The acute phase of the inflammatory response involves an increase in the concentrations of different plasma proteins that include fibrinogen (Fbg) and multiple proinflammatory mediators. In parallel, neutrophil activation is thought to play a crucial role in several inflammatory conditions, and it has been recently demonstrated that Fbg specifically binds to the α-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 FcγRIII. 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.
Fibrinogen (Fbg)5 is a complex dimeric protein with each subunit composed of three nonidentical polypeptide chains (α, β, and γ). It has been demonstrated that Fbg binds to β2 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 γ 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 (FcγRIII) (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.
Materials and Methods
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 FcγRII (CD32), R-PE-conjugated mouse mAb 3G8 (IgG1) anti-human FcγRIII (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 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.
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 × 105 leukocytes, were placed into tubes containing medium or 50 ng/ml of TNF-α 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 × 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 × 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 × 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 FcγR, FcγRII, and FcγRIII (CD32 and CD16, respectively) and CD11b (Mac-1) on neutrophils was performed by direct immunofluorescence flow cytometry. For analysis of FcγRII and FcγRIII 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 FcγRs 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 × 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 × 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%.
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 × 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.
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.
Human Fbg induces transient rise in [Ca2+]i in purified neutrophils
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.
Human Fbg induces neutrophil degranulation
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.
Human Fbg induces modulation of cell surface expression of FcγRs and CD11b on purified neutrophils
Considering that FcγRs and β2 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 FcγRII, FcγRIII, 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 FcγRIII, although some individuals showed a maximal decrease at earlier times (data not shown). Subsequently, aliquots of cells (5 × 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 FcγRIII 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 FcγRIII expression. Meanwhile, FcγRII 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).
When CD11b was evaluated by flow cytometry, we found that incubation with hFbg (2 mg/ml) induced an increase in this molecule on purified PMN, reaching a plateau from 30 up to 60 min (Fig. 4).
Human albumin (20 mg/ml) did not induce any alteration in FcγRs or CD11b surface expression.
Human Fbg enhances neutrophil functional activity
Since hFbg incubation in vitro modified the surface expression of FcγRIII and CD11b, two receptors directly or indirectly involved in FcγR-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. 5 b). However, at suboptimal Ab concentrations for target sensitization (3 ng), ADCC was not modified upon hFbg incubation.
Human Fbg delays spontaneous neutrophil apoptosis
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%.
Human albumin at physiological concentrations was assayed in parallel to rule out a nonspecific protein effect, and preincubation of the cells with anti-CD11b abolished the hFbg effect on spontaneous apoptosis. The mAb anti-CD11b at a saturating concentration had no effect on neutrophil apoptosis. In contrast, cross-linking of CD11b by addition of a second (Fab′)2 anti-mouse Ab significantly promoted spontaneous apoptosis (Fig. 6).
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-α, 340 ± 37 (not significant vs after PMN purification); n = 6). Then hFbg was assayed in both conditions, resting and TNF-α-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-α. 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).
Another biologically relevant question was whether specific fibrinogen cleavage products retain the activating capacity of hFbg. In consequence, purified neutrophils were treated with autologous plasma or hFbg previously incubated with 0.05 U/ml of thrombin for 60 min to ensure the total conversion of fibrinogen to fibrin (36). When CD66b membrane expression was measured we observed that pretreatment with thrombin efficiently abrogated hFbg or plasma effects (MFI (mean ± SEM): control, 225 ± 36; hFbg, 504 ± 42 (p < 0.001 vs control); hFbg plus thrombin, 240 ± 34 (p < 0.001 vs the respective treatment without thrombin); plasma, 468 ± 35 (p < 0.001 vs control); plasma plus thrombin, 186 ± 22 (p < 0.001 vs the respective treatment without thrombin); n = 6;). We therefore conclude that cleavage products derived from hFbg or plasma were not able to modulate CD66b expression on the PMN surface.
In this paper we report a novel effect of soluble Fbg that contributes to the sequential activation of circulating neutrophils. Although β2 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-κB 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-α pretreatment (35). These results are in agreement with previous reports that show that β2 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 β2 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-α, 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 β2-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 β2 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 FcγRIII, while the expression of FcγRII was not modified. It has been previously shown that proinflammatory agents, such as FMLP and PMA, can induce similar FcγRIII down-regulation in a secretory event involving the proteolytic cleavage of the receptor (52, 53). Moreover, an elevated plasma soluble FcγRIII 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 FcγRIII down-regulation could suggest a decrease in Fcγ-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 FcγRs to mediate a number of neutrophil responses after engagement of FcγR 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 Fcγ-dependent reactions. Considering the enhancement of these functions induced by hFbg, while the loss of FcγRIII 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 FcγRIII could affect neutrophil cytotoxicity when it was conducted against targets suboptimally sensitized. This is in good agreement with several reports, which have suggested that FcγRIII, a glycosylphosphatidyl inositol moiety, may contribute to efficient ligand binding and internalization. The high density of FcγRIII and its predicted fast mobility in the membrane bilayer may favor capture of immune complexes, while FcγRII 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.
We thank Marta Felippo, Nora Galassi, and Norma Riera for their excellent technical assistance, and Christiane Dosne Pasqualini for reviewing the manuscript. We also thank Fundación de la Hemofilia and Academia Nacional de Medicina for the use of the FACScan flow cytometer, and Sergio Fridman, Viviana Pressiani, and Antonio Fernández from the Department of Hemotherapy, Centro de Educación Médica e Investigación Clinica, for blood samples.
This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Tecnológicas, Fundación Antorchas and Fundación Alberto J. Roemmers, and Agencia Nacional de Promoción Científica y Tecnológica (Buenos Aires, Argentina).
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.