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Fibrinogen Activates NF-B Transcription Factors in Mononuclear Phagocytes¹

Robert G. Sitrin^2,*, Pauline M. Pan^*, Sujata Srikanth^* and Robert F. Todd, III

^* Pulmonary and Critical Care Medicine Division and Hematology-Oncology Division, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109

	Abstract

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Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10–100 µg/ml)/Mn²⁺ (50 µM) for 2 h were examined by electrophoretic mobility shift assay. NF-B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn²⁺ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-B.

	Introduction

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Leukocytes utilize a complex array of adhesion proteins as they respond to chemotaxins and emigrate from the vascular space into inflammatory foci (reviewed in Refs. 1–3). This sequence of events has been characterized extensively, although important mechanisms at every phase of this process remain undefined. Under conditions of shear stress, selectins initiate contact with the endothelium, producing rolling adhesion, followed by firm adhesion, mediated in large part by ß₂ integrins (3). The intensity of an inflammatory reaction is determined by both the extent of leukocyte recruitment and the proinflammatory actions of the migrating leukocytes. Accordingly, the coupling mechanisms that integrate leukocyte recruitment with signaling pathways for cellular activation have received considerable attention. Clearly, leukocyte activation signaling can proceed in nonadherent cells, but engagement of adhesion proteins modifies the activation process (4, 5). For example, monocyte tethering to endothelial cell P-selectin may not only immobilize the cell but also serve as a potent costimulus for secretion of monocyte chemotactic protein-1 and TNF- in association with activation of NF-B transcription regulatory factors (6). Engagement of ß₁ integrins can trigger activation signaling (7), although the capacity for activation signaling directly through ß₂ integrins is less clearly defined (5). CR3 (Mac-1, _Mß₂, CD11b/CD18), a ß₂ integrin critically involved in adhesion, locomotion, chemotaxis, and phagocytosis, serves as a receptor for a diverse group of ligands, including iC3b, ICAM-1, bacterial LPS, ß glucan, coagulation factor X, and fibrinogen (5, 8, 9). Fibrinogen is also a ligand for a limited number of other adhesion proteins, including the ß₂ integrin CR4 (p150/95, CD11c/CD18), _vß₃, and platelet GPIIb/IIIa (5, 10). Interactions between leukocytes and fibrinogen/fibrin may have special importance, since leukocytes commonly encounter provisional matrices rich in fibrinogen and fibrin at sites of inflammation. There is considerable evidence that the products of coagulation and fibrinolysis profoundly affect many facets of inflammatory reactions, wound healing, and fibrosis (11, 12, 13). Likewise, it is well established that contact with fibrinogen or fibrin matrix can dramatically alter the expression of proinflammatory factors such as TNF-, IL-1ß, reactive oxygen intermediates, and possibly IL-8 in vitro (12, 14, 15, 16, 17). However, the precise pathways by which fibrinogen and its derivatives modulate leukocyte function are far less understood. One plausible mechanism by which activation signaling through fibrinogen/fibrin could broadly influence leukocyte function is by activation of pleiotropic transcription regulatory proteins. NF-B is a particularly important candidate, since its activation affects a broad array of immediate-early gene products, including TNF-, ILs, chemokines, and colony stimulating factors (reviewed in Refs. 18–21). NF-B activation has been demonstrated by engagement of other adhesion proteins such as ß₁ integrins, but not by fibrinogen binding or by Ab ligation of ß₂ integrins (22). In this study, we sought to determine whether fibrinogen could trigger activation of NF-B and other transcription regulatory proteins in mononuclear phagocytes. We demonstrate selective activation of NF-B and AP-1 by fibrinogen binding in PMA-differentiated U937 cells. Moreover, it is shown that fibrinogen-triggered NF-B activation is sufficient to serve as a potent stimulus for NF-B-dependent gene transcription.

	Materials and Methods

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Reagents

Plasminogen-free fibrinogen (>95% purity) was obtained from Calbiochem (La Jolla CA). LPS from Escherichia coli 0111:B4 was obtained from Sigma, St. Louis MO. Where appropriate, all reagents used during cell stimulation were pretreated with End-X beads (Associates of Cape Cod, Woods Hole, MA) to adsorb any contaminating endotoxins. This pretreatment yielded reagents that contained less than 0.01 endotoxin U/ml, as determined by a chromogenic Limulus amebocyte lysate assay (Associates of Cape Cod). The anti-ß₂ integrin mAbs included anti-CD18 mAb (TS1/18; IgG1), anti-CD11b mAbs (clone 44 (IgG2a), and clone OKM1 (IgG2b), generously provided by Dr. Thomas Mercolino, Ortho Diagnostic Systems, Raritan NJ), anti-CD11c mAbs (clone 3.9 (IgG1) Leinco Technologies, Ballwin, MO, and clone 2E1 (IgG2b)). Isotype matched control mAbs were obtained from Leinco, and ICN, Costa Mesa, CA. The _vß₃ mAb (clone LM 609; IgG1) was obtained from Chemicon International, Temecula, CA. Anti-p65, p50, and c-Rel Abs were obtained from Santa Cruz Biotechnologies, Santa Cruz, CA. NF-B oligonucleotides were prepared by the DNA Core Facility, University of Michigan Medical Center.

Cell culture and stimulation

The U937 monocytic leukemia cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were propagated in 75-cm² polystyrene flasks in standard medium consisting of RPMI 1640 (Life Technologies, Grand Island, NY) with penicillin (100 U/ml), streptomycin (100 µg/ml), gentamicin (100 µg/ml), glutamine (2 mM), and 5% FBS (HyClone, Logan, UT). To induce morphologic and functional differentiation, the U937 cells were transferred to 1000-ml Teflon tissue culture bags (Baxter Healthcare, Deerfield, IL; 5 x 10⁷ cells in 500 ml of medium) for stimulation with 0.5 nM PMA (Sigma) in nonadherent culture and incubated at 37°C, 5% CO₂ for 48 h. The cells were then washed and resuspended in 20 mM HEPES/140 mM NaCl/2 mg/ml glucose/10 µg/ml polymyxin B, with additives as indicated, and returned to 15-ml Teflon tissue culture bags (American Fluoroseal, Columbia, MD) until the cells were harvested to prepare samples for EMSA³ or CAT assays.

Immunofluorescence flow cytometry

Cells were resuspended in staining buffer (PBS with 1% BSA, 0.1% sodium azide, pH 7.4) and incubated with the relevant primary mAb for 30 min, 4°C, followed by R-phycoerythrin-conjugated goat anti-mouse IgG (30 min, 4°C). Immunofluorescence flow cytometric analysis was performed with a Coulter electronics EPICSC flow cytometer with a logarithmic amplifier (Coulter, Miami, FL; Flow Cytometry Core Facility, University of Michigan Medical Center). The percent of positive cells was determined, using cells stained in parallel with an irrelevant isotype-matched primary Ab as background controls.

EMSA

Activation of NF-B transcription factors was measured by EMSA (adapted from 23 . Nuclear proteins were extracted by the method of Schreiber et al. (24) with minor modifications. After a preliminary wash in Tris-buffered saline, the cells were pelleted and washed twice in 10 mM HEPES, pH 7.9, with 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF, and incubated in a 700-µl volume at 0°C for 15 min. Five microliters of 1% Nonidet P-40 was then added, and the nuclei were pelleted at 15,000 x g. The pellet was resuspended in 75 µl of 20 mM HEPES, pH 7.9, with 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, and incubated for 30 min at 4°C. The nuclei were pelleted once again at 15,000 x g, 4°C, and the supernatant was stored at -70°C. Nuclear extracts (5–10 µg protein in 10 µl) were incubated for 15 min, 25°C, with 10 µl reaction mixture (100 mM Tris Cl, pH 7.5, 1 M NaCl, 50 mM DTT, 10 mM EDTA, 40% glycerol, 1 mg/ml BSA (nuclease free), 50 ng/ml double-stranded poly(dI·dC)), and incubated with ³²P end-labeled NF-B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG-3') (6), or AP-1 consensus oligo (5'-CGC TTG ATG AGT CAG CCG GAA-3'), SP1 consensus oligo, (5'-ATT CGA TCG GGG CGG GGC GAG C-3'), or CREB consensus oligo (5'-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3') (Santa Cruz Biotechnologies). The reaction mixture was run on a 4% PAGE gel in high ionic strength buffer (0.05 M Tris, pH 8.5, 380 mM glycine, 2 mM EDTA), and developed with a Molecular Dynamics PhosphorImager (Sunnyvale, CA). Binding of nuclear protein(s) to the labeled oligonucleotide was demonstrated by visualizing discrete bands of DNA-protein complexes with retarded mobility in the PAGE gel. Binding of labeled oligonucleotides was shown to be sequence-specific by demonstrating that binding was 1) blocked by an excess of unlabeled oligonucleotide and 2) unaffected by an excess of a control oligonucleotide of identical length and base composition, but a randomized sequence. All the key findings shown were confirmed with EMSA of the nuclear extracts prepared from at least two independently run experiments.

Chloramphenicol acetyltransferase (CAT) assays

U937 cells were harvested during logarithmic growth and transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-B sites) linked to a CAT reporter, which was generously provided by Dr. Gary Nabel, Howard Hughes Medical Institute, University of Michigan (25). Transfection was achieved by electroporation, using 15 µg of DNA for 10⁷ cells at 300 V and 1000 µF. The cells were returned to culture for 24 h, at which time they were transferred to 15-ml Teflon culture bags and stimulated either with PMA (0–5 nM) ± fibrinogen (50 µg/ml) for 24 h, or fibrinogen (50 µg/ml) with Mn²⁺ (50 µM) for 2 h, in serum-free medium (Mac-SFM, Life Technologies). Control cells cultured in parallel were mock transfected or transfected with the HIV-1-CAT construct and cultured in medium supplemented with 5% FBS. As a further control, cells were also transfected with a B mutant HIV-1-CAT plasmid and stimulated with PMA ± fibrinogen as above. After culture for 24 h, cell lysates were adjusted for protein content and assayed for CAT activity as described previously (26). Briefly, lysates were incubated with 0.025 µCi of D-threo-[dichloroacetyl-1-¹⁴C] chloramphenicol (Amersham, Arlington Heights, IL) and 1.38 mM acetyl coenzyme A at 37°C for 24 h. The chloramphenicol was separated from its acetylated products by thin layer chromatography (TLC). The TLC plates were then analyzed with a PhosphorImager, and the percentage of acetylation was determined with Image Quant software (Molecular Dynamics).

	Results

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PMA-induced differentiation of U937 cells

Preliminary experiments verified that there was weak NF-B activation in undifferentiated U937 cells in response to LPS (not shown). Therefore, U937 cells were induced to differentiate by stimulation with a low concentration of PMA (0.5 nM) for 48 h. Under these conditions, nuclear NF-B activity remained very low, provided that the cells were maintained in nonadherent culture (Fig. 1), whereas contact with any adhesive surfaces such as polystyrene induced significant NF-B activation (not shown). In addition, the PMA-differentiated cells became quite responsive to LPS, demonstrating a strong NF-B response (Fig. 1).

View larger version (69K): [in this window] [in a new window]   FIGURE 1. Fibrinogen-induced NF-B activation. A, The inset (right) demonstrates representative EMSA assays, using 32P-NF-B consensus oligonucleotide. The unbound oligo is seen at the leading edge, and the regions containing discrete bands of NF-B DNA-protein complexes (bracketed) are detailed (left). The unstimulated cells show minimal NF-B binding proteins, while fibrinogen (50 µg/ml) with Mn2+ (50 µM) for 2 h induced substantial NF-B activation, even in comparison with stimulation with LPS (100 ng/ml) for 2 h. The band of NF-B-specific binding (demonstrated in Fig. 3A) is indicated by the arrowhead. The lower band (*), which varied in intensity between experiments, and other bands outside the bracketed area represented nonspecific oligo binding, since they were blocked effectively by unlabeled oligo with a randomized sequence (not shown). B, Dose-response relationship between the concentration of fibrinogen (10–100 µg/ml) with Mn2+ (50 µM). A detailed view of the EMSA assay is shown, as in A. C, Time course of fibrinogen-induced NF-B activation, indicating a prominent response at 1 and 2 h, in contrast to the later peak observed at 2 h of LPS-induced activation. Neither stimulus induced persistence of NF-B activation extending as long as 4 h.

Fibrinogen-induced activation of NF-B

PMA-differentiated U937 cells were stimulated with fibrinogen (50 µg/ml) in nonadherent culture for 2 h, at which time nuclear proteins were extracted for EMSA assay. Where indicated, the medium was supplemented with MnCl₂ (50 µM) to ensure that cation-dependent integrins were optimally activated (27). As shown in Figure 1A, fibrinogen induced substantial NF-B activation in the presence of Mn²⁺, even in comparison with LPS stimulation, which was included as a positive control. The effect of fibrinogen was dose related, since NF-B activation was first evident with as little as 10 µg/ml, with a substantial response occurring with 25 µg/ml, a maximal response with 50 µg/ml, and a plateau effect extending through 100 µg/ml (Fig. 1B). Time course studies indicate that fibrinogen-induced NF-B activation was rapid and transient, with a maximal response after 1 and 2 h of stimulation, and a prompt return to baseline within 4 h. By contrast, LPS-induced NF-B activation was somewhat slower to evolve, with a maximal response developing within the 1- and 2-h time points. Like the response to fibrinogen, NF-B activation then returned to baseline within 4 h (not shown).

All these experiments were conducted in the presence of polymyxin B to block the effects of endotoxin, even though the reagents or media contained no more than 0.01 EU/ml. This corresponded to less than 1 ng/ml of the LPS used as a positive control, an amount that produces minimal, if any, NF-B activation (not shown). It was also confirmed that polymyxin B did not affect the NF-B activation produced by fibrinogen but significantly reduced the magnitude of the response to LPS (100 ng/ml) (Fig. 2). In addition, LPS-induced NF-B activation was unaffected by the absence of Mn²⁺ (not shown), while Mn²⁺ was necessary for fibrinogen stimulation (Fig. 5; see below). Finally, fibrinogen activated NF-B in undifferentiated U937 cells (Fig. 8; see below), while, as noted above, LPS produced a weak NF-B response unless the cells were pretreated with PMA. These distinctions confirm that LPS contamination was not responsible for fibrinogen-induced NF-B activation.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Elimination of endotoxin contamination as a cause of fibrinogen-induced NF-B activation. Fibrinogen-induced NF-B activation is not suppressed by inclusion of polymyxin B (10 µg/ml) in the medium, while NF-B activation in response to LPS (100 ng/ml) is significantly reduced. Detailed views of EMSA assays are shown as in Figure 1, with the band of NF-B-specific binding indicated by the arrowhead.

View larger version (118K): [in this window] [in a new window]   FIGURE 5. Fibrinogen-induced NF-B activation is dependent on the presence of Mn2+. Detailed views of EMSA assays are shown as in Figure 1, with the band of NF-B-specific binding indicated by the arrowhead. NF-B activation was induced only by fibrinogen (50 µg/ml) in the presence of Mn2+ (50 µM), and not by either fibrinogen or Mn2+ alone.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. Effects of PMA and fibrinogen on NF-B activation. Cells (without prior differentiation with PMA) were stimulated with PMA (5.0 and 0.5 nM) for 2 h, with fibrinogen (50 µg/ml)/Mn2+ (50 µM) for 2 h, or with the combination of PMA (0.5 nM) + fibrinogen (50 µg/ml)/Mn2+ (50 µM) for 2 h, and assayed by EMSA using the consensus NF-B oligonucleotide. PMA alone induced a significant degree of NF-B activation, as did fibrinogen (50 µg/ml)/Mn2+ (50 µM) in undifferentiated cells. The combination of PMA and fibrinogen yielded a level of NF-B activation that was only marginally greater than fibrinogen/Mn2+ alone.

Characterization of fibrinogen-induced NF-B proteins

Experiments were next performed to characterize the specific NF-B proteins induced by fibrinogen. First, cold competition studies were used to determine which protein-DNA complexes on EMSA are sequence specific. For both LPS- and fibrinogen-stimulated cells, a single complex was formed, with the labeled NF-B consensus oligo, that was completely blocked by an excess of cold oligo but was unaffected by a cold oligo with a randomized sequence (Fig. 3A).

View larger version (96K): [in this window] [in a new window]   FIGURE 3. Characterization of fibrinogen-induced NF-B proteins. A, Cold competition of EMSA assays of nuclear protein extracts prepared from fibrinogen/Mn2+ or LPS-stimulated cells. Excess unlabeled NF-B oligo completely abrogated formation of complexes with the 32P-oligo, indicated by the arrowhead. By contrast, cold competition with an excess of unlabeled random sequence control oligo had no effect on the NF-B-specific complex. B, Ab supershifting. Nuclear protein extracts prepared from cells stimulated with fibrinogen/Mn2+ (left) or LPS (right) were incubated with 32P-NF-B oligo in the presence of Abs reactive against the p50 and p52 forms (anti-p50), the p65(Rel A) and c-Rel (anti-p65) forms, or both. The NF-B-specific complex (arrowheads) is composed of a mixture of NF-B forms, as the lower portion of the band is removed by the anti-p50 Ab, while the more prominent upper portion of the band is removed by the anti-p65 Ab, and both Abs together eliminate the band almost completely.

Selectivity of NF-B activation

To determine whether fibrinogen/Mn²⁺ broadly induces activation of transcriptional regulatory factors, EMSA assays were also performed using consensus oligonucleotides for AP-1, SP1, and CREB. As shown in Figure 4, fibrinogen/Mn²⁺ produced a substantial increase in AP-1 activation that was highly consistent but not as prominent as the effect produced by LPS. However, fibrinogen/Mn²⁺ did not affect binding of SP1 or CREB, even though activation of both factors was evident in nuclear extracts of LPS-stimulated cells. These findings indicate that signaling through fibrinogen/Mn²⁺ yields a limited and selective array of activated transcriptional regulatory factors. Nonetheless, the broad representation of NF-B and AP-1 regulatory elements in the enhancer regions of immediate-early gene products and other proinflammatory mediators justifies the expectation that fibrinogen-induced activation of both NF-B and AP-1 families should have wide-ranging effects on expression of genes that are tightly regulated during inflammation and wound healing.

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Selectivity of NF-B activation in response to fibrinogen/Mn2+. Nuclear protein extracts prepared from 1) unstimulated control cells, 2) cells stimulated with fibrinogen/Mn2+, and 3) cells stimulated with LPS were assayed by EMSA, using consensus oligonucleotides for NF-B, AP-1, SP1, and CREB. Fibrinogen/Mn2+ increased binding to NF-B and AP-1 oligonucleotides but had no effect on SP1 or CREB, even though both of these were induced by LPS.

CR3 mediates fibrinogen-induced NF-B activation

One of the more notable properties of fibrinogen-mediated NF-B activation was that the effect was seen only in Mn²⁺-supplemented medium (Fig. 5). Fibrinogen (50 µg/ml) in HEPES-buffered saline/glucose without Mn²⁺ had no appreciable effect on NF-B activation. Likewise, the presence of MnCl₂ (50 µM) alone did not induce NF-B activation. Preliminary experiments indicated that the 50 µM concentration of MnCl₂ was optimal for supporting NF-B activation (not shown). This was quite similar to the Mn²⁺ concentration that maximally activates LFA-1 in the same buffer system (29), in keeping with the suggestion that the requirement for Mn²⁺ reflected the conditions that optimized ligation to fibrinogen-binding integrins.

Immunofluorescence flow cytometry was performed to assess the expression of integrins capable of serving as fibrinogen receptors, namely CR3 (CD11b/CD18), CR4 (CD11c/CD18), and _vß₃. GPIIb/IIIa (CD41b/CD61), which shares epitope(s) and function with CR3, was not assessed, since it is expressed by platelets and megakaryocytes and not by mononuclear phagocytes (30). Using clone 44 anti-CD11b mAb, it was determined that 62 ± 11% of PMA-stimulated cells expressed CR3 (mean ± SEM), while CR4 expression, although significant, was limited to 34 ± 7% of cells (using clone 3.9 anti-CD11c mAb). The staining intensities for both CD11b and CD11c were broadly distributed, so these percentages may be underestimates, excluding cells with relatively low levels of integrin expression and including only those with fluorescence intensities clearly exceeding control cells stained with irrelevant primary Abs. There was negligible expression of _vß₃ (1.5 ± 0.3% positive cells). Finally, expression of the urokinase receptor (uPAR; CD87) was also assessed, since uPAR associates with many integrins, including CR3 and CR4, and this association strongly influences integrin-mediated adhesion, including CR3-mediated adhesion of human monocytes to fibrinogen (31, 32, 33). Detectable uPAR was found on 71 ± 10% of cells, confirming their availability for interactions with integrins. The presence of these two fibrinogen-binding ß₂ integrins, coupled with a requirement for a divalent cation (Mn²⁺), which is characteristic of ß₂ integrins, indirectly implicated CR3 and/or CR4 as candidate receptors responsible for fibrinogen-induced NF-B.

Confirmatory studies were next performed to determine which integrin(s) might be responsible for fibrinogen-induced NF-B activation by blocking the response selectively with a series of mAbs. In these experiments, the cells were pretreated with mAb (100 µg/ml) for 30 min before adding fibrinogen/Mn²⁺. Monoclonal Abs were also added with the fibrinogen/Mn²⁺ to maintain the concentration at 100 µg/ml throughout the 2-h incubation before nuclear protein extraction. As shown in Figure 6, the addition of either an anti-CD18 mAb (clone TS1/18) or an anti-CD11b mAb (clone OKM1) completely abrogated the NF-B response to fibrinogen/Mn²⁺. Control mAbs had no effect on NF-B activation. Likewise, the anti-CD11c mAb (clone 2E1) was ineffective, suggesting that CR3, and not CR4, is the receptor predominately responsible for fibrinogen-induced NF-B activation. It is interesting to note that while OKM1 blocked NF-B activation completely, clone 44, an anti-CD11b mAb that reacts with an epitope located in the I domain, had no effect at all (not shown). This agrees closely with prior studies indicating that OKM1 preferentially blocks binding of soluble fibrinogen to CR3, while I-domain Abs such as clone 44 preferentially block cellular adhesion to immobilized fibrinogen (8, 9, 34).

View larger version (66K): [in this window] [in a new window]   FIGURE 6. Blockade of fibrinogen-induced NF-B activation with anti-CD11b/CD18 Abs. Monoclonal Abs against CD18 (TS1/18) and CD11b (OKM1) block NF-B activation in response to fibrinogen/Mn2+. The anti-CD11c mAb (2E1) did not inhibit NF-B activation. Likewise, none of the isotype-matched control mAbs had any effect (only the IgG1 control is shown). In all instances, cells were pretreated with 100 µg/ml concentrations of mAb, which was maintained throughout subsequent stimulation with fibrinogen/Mn2+. Detailed views of EMSA assays are as shown in previous figures, with the bands of NF-B-specific binding indicated by the arrowheads.

To determine whether stimulation with fibrinogen is sufficient to modulate NF-B-driven gene transcription, U937 cells were transfected with an HIV-1 enhancer-CAT construct, as described in Materials and Methods. Prior studies have shown that transcription of this construct is refractory to further modulation once the cells are differentiated into a more mature phenotype (25). Accordingly, these experiments were performed without pretreating the cells with PMA. Preliminary experiments confirmed that control cells (transfected, but otherwise unstimulated) expressed very low levels of CAT activity minimally above the background activities of mock-transfected cells (electroporated, no plasmid), indicating that transfection with the NF-B/CAT plasmid alone resulted in little cellular activation (not shown). Figure 7A shows that stimulation with PMA alone for 24 h had only a limited effect on CAT activity. Only the highest concentration of PMA, 5 nM, produced a statistically significant increase in CAT activity (2.6-fold), relative to unstimulated controls. By contrast, cells stimulated with fibrinogen (50 µg/ml), in the presence of Mn²⁺ (50 µM) for only 2 h and then returned to serum-free medium for the remainder of a 24-h culture, yielded a statistically significant increase in CAT expression, 3.2-fold relative to unstimulated controls (Fig. 7A). Experiments were also performed to determine whether fibrinogen was an effective costimulus with PMA in inducing CAT expression. In these experiments, cells were cultured with varying concentrations of PMA ± fibrinogen for 24 h in serum-free medium. Mn²⁺ was omitted from these experiments since concurrent stimulation with PMA was deemed adequate to activate fibrinogen-binding integrins (35). As shown in Figure 7B, costimulation with fibrinogen significantly enhanced CAT activity for all concentrations of PMA tested. The peak effect of fibrinogen was seen with 0.5 nM PMA, producing 5.7-fold the CAT activity achieved by stimulation with PMA alone. This protocol was repeated with cells transfected with a HIV-1 enhancer-CAT construct with both NF-B sites inactivated (Fig. 7B). Under these circumstances, fibrinogen costimulation did not influence CAT expression at all, indicating that the effect is clearly NF-B dependent. Finally, we wished to determine whether other serum proteins could serve the same costimulatory function as fibrinogen. Therefore, cells transfected with the NF-B-intact HIV enhancer-CAT construct were stimulated with PMA (0.5 nM) with and without 5% FBS. The addition of serum had no costimulatory effect on CAT expression (Fig. 6B), indicating that fibrinogen has a selective effect on NF-B-dependent gene expression that is not shared broadly by other circulating proteins.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. A, NF-B-dependent transcriptional regulation by PMA and fibrinogen/Mn2+. Cells transfected with the wild-type HIV-1/CAT plasmid were stimulated with PMA (0.05–5.0 nM) for 24 h, or with fibrinogen (50 µg/ml)/Mn2+ (50 µM) for 2 h, followed by incubation in serum-free medium for 22 h. CAT activities of the cell lysates were normalized to the activities of transfected, but otherwise unstimulated, controls. PMA induced a dose-dependent increase in CAT activity that reached statistical significance only with the 5.0-nM concentration (2.6-fold). Fibrinogen/Mn2+ induced a significant 3.2-fold increase in CAT activity. One sample, two-tailed Student’s t tests were used to demonstrate that CAT activities were 100% of control. * p < 0.02, n = 4. B, left, Effects of stimulating with fibrinogen (50 µg/ml)/Mn2+ (50 µM) concurrently with PMA (0.05–5.0 nM) for 24 h. Cells were transfected with the wild-type (wt) HIV-1 LTR/CAT construct. CAT activities of cell lysates are normalized to controls stimulated with PMA alone (without fibrinogen/Mn2+). Costimulation with fibrinogen/Mn2+ significantly augmented CAT activities at all PMA concentrations. n = 7. Middle, Costimulation with fibrinogen (50 µg/ml)/Mn2+ (50 µM) had no effect on PMA-driven CAT activity when cells were transfected with a HIV-1 LTR/CAT construct bearing inactivated NF-B sites (NF-B (-)). n = 3. Right, FBS (5%) had no effect as a costimulus with PMA (0.5 nM), using wt HIV-1 LTR/CAT-transfected cells. n = 7. Statistical analysis was performed as described in A. * p < 0.05.

	Discussion

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These studies demonstrate that fibrinogen is capable of triggering substantial activation of NF-B transcription regulatory factors in undifferentiated and PMA-differentiated U937 mononuclear phagocytes. The composition of NF-B-binding proteins closely resembled the profile induced by activation with LPS, in that dimers containing p65 and c-REL were the dominant forms, with a minor contribution by dimers composed of p50 and p52 (Fig. 3B). Under the conditions employed, fibrinogen was able to produce this response only in the presence of Mn²⁺. which suggested that an integrin adhesion protein serves as the fibrinogen receptor. Many prior studies have indicated that CR3 (Mac-1; CD11b/CD18), a ß₂ integrin, is the dominant fibrinogen-binding receptor on mononuclear phagocytes (8, 9, 36), while another ß₂ integrin, CR4 (150/95; CD11c/CD18), can also bind fibrinogen (10). Flow cytometry experiments indicate that the PMA-differentiated U937 cells used in most experiments in the present study expressed predominately CR3 (62% of cells), although there was an appreciable representation of CR4 as well (34% of cells). The use of blocking mAbs further verify that CR3 is the dominant, if not sole, receptor responsible for fibrinogen-induced NF-B activation. The NF-B response to fibrinogen/Mn²⁺ was completely negated by Abs to CD18 and CD11b. By contrast, fibrinogen-induced NF-B activation was preserved in the presence of anti-CD11c mAb. The absence of a blocking effect with a single Ab is not itself convincing, but, together with the absence of any residual NF-B activation in the presence of anti-CD11b mAb, it is unlikely that CR4 contributed significantly to fibrinogen-induced NF-B activation. Given the overlap in ligand specificities and the structural similarities between CR3 and CR4, these functional dissimilarities are striking and offer the opportunity to further explore the distinct roles these integrins may play in adhesion/activation coupling. Alternatively, it is possible that the level of CR4 expression achieved under these experimental conditions was simply insufficient to mediate NF-B activation.

The functional implications of this NF-B activation was demonstrated by the markedly enhanced transcriptional activity of the HIV-1 LTR-CAT construct in response to fibrinogen, both as a lone stimulus (3.2-fold the activity of unstimulated controls; Fig. 7) and as a potent costimulus with PMA, where the resulting CAT activities were enhanced by fibrinogen at all PMA concentrations tested, optimally augmenting CAT expression by 5.7-fold. None of these effects were observed when the cells were transfected with a construct lacking functional NF-B sites. While it is certainly possible that both PMA and fibrinogen may have effects other than NF-B activation that enhance expression of the HIV-1 LTR-CAT construct, it is clear that the effect is entirely dependent on the functionality of NF-B.

There are a few features of the model used in the EMSA experiments (PMA-pretreatment of U937 cells for 48 h) that merit specific comment. Preliminary experiments demonstrated that untreated U937 cells exhibited poor NF-B activation over 2 h in response an archetypal agonist such as LPS. Prior work has shown that pretreating U937 cells with PMA for 24 h induces sustained and constitutive NF-B (25). The key methodologic difference in our study is that the U937 cells were maintained in strictly nonadherent culture throughout this incubation. Preliminary experiments showed that PMA-pretreated U937 cells were exquisitely sensitive to even transient exposure to any adhesive surface (including protein-coated or uncoated plastic), resulting in near-maximal NF-B activation. This indicates that the emergence of constitutive NF-B expression during PMA-induced differentiation is itself critically dependent on cellular adhesion. The very low baseline expression of nuclear NF-B proteins, combined with a differentiated phenotype (with relatively high levels of integrin expression) and the preserved responsiveness to subsequent stimulation (fibrinogen/Mn²⁺ or LPS), make this model a highly attractive one for studying the effects of integrins on gene expression in mononuclear phagocytes. Since adhesion to fibrinogen-coated flasks produced the same high levels of NF-B activation as contact with uncoated or albumin-coated plastic (not shown), the model is limited in that it is applicable only to studying soluble counterligands and precluded us from examining the effects of immobilized fibrinogen. Another particularly interesting feature of this model is that fibrinogen induces NF-B activation even while the cells are kept meticulously in nonadherent conditions, in striking contrast to prior studies suggesting that integrin-mediated activation signaling requires integrin cross-linking, cellular adhesion, and cytoskeletal rearrangement (5). We cannot dismiss the possibility that intercellular contact, which can be enhanced by fibrinogen, also contributed to NF-B activation (36). However, the cells were routinely examined after fibrinogen treatment, and the cells emerged from these cultures in uniform suspensions. Homotypic aggregation was not observed (not shown).

The concept that leukocyte integrins can serve as conduits for activation signaling first emerged from observations that these proteins can sample the extracellular environment for extracellular matrix (ECM) proteins, adjacent cells, LPS, and microbial pathogens, and, through transmembrane domains, can provide a physical link to the cytoskeleton and thereby to the machinery of gene expression (4, 5). Subsequent studies have reinforced this concept by demonstrating that integrin ligation can engage various pathways of intracellular signal transduction. Many integrins can activate the Na⁺/H⁺ antiporter with a resultant increase in intracellular pH (5). Most integrins tested, including CR3, can also trigger an increase in the concentration of intracellular calcium (5). Ab binding of ß₁ integrins has been shown to trigger immediate-early gene expression and NF-B activation (7, 22), but a similar approach to signaling through ß₂ integrins was unsuccessful (7). Thus far, evidence for activation signaling through ß₂ integrins has been indirect or limited to changes in early signaling mediators such as intracellular calcium. Contact with fibrinogen-related proteins can induce or enhance expression of immediate-early genes in mononuclear phagocytes, including TNF-, IL-1ß, and tissue factor (12, 14, 15). All these proteins are subject to regulation by NF-B, but NF-B activation was not demonstrated directly in any of these studies. However, recent work has shown that CR3-transfected fibroblasts are capable of NF-B activation in response to LPS (37).

Local activation of coagulation and fibrinolytic pathways, virtually ubiquitous at inflammatory foci, is deeply insinuated in the pathogenesis of inflammatory tissue injury and repair. Fibrinogen and its derivative products are involved in the adhesion, spreading, and movement of many cell types involved in wound repair, including fibroblasts, endothelial, cells, and epithelial cells (38, 39, 40). It is also expected that leukocytes will encounter fibrinogen-related proteins at virtually every phase of the inflammatory response. Circulating monocytes would certainly be exposed to high concentrations of fibrinogen in plasma. Monocytes do express some nuclear NF-B constitutively (41), which is clearly not maximal, and monocytes retain responsiveness to fibrinogen in vitro (12, 14, 15, 16). Consistent with the results of the present study (Fig. 5), it is possible that monocyte CR3 is relatively incapable of mediating NF-B activation until activated by exposure to chemoattractants, early adhesion, or other signals. These and other factors that may modulate monocyte responsiveness to plasma fibrinogen certainly merit future study. Fibrin and platelet deposits accumulate on injured endothelium, particularly under shear stress, providing an adhesive surface for activated leukocytes (42). Fibrinogen can significantly enhance adhesive reactions between monocytes and endothelial cells by acting as a bridge molecule between the CR3 of a mononuclear phagocyte and endothelial ICAM-1 (36). Extravascular fibrinogen is rapidly converted into fibrin deposits that persist as prominent features of many acute and chronic inflammatory reactions. Fibrinogen may gain access to the extravascular compartment either by exudation of circulating fibrinogen or by frank hemorrhage, which has been shown to induce NF-B activation in vivo (43). Fibrinogen can also be synthesized locally by epithelial cells (44). Leukocytes contacting fibrin-rich matrices may be directed to alter production of proinflammatory cytokines (12, 15), and, in addition, fibrin degradation products can amplify the inflammatory reaction by serving as chemotaxins (45, 46, 47). In the present studies, all experiments were performed with soluble fibrinogen, so it will be necessary to determine whether its derivative forms expected at sites of inflammation, including fibrin monomer, insoluble fibrin polymer, and fibrin degradation products, are equally capable of activating NF-B and AP-1. The fibrinous stroma also influences the architecture of inflamed tissue by limiting hemorrhage, entrapping leukocytes, and providing a provisional matrix for the ingrowth of fibroblasts, and evidence is mounting that impaired local fibrinolysis favors the development of excessive fibrosis rather than restoration of normal tissue structure (13). The results of the present study add to the potential mechanisms by which local fibrin deposition can alter the course of inflammation and healing. The broad array of genes that are regulated by NF-B factors include a host of cytokines that participate in leukocyte recruitment and activation, including ILs (IL-1ß, IL-6, IL-12), TNF-, chemokines (IL-8, growth-related oncogene protein (Gro)-, -ß, and -, macrophage inflammatory protein (MIP)-1, monocyte chemotactic protein (MCP)-1), inducible nitric oxide synthase (iNOS), adhesion proteins (ICAM-1, VCAM-1), and viruses (HIV, cytomegalovirus (CMV), and adenovirus) (18, 21, 48). Thus, one can easily hypothesize that the same fibrinogen-integrin interaction that facilitates immobilization and recruitment of leukocytes can, by triggering NF-B activation, augment the production of cytokines, regulate expression of adhesion proteins, and, possibly, contribute to the persistence and proliferation of viruses. Indeed, prior studies have shown that HIV-infected leukocytes exhibit enhanced expression of CD11b, increased adhesion, and enhanced replication of HIV when cells adhere to specific components of the extracellular matrix (49, 50, 51). It is well recognized that HIV replication can be enhanced by the effects of opportunistic infections, and local deposition of fibrinogen-related proteins at these sites of infection provide yet another potentially important stimulus by which this may occur. Lastly, NF-B factors have been implicated in both the induction and prevention of apoptosis, suggesting that fibrinous stroma may influence the longevity of leukocytes and parenchymal cells in inflamed or healing tissues (52).

In summary, this study demonstrates that U937 mononuclear phagocytes react to exposure to fibrinogen in vitro with prompt activation of pleiotropic transcription regulatory factors, NF-B and AP-1. The NF-B response is mediated by CR3 but does not require cellular adhesion. Finally, fibrinogen, either alone or in concert with PMA, is capable of supporting substantially enhanced transcriptional activity of a NF-B-regulated gene. Fibrinogen-related proteins are already believed to be important participants in leukocyte trafficking and tissue remodeling and now can also be implicated in the mechanisms whereby altered coagulation/fibrinolysis homeostasis in inflamed tissue influences activation signaling of mononuclear phagocytes.

	Acknowledgments

 
We thank Laura Mayo-Bond for preparation of mAbs.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL53283, CA 39064, and CA42246.

² Address correspondence and reprint requests to Dr. Robert G. Sitrin, 6301 MSRB III, Box 0642, 1150 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109. E-mail address:

³ Abbreviations used in this paper: EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element-binding protein; LTR, long terminal repeat; Fbg, fibrinogen; wt, wild-type.

Received for publication December 11, 1997. Accepted for publication March 25, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Albelda, S., C. Smith, P. Ward. 1994. Adhesion molecules and inflammatory injury. FASEB J. 8:504.[Abstract]
2. Beekhuizen, H., R. van Furth. 1993. Monocyte adherence to human vascular endothelium. J. Leukocyte Biol. 54:363.[Abstract]
3. Schleiffenbaum, B., J. Fehr. 1996. Regulation and selectivity of leukocyte emigration. J. Lab. Clin. Med. 127:151.[Medline]
4. Miyamoto, S., S. Akiyama, K. Yamada. 1995. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267:883.[Abstract/Free Full Text]
5. Rosales, C., R. Juliano. 1995. Signal transduction by cell adhesion receptors in leukocytes. J. Leukocyte Biol. 57:189.[Abstract]
6. Weyrich, A., T. McIntyre, R. McEver, S. Prescott, G. Zimmerman. 1995. Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and TNF- secretion. J. Clin. Invest. 95:2297.
7. Yurochko, A., D. Liu, D. Eierman, S. Haskill. 1992. Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction. Proc. Natl. Acad. Sci. USA 89:9034.[Abstract/Free Full Text]
8. Altieri, D., F. Agbanyo, J. Plescia, M. Ginsberg, T. Edgington, E. Plow. 1990. A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac-1 (CD11b/CD18). J. Biol. Chem. 265:12119.[Abstract/Free Full Text]
9. Wright, S., J. Weitz, A. Huang, S. Levin, S. Silverstein, J. Loike. 1988. Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen. Proc. Natl. Acad. Sci. USA 85:7734.[Abstract/Free Full Text]
10. Loike, J., B. Sodeik, L. Cao, S. Leucona, J. Weitz, P. Detmers, S. Wright, S. Silverstein. 1991. CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A chain of fibrinogen. Proc. Natl. Acad. Sci. USA 88:1044.[Abstract/Free Full Text]
11. Sitrin, R.. 1992. Plasminogen activation in the injured lung: pulmonology does not recapitulate hematology. Am. J. Resp. Cell Mol. Biol. 6:131.
12. Perez, R., J. Roman. 1995. Fibrin enhances the expression of IL-1ß by human peripheral blood mononuclear cells. J. Immunol. 154:1879.[Abstract]
13. Eitzman, D., R. McCoy, X. Zheng, W. Fay, T. Shen, D. Ginsburg, R. Simon. 1996. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J. Clin. Invest. 97:232.[Medline]
14. Fan, S., T. Edgington. 1991. Coupling of the adhesive receptor CD11b/CD18 to functional enhancement of effector macrophage tissue factor response. J. Clin. Invest. 87:50.
15. Fan, S., T. Edgington. 1993. Integrin regulation of leukocyte inflammatory functions: CD11b/CD18 enhancement of the TNF- responses of monocytes. J. Immunol. 150:2972.[Abstract]
16. Trezzini, C., B. Schüepp, F. Maly, T. Jungi. 1991. Evidence that exposure to fibrinogen or to antibodies directed against Mac-1 (CD11b/CD18; CR3) modulates human monocyte effector functions. Brit. J. Haematol. 77:16.[Medline]
17. Pakianathan, D.. 1995. Extracellular matrix proteins and leukocyte function. J. Leukocyte Biol. 57:699.
18. Blackwell, T., J. Christman. 1997. The role of nuclear factor- B in cytokine gene regulation. Am. J. Respir. Cell Mol. Biol. 17:3.[Abstract/Free Full Text]
19. Baldwin, A.. 1996. The NF-B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
20. Grilli, M., J. J.-S. Chiu, M. Lenardo. 1993. NF- B and Rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:1.[Medline]
21. Müller, J. M., H. W. L. Ziegler-Heitbrock, P. A. Baeuerle. 1993. Nuclear factor kappa B, a mediator of lipopolysaccharide effects. Immunobiology 187:233.[Medline]
22. Lin, T., C. Rosales, K. Mondal, J. Bolen, S. Haskill, R. Juliano. 1995. Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells: a possible signaling role for the Syk tyrosine kinase. J. Biol. Chem. 270:16189.[Abstract/Free Full Text]
23. Chodosh, L. A. 1989. Mobility shift DNA-binding assay using gel electrophoresis. In Current Protocols in Molecular Biology, Vol. 2. A. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds. Greene Publishing and Wiley Interscience, New York, p. 12.2.1.
24. Schreiber, E., P. Matthias, M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini extracts" prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
25. Griffin, G., K. Leung, T. Folks, S. Kunkel, G. Nabel. 1989. Activation of HIV gene expression during monocyte differentiation by induction of NF-B. Nature 339:70.[Medline]
26. Schmid, R., N. Perkins, C. Duckett, P. Andrews, G. Nabel. 1991. Cloning of an NF-B subunit which stimulates HIV transcription in synergy with p65. Nature 352:733.[Medline]
27. Altieri, D.. 1991. Occupancy of CD11b/CD18 (Mac-1) divalent ion binding site(s) induces leukocyte adhesion. J. Immunol. 147:1891.[Abstract]
28. Oeth, P., G. Parry, C. Kunsch, P. Nantermet, C. Rosen, N. Mackman. 1994. Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a B-like site. Mol. Cell. Biol. 14:3772.[Abstract/Free Full Text]
29. Dransfield, I., C. Cabanas, A. Craig, N. Hogg. 1992. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116:219.[Abstract/Free Full Text]
30. De Nichilo, M., D. Shafren, W. Carter, M. Berndt, G. Burns, A. Boyd. 1996. A common epitope on platelet integrin _IIbß₃ (glycoprotein IIbIIIa; CD41b/CD61) and _mß₂ (Mac-1; CD11b/CD18) detected by a monoclonal antibody. J. Immunol. 156:284.[Abstract]
31. Sitrin, R., R. Todd, H. Petty, T. Brock, S. Shollenberger, E. Albrecht, M. Gyetko. 1996. The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. J. Clin. Invest. 97:1942.[Medline]
32. III Todd, R., H. Petty. 1997. ß₂ (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors. J. Lab. Clin. Med. 129:492.[Medline]
33. Wei, Y., M. Lukashev, D. Simon, S. Bodary, S. Rosenberg, M. Doyle, H. Chapman. 1996. Regulation of integrin function by the urokinase receptor. Science 273:1551.[Abstract]
34. Diamond, M., J. Garcia-Aguilar, J. Bickford, A. Corbi, T. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J. Cell Biol. 120:1031.[Abstract/Free Full Text]
35. Hogg, N., J. Harvey, C. Cabanas, R. Landis. 1993. Control of leukocyte integrin activation. Am. Rev. Respir. Dis. 158:S55.
36. Languino, L., J. Plescia, A. Duperray, A. Brian, E. Plow, J. Geltosky, D. Altieri. 1993. Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1 dependent pathway. Cell 73:1723.
37. Ingalls, R., M. Amin Arnaout, D. Golenbock. 1997. Outside-in signaling by lipopolysaccharide through a tailless integrin. J. Immunol. 159:433.[Abstract]
38. Brown, L., N. Lanir, J. McDonagh, K. Tognazzi, A. Dvorak, H. Dvorak. 1993. Fibroblast migration in fibrin gel matrices. Am. J. Pathol. 142:273.[Abstract]
39. Bunce, L., L. Sporn, C. Francis. 1992. Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15–42 of the ß chain. J. Clin. Invest. 89:842.
40. Donaldson, D., J. Mahan, D. Amrani, J. Hawiger. 1989. Fibrinogen-mediated epidermal cell migration: structural correlates for fibrinogen function. J. Cell. Sci. 94:101.[Abstract/Free Full Text]
41. Frankenberger, M., A. Pforte, T. Sternsdorf, B. Passlick, P. Baeuerle, H.W. Löms Ziegler-Heitbrock. 1994. Constitutive nuclear NF-B in cells of the monocyte lineage. Biochem. J. 304:87.
42. Kuijper, P., H. GallardoTorres, J. Lammers, J. Sixma, L. Koenderman, J. Zwaginga. 1997. Platelet and fibrin deposition at the damaged vessel wall: cooperative substrates for neutrohil adhesion under flow conditions. Blood 89:166.[Abstract/Free Full Text]
43. Shenkar, R., E. Abraham. 1997. Hemorrhage induces rapid in vivo activation of CREB and NF-B in murine intraparenchymal lung mononuclear cells. Am. J. Respir. Cell Mol. Biol. 16:145.[Abstract]
44. Simpson-Haidaris, P.. 1997. Induction of fibrinogen biosynthesis and secretion from cultured pulmonary epithelial cells. Blood 89:873.[Abstract/Free Full Text]
45. Leavell, K., M. Peterson, T. Gross. 1996. The role of fibrin degradation products in neutrophil recruitment to the lung. Am. J. Respir. Cell Mol. Biol. 14:53.[Abstract]
46. Kazura, J., J. Wenger, R. Salata, A. Budzynski, G. Goldsmith. 1989. Modulation of polymorphonuclear leukocyte microbicidal activity and oxidative metabolism by fibrinogen degradation products D and E. J. Clin. Invest. 83:1916.
47. Skogen, W., R. Senior, G. Griffen, G. Wilner. 1988. Fibrinogen-derived peptide B ß 1–42 is a multidomained neutrophil chemoattractant. Blood 71:1745.
48. Shu, H., A. Agranoff, E. Nabel, K. Leung, C. Duckett, A. Neish, T. Collins, G. Nabel. 1993. Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-B subunits in endothelial and epithelial cells. Mol. Cell. Biol. 13:6283.[Abstract/Free Full Text]
49. Dhawan, S., M. Vargo, M. Meltzer. 1992. Interactions between HIV-infected monocytes and the extracellular matrix: increased capacity of HIV-infected monocytes to adhere to and spread on extracellular matrix associated with changes in extent of virus replication and cytopathic effects in infected cells. J. Leukocyte Biol. 52:62.[Abstract]
50. Palmer, S., A. Hamblin. 1993. Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity. Clin. Exp. Immunol. 93:344.[Medline]
51. Schnapp, L., Y. Wang, A. Mosoian, P. Klotman, M. Klotman. 1997. Role of adhesion in HIV replication. Am. J. Respir. Crit. Care Med. 155:A15.
52. Baeuerle, P., D. Baltimore. 1996. NF-B: ten years after. Cell 87:13.[Medline]

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