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Keywords:

  • neutrophil;
  • chemotaxis;
  • integrin signaling

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Futhermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.

Abbreviations
Alt-C

alternagin-C

ECM

extracellular matrix

fMLP

N-formyl-methionyl-leucyl-phenylalanine peptide

ADAM

a disintegrin and metalloproteinase

MEK

Map kinase kinase

ERK

extracellular signal-regulated kinase

MAP

mitogen activated protein

PI3-kinase

phosphatidylinositol 3-kinase

The recruitment of polymorphonuclear neutrophils to sites of inflammation and tissue injury requires rolling on the vessel walls and subsequent migration through the vascular endothelium. Migration involves multiple neutrophil adhesion receptors, such as l-selectin for rolling and integrins for adherence and locomotion [1,2]. These adhesion receptors have counter-receptors on endothelial cells and also specific ligands that are extracellular matrix (ECM) proteins [3].

Integrins are comprised of noncovalently linked α and β chains that can associate in various combinations and thus determine the ligand-binding specificities of the intact heterodimer [4,5]. On the other hand, binding of integrins to the ECM is mediated by an integrin-recognition RGD mogif found on some ECM components such as fibronectin, vitronectin and fibrinogen [6]. Integrin–ligand binding and receptor clustering initiate a signaling cascade that involves receptor activation, increase in tyrosine kinase activity and protein phosphorylation, and reorganization of the actin cytoskeleton [5,7]. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is localized to focal adhesion sites upon clustering of integrins [7,8]. Focal adhesions contain a number of specialized cytoplasmic proteins, including talin, vinculin, α-actinin and paxillin that regulate actin cytoskeleton organization [7,9]. Focal adhesions also trigger various signal transduction events, including the activation of Src-family kinases, guanine nucleotide exchange factors, Ras-family proteins, mitogen activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-kinase) [10–12].

A significant development in the study of integrin–ligand interactions was the discovery, originally in snake venoms, of disintegrins. These peptides represent a family of cysteine-rich proteins isolated from snake venoms and are known to inhibit cell-matrix and cell–cell interactions mediated by integrins [13–15]. Most disintegrins contain an RGD/KGD sequence within an amino acid hairpin loop maintained by disulfide bridges, and are very potent inhibitors of platelet aggregation as well as cell–ECM interactions involved in tumor cell metastasis and angiogenesis [16,17]. In mammalian tissues, ‘a disintegrin and metalloproteinase’ (ADAM) proteins have been described to mediate important roles in many pathophysiological processes, including tissue development, tumor cell adhesion and inflammatory responses [18]. ADAMs are cell membrane-anchored proteins that contain metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane and cytoplasmic domains [18]. However, the physiological role of the disintegrin and cysteine-rich domains in ADAMs is not well understood.

We previously reported that jarastatin, a new RGD-disintegrin isolated from Bothrops jararaca venom, inhibited human neutrophil migration in vivo and in vitro induced by chemoattractants, and promoted actin cytoskeleton reorganization [19]. Interestingly, jarastatin is a potent chemotactic for neutrophils in vitro[19]. We also demonstrated that jarastatin and two known monomeric RGD-disintegrins, kistrin and flavoridin, affected human neutrophil chemotaxis by triggering intracellular signaling pathways mediated by integrin activation, despite kistrin and flavoridin not being chemotactic to neutrophils [20].

Recently, the disintegrin-like domain of a novel metalloproteinase (alternagin) isolated from Bothrops alternatus snake venom was purified and named alternagin-C (Alt-C) [21]. This disintegrin-like domain has an additional cysteine-rich domain, which is not found in RGD-disintegrins, and the RGD mogif is replaced by an ECD sequence. Furthermore, Alt-C was shown to be a potent inhibitor of collagen-induced adhesion by blockage of α2β1 integrin [21].

We have evaluated the effects of Alt-C on human neutrophil chemotaxis in vitro and its ability to trigger intracellular signaling pathways mediated by integrin activation. We also examined the effect of a cyclic oligopeptide corresponding to a conserved fragment containing the ECD sequence in the disintegrin-like domain. The present study demonstrates that Alt-C has a chemotactic activity on neutrophils and this effect involves actin cytoskeleton rearrangement, FAK, PI3-kinase and Erk-2 activities. Moreover, we found that ECD peptide is also a potent chemotactic and that it is able to inhibit Alt-C activity.

Disintegrin-like domain

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Alternagin-C, the processed disintegrin domain of alternagin, was isolated from Bothrops alternatus venom and purified as described previously [21]. The cyclic peptide corresponding to the disintegrin loop with the ECD sequence (CRASMSECDPAEH-NH2) was a gift from M. Juliano (Department Biofísica, UNIFESP, SP, Brazil). Alt-C and ECD peptide were diluted in sterile distilled water and stored at −20 °C until use.

Isolation of human neutrophils

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Human neutrophils were isolated from 0.5% (w/v) EDTA-treated peripheral venous blood of healthy volunteers, with previous agreement, using a four-step discontinuous Percoll (Amersham Pharmacia Biotech, San Francisco, CA) gradient [22]. Erythrocytes were removed by hypotonic lysis. Isolated neutrophils (98% purity), estimated to be > 96% viable by trypan blue exclusion, were resuspended in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO).

Neutrophil chemotaxis assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Neutrophil chemotaxis was assayed in a 48-well Boyden chamber (Neuroprobe microchemotaxis system) using a 5 µm poly(vinyl propylene)-free polycarbonate filter as described previously [19]. For chemotaxis assays, the chemotactic stimuli, N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP, 100 nm; Sigma) and different concentrations of Alt-C (0.1–1000 nm) or ECD peptide (1–1000 nm) diluted in RPMI medium, were added to the bottom wells of the chamber. Cells suspended in RPMI medium (106 cells·mL−1) were added (50 µL) to the top wells of the Boyden chamber and allowed to migrate for 60 min at 37 °C in a 5% (v/v) CO2 atmosphere. In some experiments, neutrophils were pretreated (5 min at 37 °C) with Alt-C (100 nm) or ECD-peptide (0.1–1000 nm) and allowed to migrate in the Boyden chamber toward fMLP (100 nm), Alt-C (100 nm) or ECD-peptide (1000 nm). In another set of experiments, neutrophils were preincubated with LY294002 (3 µm) or PD98059 (2 µm) (Calbiochem, San Diego, CA) for 5 min at 37 °C prior to the chemotaxis assay. After that, cells were incubated for 60 min at 37 °C in a 5% (v/v) CO2 atmosphere and the filters were removed from the chambers, fixed and stained with a Diff-Quick stain kit (Baxter Travenol Laboratories, ON, Canada). Neutrophils that had migrated through the membrane were counted under light microscopy (×100 objective) on at least five random fields. Results, expressed as the number of neutrophils per field, were representative of three different experiments performed in triplicate for each sample. Neutrophil migration toward RPMI-1640 medium alone (random movement) was used as a negative control.

Immunocytochemistry and cytochemistry assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Neutrophils (1 × 106 cells·mL−1) were incubated with 100 nm Alt-C for 5 min at 37 °C and 5% (v/v) CO2 atmosphere. Cells were then cytocentrifuged at 480 gand fixed with NaCl/Pi containing 4% (v/v) paraformaldehyde and 4% (w/v) sucrose for 20 min at room temperature. Cells were permeabilized in NaCl/Pi containing 0.1% Triton X-100 for 5 min, washed with NaCl/Pi and incubated with biotin-conjugated anti-phosphotyrosine Ig (1 : 50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Subsequently, cells were incubated with streptavidin-conjugated fluorescein isothiocyanate (1 : 50 dilution; Caltag Laboratories, Burlingame, CA) for 1 h at room temperature. To evaluate the effect of Alt-C on actin cytoskeleton network, cells were also labeled with tetramethyl rhodamine isothiocyanate (TRITC)-labelled phalloidin (1 : 1000 dilution; Sigma) for 2 h at room temperature. Slides were mounted using a solution of 20 mm propyl gallate and 20% (v/v) glycerol in NaCl/Pi. Microscopic analysis of fluorescent images was done using an epifluorescence microscope (Olympus BX40, Tokyo, Japan) equipped with appropriate filters and using ×100 oil-immersion objetives. Image capturing was performed with a cooled-charged-coupled device camera (Photometrics, Tucson, AR). Fluorescence intensity from original images was analysed by image-pro plus 4.0 (Media Cybernetics) and grey images were taken using Adobe photoshop software.

Preparation of nuclear extracts

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Nuclear extracts were obtained as described previously [20]. Briefly, neutrophils (5 × 106 cells·mL−1) were incubated with Alt-C (100 nm) for 1 h at 37 °C in a 5% (v/v) CO2 atmosphere. Cells were lysed in ice-cold buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiotreitol and 0.5 mm phenylmethane-sulfonyl fluoride) and after a 15 min of incubation on ice, NP-40 was added to a final concentration of 0.5% (v/v). Nuclei were collected by centrifugation (1810 g; 5 min at 4 °C). The nuclear pellet was suspended in ice-cold buffer C (20 mm Hepes, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiotreitol, 1 mm phenylmethanesulfonyl fluoride, 1 µg·mL−1 pepstatin, 1 µg·mL−1 leupeptin, 20% (v/v) glycerol) and incubated for 30 min. Nuclear proteins were collected in the supernatant after centrifugation (12 000 g; 10 min at 4 °C).

Immunoprecipitation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Neutrophils (5 × 106 cells·mL−1) were incubated with Alt-C (100 nm) for 5 min at 37 °C in a 5% (v/v) CO2 atmosphere. Cells were lysed in 50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1.5 mm MgCl2, 1.5 mm EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 10 µg µ−1L aprotinin, 10 µg µ−1L leupeptin, 2 µg·µL−1 pepstatin and 1 mm phenylmethanesulfonyl fluoride. Lysates (2 µg·µL−1) were incubated overnight at 4 °C with anti-FAK Ig (1 : 200; Santa Cruz Biotechnology). Then, protein A/G-agarose (20 µL·mg protein−1; Santa Cruz Biotechnology) was added and the samples were incubated at 4 °C under rotation for 2 h. The content of phosphorylated FAK and PI3-kinase associated with FAK was analyzed by Western blotting as subsequently described.

Immunoblotting analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

The total protein content in the cell extracts was determined by the Bradford method [23]. Cellular proteins (30 µg) were subjected to 12% (w/v) SDS/PAGE, transferred to poly(vinylidene difluoride) filters (PVDF Hybond-P, Amersham Pharmacia Biotech) and blocked with Tween/TBS [20 mm Tris/HCl, pH 7.5, 500 mm NaCl, 0.1% (v/v) Tween-20] containing 1% (w/v) bovine serum albumin. Primary antibodies used in Western analysis were anti-actin (diluted 1 : 500; Santa Cruz Biotechnology); anti-phosphotyrosine (diluted 1 : 200; Santa Cruz Biotechnology); anti-FAK (diluted 1 : 1000); anti-(PI3-kinase p85 subunit) (diluted 1 : 1000; Santa Cruz Biotechnology) or anti-Erk-2 (1 : 1000; Santa Cruz Biotechnology). The poly(vinylidene difluoride) filters were next washed three times with Tween/TBS, followed by 1 h incubation with the appropriate secondary antibody conjugated to biotin (Santa Cruz Biotechnology). Then, the filters were incubated with streptavidin-conjugated horseradish peroxidase (diluted 1 : 1000; Caltag Laboratories). Immunoreactive proteins were visualized by 3,3′-diaminobenzidine (Sigma) staining. The bands were quangified by densitometry, using scion image software (Scion Co, Frederick, MD, USA).

Effect of Alt-C on human neutrophil chemotaxis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

To evaluate the effect of Alt-C as a direct chemotatic stimulus for human neutrophils in vitro, the cells were allowed to migrate toward different concentrations of Alt-C (0.1–1000 nm). Figure 1A shows that Alt-C induced significant chemotaxis of neutrophils in a concentration-dependent manner. The chemotatic activity of Alt-C (100 nm) was similiar to fMLP (100 nm), a classic chemotactic agent.

image

Figure 1. Effect of Alt-C on human neutrophil chemotaxis. (A) Alt-C induces chemotaxis in a dose-dependent manner. The cells were allowed to migrate in a Boyden chamber toward medium alone (RPMI, control, ▮), fMLP (100 nm, •) or Alt-C (▴). Data shows mean ± SD from three independent experiments. *P < 0.05 compared to control. (B) Alt-C inhibits fMLP- and Alt-C-induced chemotaxis. Cells were incubated in presence or absence of Alt-C (100 nm) for 5 min at 37 °C. Neutrophil chemotaxis was evaluated in a Boyden chamber toward medium alone (RPMI), fMLP (100 nm) or Alt-C (100 nm). Data shows mean ± SD from three independent experiments. *P < 0.05 compared to control.

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We also examined the effect of alternagin-C on neutrophil chemotaxis induced by fMLP and by itself. Neutrophils were pretreated with Alt-C (100 nm) for 5 min, and the cells were allowed to migrate toward fMLP or Alt-C in a Boyden chamber (directional cell movement). In the presence of Alt-C, the chemotactic response of neutrophils to fMLP was significantly inhibited, and we observed the same effect in response to Alt-C (Fig. 1B). In addition, pretreatment of neutrophils with Alt-C did not affect the random cell movement.

Effect of ECD-peptide on human neutrophil chemotaxis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

To better understand the mechanism of action of Alt-C, the activity of a synthetic peptide containing the ECD sequence was examined in chemotaxis assays. This peptide was synthesized based on the disintegrin-like domain and cyclized by a disulfide bond between the two cysteines. Cells were allowed to migrate toward different concentrations of ECD-peptide (1–1000 nm), placed in the bottom wells of the Boyden chamber. The ECD-peptide showed a chemotactic effect only at the highest dose used (Fig. 2A; 1000 nm).

image

Figure 2. Effect of ECD-peptide on Alt-C-induced human neutrophil chemotaxis. (A) ECD-peptide induces chemotaxis. The cells were allowed to migrate in a Boyden chamber toward medium alone (RPMI, control, ▮), fMLP (100 nm, •) or ECD (◆). Data shows mean ± SD from three independent experiments. *P < 0.05 in comparison with control. (B) ECD-peptide inhibits Alt-C-induced chemotaxis. Neutrophils were preincubated in presence or absence of ECD-peptide (0.1–1000 nm) for 5 min at 37 °C and then allowed to migrate in a Boyden chamber toward medium alone (RPMI, control, ▮) or Alt-C (100 nm, ▴). Data shows mean ± SD from three independent experiments. *P < 0.05 in comparison with Alt-C-incuced chemotaxis. (C) ECD-peptide inhibits fMLP-induced chemotaxis. Cells were incubated in absence or presence of ECD-peptide (1000 nm) for 5 min at 37 °C. Neutrophil chemotaxis was evaluated in a Boyden chamber toward medium alone (RPMI) or fMLP (100 nm). Data shows means ± SD from three independent experiments. *P < 0.05 compared to random movement.

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The inhibition of a chemotactic effect by prior exposure to structurally related and unrelated chemotactic factors has been already described for known neutrophil activators [19,20]. In this regard, the effect of ECD-peptide on neutrophil chemotaxis induced in vitro by Alt-C or fMLP was also investigated. In the presence of ECD-peptide, neutrophil chemotaxis in response to Alt-C (100 nm) was significantly inhibited at all studied doses (Fig. 2B). When the random cell movement was analysed in the presence of ECD-peptide, no alterations were observed (Fig. 2C). However, the chemotactic response of neutrophils to fMLP was completely blocked by ECD-peptide (Fig. 2C).

Alt-C-induced rearrangement of neutrophil actin network

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

It has been reported that some neutrophil activities, such as chemotaxis and phagocytosis, are mediated by cytoskeletal actin polymerization [24]. Neutrophils were incubated with Alt-C (100 nm) and the alterations of neutrophil actin cytoskeleton network were analyzed by TRITC–phalloidin staining. Figure 3 shows that Alt-C was able to induce a marked increase in the F-actin content in these cells (Fig. 3B) when compared with nonstimulated cells (Fig. 3A). When the fluorescence intensity was measured, Alt-C induced 77% more actin polymerization in comparison to the control (nontreated cells: 30.10 ± 4.35; treated cells: 53.41 ± 6.02; in arbitrary units, P < 0.05) (Fig. 3C). This suggests that the motile activities of neutrophils induced by Alt-C are driven by actin cytoskeleton dynamic rearrangement.

image

Figure 3. Alt-C increases actin cytoskeleton polymerization on human neutrophils. Neutrophils were incubated with Alt-C (100 nm) for 5 min at 37 °C and actin filaments were stained with TRITC-phalloidin for 2 h at room temperature. (A) Control and (B) Alt-C incubated neutrophils labelled with TRITC-phalloidin. (C) Fluorescence intensity with mean ± SD from three independent experiments. *P < 0.05 compared to control values.

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Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

We evaluated the involvement of tyrosine kinase pathways in the neutrophil activation induced by Alt-C. The alterations in protein tyrosine phosphorylation were analyzed by immunocytochemistry. As shown in Fig. 4B, Alt-C (100 nm) was able to increase the content of phosphotyrosine when compared with nonstimulated neutrophils (Fig. 4A). The immunoreactivity to phosphotyrosine was 80% greater in cells treated with Alt-C when compared to the control (nontreated cells: 51.56 ± 8.16; treated cells: 93.00 ± 10.05; in arbitrary units, P < 0.05) (Fig. 4C).

image

Figure 4. Involvement of tyrosine kinase activity in the effect of Alt-C on human neutrophils. Neutrophils were incubated with Alt-C (100 nm) for 5 min at 37 °C, permeabilized with 0.1% (v/v) Triton X-100, incubated with anti-phosphotyrosine Ig conjugated to biotin followed by streptavidin-conjugated fluorescein isothiocyanate. (A) Control and (B) Alt-C incubated FITC-labelled neutrophils. (C) The measure of fluorescence intensity with mean ± SD from three independent experiments. *P < 0.05 compared to control values.

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Alt-C induced FAK and PI3-kinase activation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Focal adhesion contacts are comprised by integrins, cytoskeletal proteins and FAK association followed by FAK autophosphorylation and activation [7]. By immunoprecipitation of FAK, we determined its activation by the increase in the content of phosphotyrosine. As shown in Fig. 5A, incubation of neutrophils with Alt-C (100 nm) was able to increase FAK phosphorylation. The tyrosine phosphorylation of FAK generates docking sites for several proteins containing Src homology 2 (SH2) domains, as the p85 regulatory subunit of PI3-kinase [25]. PI3-kinase activation can modulate some cellular responses such as cell migration [26]. Activation of PI3-kinase was evaluated by its p85 subunit association with FAK in cells treated with Alt-C. Figure 5B shows that Alt-C induced an increase in the PI3-kinase association with FAK.

image

Figure 5. Involvement of FAK and PI3-kinase in the effect of Alt-C on human neutrophils. (A) Alt-C induces FAK activation. FAK phosphorylation was determined in neutrophils incubated with Alt-C (100 nm) for 5 min at 37 °C in the presence or absence of LY294002 (3 µm). Cell lysates were immunoprecipitated with anti-FAK Ig and blotted with either anti-phosphotyrosine or anti-FAK Igs. IP, immunoprecipitation; WB, Western blotting. Blots were analyzed by densitometry and the content of phosphorylated FAK was expressed in densitometric units. Data show mean ± SD from three independent experiments. *P < 0.05 compared to cells incubated with medium alone (Control) (B) Alt-C increases FAK-associated PI3-kinase. Cell lysates of neutrophils incubated with Alt-C (100 nm) for 5 min at 37 °C in the presence or absence of LY294002 (3 µm) were immunoprecipitated with anti-FAK Ig and then blotted with anti-FAK or anti-PI3-kinase p85 subunit Igs. IP, immunoprecipitation; WB, Western blotting. Blots were analyzed by densitometry and the content of PI3-kinase associated to FAK was expressed in densitometric units. Data show mean ± SD from three independent experiments. *P < 0.05 compared to cells incubated with medium alone (Control). (C) Alt-C-induced chemotaxis is reduced by a PI3-kinase inhibitor. Neutrophils were preincubated for 5 min at 37 °C with LY294002 (3 µm) and then allowed to migrate in a Boyden chamber toward Alt-C (100 nm). Data show mean ± SD from three independent experiments. *P < 0.05 compared to control.

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Confirming the involvement of the PI3-kinase pathway in the chemotactic effect of Alt-C on human neutrophil chemotaxis, a specific PI3-kinase inhibitor, LY294002, completely blocked Alt-C-induced chemotaxis (Fig. 5C).

In addition to the determination of FAK and PI3-kinase activation in response to Alt-C-induced chemotaxis, we analyzed the effect of PI3-kinase inhibitor on FAK phosphorylation and FAK-associated PI3-kinase upon stimulation with Alt-C. As shown in Fig. 5A,B, the levels of FAK phosphorylation and PI3-kinase association with FAK in LY294002-treated cells were similar to those in control cells.

Alt-C-induced activation of Erk-2

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

It has been reported that some disintegrins are able to increase the activation and translocation of Erk-2 from the cytoplasm to the nucleus [20]. Here, we investigated whether Alt-C would be able to induce nuclear translocation of Erk-2 in human neutrophils. Figure 6A shows that Erk-2 could be observed in nuclear extracts from control cells. However, incubation of neutrophils with Alt-C (100 nm) for 1 h induced a significant increase on Erk-2 nuclear translocation when compared to nontreated cells (Fig. 6A).

image

Figure 6. Involvement of Erk-2 activity in the effect of Alt-C on human neutrophils. (A) Alt-C induces Erk-2 nuclear translocation. Neutrophils were incubated with medium alone (Control) or with Alt-C (100 nm) for 1 h at 37 °C in the presence or absence of LY294002 (3 µm) or PD98059 (2 µm). The nuclear content of Erk-2 was determined by immunoblotting using an anti-Erk-2 Ig and quangified by densitometry. WB, Western blotting. Data show mean ± SD from three independent experiments. *P < 0.05 compared to cells incubated with medium alone (Control). (B) Alt-C-induced chemotaxis is reduced by a MEK inhibitor. Neutrophils were preincubated for 5 min at 37 °C with PD98059 (2 µm) and then allowed to migrate in a Boyden chamber toward Alt-C (100 nm). Data show mean ± SD from three independent experiments. *P < 0.05 compared to control. The # indicates that LY294002 significantly increased Alt-C-induced Erk-2 nuclear translocation (P < 0.05).

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FAK activation mediates the Ras/Raf/MAP kinase kinase (MEK) signal transduction cascade leading to Erk-2 activation and regulating cell motility [27]. To investigate the involvement of the Ras/Raf/MAP kinase pathway in the effects of Alt-C on neutrophil chemotaxis, cells were preincubated with PD98059, a MEK inhibitor, and then allowed to migrate toward Alt-C. Figure 6B demonstrates that chemotaxis induced by Alt-C was significantly inhibited by PD 98059 (24% inhibition).

As shown in Fig. 6A, preincubation of neutrophils with PD98059 abolished Erk-2 nuclear translocation. To provide further insight into the regulation of Alt-C-induced Erk-2 activation, we determined whether PI3-kinase activity is a prerequisite for Erk-2 activation. Figure 6A shows that nuclear translocation of Erk-2 in response to Alt-C was significantly increased by 38% in neutrophils by exposure to PI3-kinase inhibitor.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References

Cell adhesion to the ECM is primarily mediated by the binding of cell surface integrins to the RGD mogif found on ECM proteins [6]. Disintegrins mostly express an RGD sequence at an integrin-binding loop. The type and position of the amino acids flanking the RGD mogif determine the selectivity of disintegrin interaction with integrin [15,18]. The understanding of the precise mechanism of action and structure of disintegrins will provide information about adhesive ligands and their integrin receptors. We previously reported that RGD-disintegrins interfered with neutrophil chemotaxis induced by chemoattractants [19,20]. It has been postulated that disintegrins are approximately 1000-fold more potent than linear RGD-containing peptides, being determined by the conformation of the RGD amino acid sequence within their structures [13–15]. In the present study we have investigated the effects of a disintegrin-like protein, Alt-C, on neutrophil activation and function. Alt-C has been described to inhibit collagen-induced adhesion of cells expressing α2β1 integrin in a dose-dependent manner [21]. Alt-C also has a cysteine-rich disulfide-bonding pattern and the primary structure containing an ECD sequence presents homology with the disintegrins [21]. The results demonstrated that Alt-C strongly induced human neutrophil chemotaxis in vitro. Furthermore, this protein inhibited chemotaxis of neutrophils induced by fMLP and by itself. We exam-ined the effect of a synthetic ECD-peptide on neutrophil chemotaxis in vitro. The peptide induced neutrophil chemotaxis and also had the ability of inhibiting Alt-C-induced chemotaxis. These data strongly suggest that the chemotactic effect of Alt-C appears to be mediated by ECD sequence conformation. It has been demonstrated that synthetic peptides having the sequence RSECD inhibit collagen-induced platelet aggregation [28]. Thus, our results indicate that Alt-C may interact with adhesive receptors on the neutrophil surface inducing cell activation and leading to desensitization of the receptor to other chemotactic stimuli after prior stimulation. These findings are in agreement with previous studies describing that neutrophil migration can be inhibited or desensitized to a given chemoattractant by prior exposure to the same agonist (homologous desensitization) or to unrelated chemotactic factors (heterologous desensitization) [20,29].

Different chemotactic and phagocytic stimuli generate dynamic alterations in the actin cytoskeleton network in neutrophils [30]. Integrins induce assembly of actin filaments and high-order structures, such as focal adhesions, in response to extracellular stimuli and during cell adhesion and migration [reviewed in 31]. We previously showed that RGD-disintegrins are able to induce the integrin activation and rearrangement of the actin cytoskeleton in human neutrophils [19,20]. Because Alt-C causes remarkable neutrophil chemotaxis, we examined whether Alt-C also produces changes in cytoskeletal F-actin. Treatment of neutrophils with Alt-C induced profound alterations in the actin network with an increase of F-actin content, suggesting that the Alt-C effect on neutrophils could involve integrin-mediated pathways.

Neutrophil functional responses that require cytoskeletal reorganization, such as adhesion to the endothelium and ECM, cell migration and phagocytosis, result in the activation of protein tyrosine kinases [31–33]. Interaction between integrins and ligands leads to a profound increase in tyrosine phosphorylation of several cellular proteins. It is well established that simple dimerization of integrins is sufficient to initiate tyrosine phosphorylation events [11]. This has been accomplished with crosslinked anti-integrin Igs, multimeric integrin ligands [34,35] as well as disintegrins, potent inducers of conformational changes in both subunits of integrins [36]. Results reported here show that Alt-C induces an increase in tyrosine kinase activity and tyrosine phosphorylation.

One of the initial events in integrin-mediated signaling is the activation of FAK, resulting in its autophosphorylation [7]. This is supported by the findings that distinct disintegrins binding to integrin stimulate FAK activity [20] and that activated FAK might mediate signal transduction in a manner similar to that of integrins. According to this, the present study demonstrated that Alt-C was able to induce an increase in phosphotyrosine content of FAK and that FAK phosphorylation may be directly involved in the activation of the migratory process in response to Alt-C. A recent report showed that FAK phosphorylation is directly required for neutrophil chemotaxis by using a dominant negative mutant of FAK [37]. Interestingly, it also has been described that FAK-deficient cells exhibit an elevated number of focal adhesions accompanied by a decrease rate of cell migration [38]. Furthermore, FAK, as a nonreceptor tyrosine kinase that associates with the cytoplasmic domain of integrins at focal adhesions, might be critical for cytoskeleton reorganization [7,9]. Earlier studies have demonstrated that two cytoskeletal proteins, paxillin and tensin, are substrates for FAK, which could account for a role of FAK in actin cytoskeleton assembly and disassembly [7,9]. In the present study we provide evidence of a link between FAK activation and rearrangement of the actin cytoskeleton in neutrophils immediatelly following stimulation with Alt-C, an ECD-disintegrin.

FAK is also considered a focal adhesion docking protein that recruits PI3-kinase and other signaling molecules to form a multimolecular complex, altering their activities [10,12,25]. In Alt-C-stimulated neutrophils, PI3-kinase was found to be associated with FAK. Therefore, it is reasonable to postulate that this association promotes PI3-kinase activation, which correlates with a variety of cellular responses to external stimuli including chemotaxis, which was completely blocked by a PI3-kinase inhibitor. These results are in agreement with previous studies showing that neutrophils lacking PI3-kinase failed to orient toward different chemotactic stimuli [39,40]. Thus Alt-C-induced neutrophil chemotaxis could be driven by PI3-kinase activation, which associates with autophosphorylated FAK through their SH2 domains.

FAK activation may also trigger the Ras signal transduction cascade [11]. Downstream signal molecules such as Erk-2 have also been implicated in the regulation of the neutrophil effector functions [41]. Our study revealed that Alt-C can induce Erk-2 activation, as observed by its translocation to the nucleus. Activation of Erk-2 is often associated with enhanced myosin light chain kinase activity and increased migration [27]. The effect of Alt-C on neutrophil chemotaxis was partially reversed by PD98059, a MEK inhibitor, supporting a role for Erk-2 in Alt-C-induced neutrophil migration. These findings suggest that activation of Erk-2 induced by Alt-C may function as a positive regulator of migration. Recently, some paradoxical findings have reported the effects of different RGD-disintegrins on cell migration supporting the role for Erk-2 as a positive or negative effector [20,42]. In addition to Alt-C-induced Erk-2 activation that accounts for its positive effect on neutrophil chemotaxis, other cellular responses may be related to this pathway. Along this line, our results also demonstrated that PI3-kinase inhibition is accompanied by an increase of Erk-2 nuclear translocation suggesting a modulatory role of PI3-kinase signaling pathway on Erk-2 activity. Studies on the expression of cytokines and chemokines and on neutrophil apoptosis are under investigation.

The present study provides evidence that Alt-C, a disintegrin-like protein presenting an ECD mogif, interacts with neutrophils promoting integrin-mediated signaling and inducing chemotaxis. Our study elucidates the mechanism of action of Alt-C, as well as establishes a potential model for the design of new therapeutic interventions in disorders involving leukocyte dysfunctions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Disintegrin-like domain
  5. Isolation of human neutrophils
  6. Neutrophil chemotaxis assay
  7. Immunocytochemistry and cytochemistry assays
  8. Preparation of nuclear extracts
  9. Immunoprecipitation
  10. Immunoblotting analysis
  11. Statistical analysis
  12. Results
  13. Effect of Alt-C on human neutrophil chemotaxis
  14. Effect of ECD-peptide on human neutrophil chemotaxis
  15. Alt-C-induced rearrangement of neutrophil actin network
  16. Involvement of tyrosine kinase pathways in neutrophil activation by Alt-C
  17. Alt-C induced FAK and PI3-kinase activation
  18. Alt-C-induced activation of Erk-2
  19. Discussion
  20. Acknowledgements
  21. References
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