induction of platelet activation
(39), cytokine production by mononuclear phagocytes
(40, 41, 42), neutrophil aggregation, and chemotaxis
(43, 44, 45). However, the effects of soluble hFbg on
inflammatory cells have only recently been investigated. In this
regard, Sitrin et al. (9) recently reported selective
activation of NF-
B and AP-1 by physiological concentrations of
soluble fibrinogen in PMA-differentiated U937 cells. Even more
interesting, our results indicate that hFbg exerts its modulatory
effect only on PMN previously primed by a purification procedure
(34) or by TNF- pretreatment (35). These
results are in agreement with previous reports that show that
2 integrins are unable to interact with their
physiological ligands in unstimulated leukocytes, a safety mechanism
that controls acute and chronic inflammatory responses
(6). We hypothesize that during an inflammatory process,
chemoattractants, early adhesion, or cytokines could prime neutrophils
in circulation or in extravascular sites, turning them into sensitive
targets for fibrinogen. On the other hand, during the fibrinolytic
cascade, fibrinogen is rapidly converted into cleavage products
(23), limiting the activation potential of Fbg-PMN
interactions. In this regard, we have also described that thrombin
completely abrogated the hFbg effect on neutrophil degranulation. Three
additional findings emphasize the importance of these studies: 1)
neutrophils activated in vitro with PMA bind 3- to 5-fold more Fbg than
the resting cells (1); 2) the deposition of Fbg on the
leukocyte surface in vivo has been demonstrated in a variety of
inflammatory responses, such as delayed-type hypersensitivity and
atherosclerosis (3); and 3) a significant correlation has
been demonstrated between increased fibrinogen levels and leukocyte
activation (plasma leukocyte elastase) in patients with peripheral
arterial disease (46).
Our studies demonstrate that soluble fibrinogen induces, in a
dose-dependent manner, a rapid and transient rise in cytosolic
[Ca2+]i in PMN. Many
prior studies have indicated that although CD11c/CD18 can also bind
hFbg (47), the 2 integrin
CD11b/CD18 (CR3, Mac-1) is the dominant fibrinogen binding receptor on
phagocytes (2, 3). In agreement with these reports, our
results show that the intracellular
[Ca2+]i mobilization
signal triggered in response to hFbg is completely abrogated by mAb
anti-CD11b, but is preserved in the presence of anti-CD11c.
Calcium mobilization has been recognized as an important mechanism in
receptor-mediated activation in neutrophils, and stimuli such as FMLP,
leukotriene B4, or IL-1, capable of inducing
degranulation and respiratory burst, are preceded by elevations of
[Ca2+]i
(48). Moreover, calcium ionophores are able to induce
neutrophil activation, as assessed by superoxide anion generation and
degranulation (49). For activation parameters, we
additionally demonstrate that hFbg induces the enhancement of CD11b and
CD66b on the PMN surface. CD11b is up-regulated within minutes of
exposure to proinflammatory substances such as TNF-, IL-8, C5a,
leukotriene B4, and platelet-activating factor
(35). However, like other integrins, CD11b must not only
increase the expression on the cell, but it must undergo a qualitative
change as well as bind to its counter-receptors (50). It
would be interesting to further determine whether hFbg induces the
expression of this activation epitope in the CD11b molecule.
Furthermore, throughout this effect, engagement of active CD11b with
fibrinogen could contribute to potentiate hFbg-mediated activation.
Neutrophil 2-integrin engagement by
extracellular matrix proteins or endothelial counter-receptors triggers
the release of granule contents and the selective up-regulation of
granule membrane molecules such as CD66b (24, 51). In
concordance with a 2 integrin-mediated
phenomenon, we found a significant and specific up-regulation of CD66b
on the neutrophil surface in response to hFbg incubation.
In contrast to the above-mentioned up-regulation of CD11b and CD66b,
hFbg induces a down-regulation of FcRIII, while the expression of
FcRII was not modified. It has been previously shown that
proinflammatory agents, such as FMLP and PMA, can induce similar
FcRIII down-regulation in a secretory event involving the
proteolytic cleavage of the receptor (52, 53).
Moreover, an elevated plasma soluble FcRIII concentration in
septic patients has been associated with in vivo neutrophil activation
(54, 55).
Considering that Ca2+ mobilization is an instantaneous reaction and that phenotype parameters were measured at the single-cell level by flow cytometric analysis, we were able to exclude hFbg adhesion to the plastic surface and PMN-PMN interactions with Fbg as a bridge as being responsible for the Fbg effects observed.
Finally, activation of PMN by hFbg resulted in both the enhancement of cytotoxic activity ADCC and the decrease in the percentage of apoptotic cells. Similarly, Watson et al. (10) have previously demonstrated that neutrophil activation before transendothelial migration in vivo and in vitro resulted in delayed apoptosis and prolonged respiratory burst activity. On the other hand, they and others have shown that resting neutrophils showed an accelerated apoptosis in response to transmigration or by Ab cross-linking of the primary anti-CD18 (55). We have also obtained such discrepant results, since the nonphysiological cross-linking of CD11b by the mAb Bear-1 and the second F(ab')2 Ab promoted spontaneous apoptosis. It has been extensively demonstrated that integrin occupancy can engage various intracellular pathways of signal transduction (56). Therefore, a possible explanation for these contrasting effects is that hFbg and Abs might generate different degrees of CD11b aggregation on the cell surface, which, in turn, might trigger different signals and transductional pathways.
Several authors have shown that delayed apoptosis is associated with
prolonged or increased functional activity, as reflected in spreading
on glass surfaces, polarization, and release of the granule enzyme
marker myeloperoxidase in response to FMLP (10). Although
FcRIII down-regulation could suggest a decrease in Fc-dependent
functions, we have shown that soluble fibrinogen was able to enhance
erythrophagocytosis and ADCC against optimally sensitized targets.
Although ADCC enhancement could reflect the minor percentage of
apoptotic cells, similar results were obtained when phagocytosis was
studied in a short term assay. It has been proposed that CD11b
cooperates with FcRs to mediate a number of neutrophil responses
after engagement of FcR with immune complexes, such as phagocytosis,
adhesion, and tyrosine phosphorylation (57, 58, 59). In
agreement with these results (57, 58, 59, 60), we have
demonstrated that anti-CD11b significantly inhibited both
Fc-dependent reactions. Considering the enhancement of these
functions induced by hFbg, while the loss of FcRIII seems to be
irrelevant, the up-regulation of CD11b could significantly contribute
to enhance phagocytosis and ADCC in a system with optimal sensitization
of target cells. Noteworthy, we found that preincubation of neutrophils
with anti-CD11b could not only block hFbg-induced enhancement of
ADCC, but also partially inhibit ADCC on resting cells. On the other
hand, the loss of FcRIII could affect neutrophil cytotoxicity when
it was conducted against targets suboptimally sensitized. This is in
good agreement with several reports, which have suggested that
FcRIII, a glycosylphosphatidyl inositol moiety, may contribute to
efficient ligand binding and internalization. The high density of
FcRIII and its predicted fast mobility in the membrane bilayer may
favor capture of immune complexes, while FcRII may have the major
role in triggering the cytotoxic signal (61).
In conclusion, we propose that neutrophil priming by chemotactic factors, cytokines, or nucleotides at inflammatory sites facilitates fibrinogen-leukocyte interactions. Hence, fibrinogen could influence the development and resolution of inflammatory foci through the regulation of neutrophil functionality.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carolina Rubel, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de elo 3081, 1425 Buenos Aires, Argentina.
3 Research Fellow, Consejo Nacional de Investigaciones Científicas y Tecnológicas.
4 Research Career, Consejo Nacional de Investigaciones Científicas y Tecnológicas.
5 Abbreviations used in this paper: Fbg, fibrinogen; hFbg, human Fbg; PMN, peripheral human neutrophils; [Ca2+]i, intracellular free calcium; MFI, mean fluorescence intensity; ADCC, Ab-dependent cellular cytotoxicity; PI, propidium iodide.
Received for publication May 30, 2000. Accepted for publication November 14, 2000.
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J. Meshki, F. Tuluc, O. Bredetean, Z. Ding, and S. P. Kunapuli Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor Am J Physiol Cell Physiol, February 1, 2004; 286(2): C264 - C271. [Abstract] [Full Text]
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, Isotype control. The thin line
indicates the control of basal CD66b expression in neutrophils
incubated with medium 1 h at 37°C. The ordinate and the abscissa
represent cell number and fluorescence intensity (FL1-H), respectively.
In g, data are the mean ± SEM MFI of
n experiments. *, Statistical significance
(p < 0.005) compared with control.
