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. 1999 Oct;98(2):197–202. doi: 10.1046/j.1365-2567.1999.00871.x

Activation of complement receptor 3 on human monocytes by cross-linking of very-late antigen-5 is mediated via protein tyrosine kinases

B M Van Den Berg 1, R Van Furth 1, W L W Hazenbos 1,*
PMCID: PMC2326914  PMID: 10540218

Abstract

Bordetella pertussis interacts with very-late antigen-5 (VLA-5) receptors on the human monocyte resulting in cross-linking of these receptors followed by activation of complement receptor 3 (CR3) and firm adhesion of B. pertussis to these monocytes. In the present study we investigated whether protein tyrosine kinases are involved in the activation of CR3 on monocytes, which was assessed by the binding of C3bi-coated erythrocytes (EC3bi). Pre-incubation of monocytes with tyrphostin-A47, a specific protein tyrosine kinase inhibitor, before adherence of the cells to an anti-VLA-5 monoclonal antibody-coated surface, or addition of tyrphostin-A47 within 10 min of the adherence to such surface, reduced the binding of EC3bi to monocytes significantly. Pre-incubation of monocytes with tyrphostin-A47 reduced the binding of B. pertussis to such monocytes as well. Inhibitors of protein kinase A and/or C had no effect on EC3bi binding to monocytes. Cross-linking of VLA-5 on monocytes resulted in tyrosine phosphorylation of several proteins. Together, these results indicate that protein tyrosine kinases are involved in the VLA-5-induced activation of CR3 on human monocytes.

INTRODUCTION

Alveolar macrophages and exudate macrophages, which are monocyte-derived cells, play an important role in the defence against Bordetella pertussis infection. There is increasing evidence that B. pertussis ingested by mouse macrophages or human monocyte-derived macrophages can survive intracellularly.1–3

Bordetella pertussis can bind to, and subsequently become ingested by human monocytes or human monocyte-derived macrophages without the requirement of opsonization.3–8 By using mutant B. pertussis strains, it has been demonstrated that binding of non-opsonized B. pertussis to human monocytes is mediated by the virulence factors, such as filamentous haemagglutinin (FHA), fimbriae and pertactin.6 Recently, we have shown that both monoclonal antibodies (mAb) against the fibronectin receptor very-late antigen-5 receptor (VLA-5; α5β1; CD49e/CD29) on monocytes and soluble fibronectin inhibit the binding of non-opsonized B. pertussis to monocytes.4 This indicates that VLA-5 serves as a receptor for B. pertussis. Fimbriae, which are stable structures that are very resistant to disruption by various denaturing agents,9 cover the bacterial surface and upon binding, cluster VLA-5 receptors on monocytes.4,5 Next complement receptor type 3 (CR3; αMβ2; CD11b/CD18) on monocytes becomes activated, allowing FHA on B. pertussis to bind more firmly to these monocytes.4–7 The signals leading to CR3 activation after cross-linking of VLA-5, however, remain to be established.

The present contribution concerns a more detailed study on the involvement of protein tyrosine kinases in the activation of CR3 after cross-linking of VLA-5 on monocytes using, for convenience, C3bi-coated erythrocytes (EC3bi) as indicator.

MATERIALS AND METHODS

Isolation of monocytes

Mononuclear cells were isolated from buffycoats from healthy donors by Ficoll–Hypaque differential centrifugation.10 Further cell purification was performed by countercurrent centrifugal elutriation in a Beckmann J2–21 M/E centrifuge using a JE-6 Elutriation Rotor (Beckmann Instruments, Palo Alto, CA) as described.11 The resulting cell suspensions contained at least 80% monocytes, as determined by Giemsa staining, of which more than 95% were viable, as determined by trypan blue exclusion. The adherence of B. pertussis and EC3bi to cells was assessed microscopically, allowing distinction between adherence to monocytes and that to lymphocytes. The results include only the adherence of bacteria or erythrocytes to monocytes.

Monoclonal antibodies

The following mAb against human cell surface proteins were used in the form of culture supernatants or of purified immunoglobulin: SAM-112 [immunoglobulin G2b (IgG2b); 500 μg/ml] against the integrin α5 subunit (CD49e) of VLA-5 (Sanbio, Uden, the Netherlands), 15A813 (IgG1; 200 μg/ml) against the integrin α4 subunit (CD49d) of VLA-4, and NKI-M914 (IgG1; 200 μg/ml) against the integrin αV subunit (CD51) of the vitronectin receptor (CLB, Amsterdam, the Netherlands), 4G10 (IgG2b; 100 μg/ml) against phosphorylated tyrosine residues (Upstate Biotechnology Inc., Lake Placid, NY). Purified mouse IgG1 and IgG2b (Pharmingen, San Diego, CA) were used as isotype-matched controls. F(ab′)2 fragments of goat anti-mouse IgG (Cappel, Durham, NC) and peroxidase-conjugated goat anti-mouse immunoglobulin (Southern Biotechnology Associates Inc., Birmingham, AL). The final concentrations used were given in the relevant experiments.

Protein kinase inhibitors

Monocytes were treated with the following protein kinase inhibitors: 450 nm staurosporine (Calbiochem, La Jolla, CA) which is a broad-spectrum inhibitor of protein kinases,15 10 μm tyrphostin-A47 (Sigma, St. Louis, MO) which selectively inhibits protein tyrosine kinase activity,16 10 μm tyrphostin-1 (Sigma), an ineffective analogue of tyrphostin-A47, 50 μm H7 (Seikagaku Koguo Co. Ltd, Tokyo, Japan) which selectively inhibits protein kinases A and C,17 and 30 μm H89 (Calbiochem) which is a selective inhibitor of protein kinase A.18 The concentrations used were those found to be optimally effective as reported in the respective references; they did not affect cell viability as determined by trypan blue exclusion (data not shown).

Adherence of B. pertussis to monocytes

Wild-type B. pertussis strain Welcome 28 (W28)19 was labelled with fluorescein isothiocyanate (FITC; Sigma), as described.1,20 Briefly, 1×108 bacteria/ml were incubated in a solution of 1 mg FITC per ml, 50 mm sodium carbonate and 100 mm NaCl (pH 9·0) for 20 min at room temperature, washed four times and resuspended in HAP medium to a final concentration of 1×108 bacteria/ml. The bacteria were kept for 30 min at 37° until use.

Adherence of B. pertussis to cultured monocytes was assessed as described4 with some minor modifications. Wells of Terasaki plates (Greiner Labortechnik, Frickenhausen, Germany) were coated with HAP medium, i.e. phosphate-buffered saline (PBS) containing 3 mm glucose, 150 nm CaCl2, 500 nm MgCl2, 0·3 U/ml aprotinin and 0·05% (w/v) human serum albumin (HSA), to which mAb against VLA-5, VLA-4, VNR, or non-specific isotype-matched antibodies were added, or not, to a final concentration of 5 μg/ml. Plates were then incubated for 1 hr at room temperature. After three washes with PBS at room temperature, 5×103 monocytes per well were allowed to adhere for 90 min at 37°. Wells were washed three times with PBS of 37° and 5×105 FITC-labelled B. pertussis was added to each well with or without mAb against VLA-5, VLA-4, VNR, or non-specific isotype-matched antibodies to a final concentration of 5 μg/ml. After incubating for 30 min at 37°, the plates were then washed five times with PBS (37°) and then fixed for 15 min with 0·05% glutaraldehyde (Polyscience Inc., Warrington, PA). After washing twice with PBS the number of bacteria adhering to 100 monocytes was counted at a magnification of ×400. In some experiments, monocytes were preincubated for 10 min at 37° with the protein tyrosine kinase inhibitor or its inactive analogue before incubation in Terasaki plates. The inhibitors remained in the medium during the entire assay. Wells of Terasaki plates were incubated with HAP medium for 1 hr at room temperature.

Adherence of EC3bi to monocytes

Sheep erythrocytes were coated with C3bi (EC3bi) as described.21 Elutriation-purified monocytes were used to avoid interference of contaminating lymphocytes. Wells of Terasaki plates (Greiner Labortechnik) were coated with HAP medium, with or without mAb against VLA-5 to a final concentration of 5 μg/ml. Plates were then incubated for 1 hr at room temperature. Next, wells were washed three times with PBS at room temperature before 5×103 monocytes per well were allowed to adhere for 90 min at 37°. After three washes with PBS at 37°, 5×105 EC3bi were added to each well with the remaining monocytes and incubated for 45 min at 37°. Plates were then washed five times with PBS (37°) and then fixed for 15 min with 0·05% glutaraldehyde (Polyscience Inc.). After washing twice with PBS the number of EC3bi cells adhering to 100 monocytes was counted at a magnification of ×400. In some experiments, monocytes were preincubated for 10 min at 37° with various protein kinase inhibitors before incubation in Terasaki plates. The inhibitors remained in the medium during the entire assay.

Assessment of tyrosine phosphorylation of proteins after cross-linking of VLA-5

After cross-linking of VLA-5 on monocytes in suspension with antibodies, the tyrosine-phosphorylated proteins of monocytes were determined as described22,23 with minor modifications. In short, purified monocytes were suspended in RPMI-1640 medium containing 10 mm HEPES to a concentration of 5×107 cells per ml. These cells were then incubated with mAb against VLA-5 to a final concentration of 5 μg/ml for 10 min at room temperature. Next, goat anti-mouse F(ab′)2 was added to a concentration of 25 μg/ml to the medium and the cells were incubated at 37° for the indicated periods. Then, to stop activation, 80 μl of the cell suspension was mixed with 30 μl of sodium dodecyl sulphate (SDS) buffer (40% SDS, 0·2 m dithiothreitol, 20%β-mercaptoethanol, 20% glycerol and 0·01% bromophenol blue in 10 mm Tris buffer, pH 7·0), followed by incubation at 100° for 5 min. Next, 10 μl cell-lysate (5 μg protein) per lane were run on an SDS–7·5% polyacrylamide gel and the proteins were electrophoretically transferred to nitrocellulose paper (Schleicher & Scheull GmbH, Dassel, Germany). These blots were incubated with 5% dried non- fat milk (Elk; Campina Melkunie bv., Eindhoven, the Netherlands) and 0·1% Tween-20 in PBS for 1 hr at room temperature to reduce non-specific binding of antibodies. After washing with PBS containing 0·1% Tween-20, the blot was incubated for 1 hr with 1 μg of antiphosphotyrosine mAb 4G10 per ml in PBS containing 0·5% Elk and 0·1% Tween-20 for 90 min at room temperature. After washing with PBS containing 0·1% Tween-20 peroxidase-conjugated goat anti-mouse immunoglobulin antibodies were added, and the blot was then incubated for 1 hr at room temperature. The binding of the antibody to tyrosine-phosphorylated proteins was assessed using ECL Western blotting detection reagents (Amersham Life Science, Buckinghamshire, UK).

Statistical analysis

Differences between the results of the various experiments were evaluated by means of a paired two-tailed t-test. Results are shown as means and standard deviation (SD).

RESULTS

Involvement of VLA-5 on the adherence of B. pertussis to monocytes

Previously we have shown that mAb against fibronectin receptor VLA-5 on monocytes and soluble fibronectin inhibit the binding of non-opsonized B. pertussis to these cells.4 To address whether other receptors with binding affinity for fibronectin are involved in the adherence of B. pertussis on monocytes, we investigated the VLA-4 receptor and the vitronectin receptor (VNR).

When monocytes, adherent to surfaces pretreated with HAP medium, were incubated with B. pertussis in the presence of mAb against VLA-5, the bacteria bound significantly less (P<0·01) in comparison with B. pertussis in the presence of HAP medium or the isotype-matched antibody control (Table 1). The presence of anti-VLA-4 or anti-VNR mAb did not reduce the adherence of B. pertussis to such monocytes, suggesting that neither the VLA-4 nor the vitronectin receptor are involved in adherence of B. pertussis to monocytes.

Table 1.

Involvement of VLA-5 on the adherence of B. pertussis to monocytes

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Monocytes cultured on surfaces coated with anti-VLA-5 mAb revealed a significantly (P<0·01) higher binding of B. pertussis (38±12 bacteria/100 cells) in comparison to monocytes cultured on surfaces without anti-VLA-5 mAb (24×8·6 bacteria/100 cells; Table 1). When monocytes were cultured on surfaces coated with anti-VLA-4, anti-VNR mAb, or isotype-matched antibody controls, the adherence of B. pertussis was not increased. Together, these results indicate that not VLA-4 and VNR, but VLA-5 is involved in the adherence of B. pertussis to monocytes.

Involvement of protein kinases in the VLA-5-induced activation of CR3 on monocytes

In monocytes cultured on surfaces coated with anti-VLA-5 mAb, CR3 become activated by cross-linking of VLA-5 receptors at the basal site of these adherent cells.24 Activated CR3 of monocytes cultured on surfaces coated with anti-VLA-5 mAb bound significantly (P<0·05) more EC3bi (43±24 EC3bi/100 cells) in comparison to CR3 of monocytes cultured on surfaces pretreated with HAP medium (31±15 EC3bi/100 cells) (Fig. 1a,b). Pre-incubation of monocytes for 10 min with the non-specific protein kinase inhibitor, staurosporin, or with the specific protein tyrosine kinase inhibitor, tyrphostin-A47, significantly inhibited the binding of EC3bi to monocytes cultured on either surfaces coated with anti-VLA-5 mAb, or pretreated with HAP medium, compared to indicated controls (Fig. 1a,b). Neither tyrphostin-1, the inactive analogue of tyrphostin-A47, nor protein kinase A and C inhibitor H7 nor the protein kinases A inhibitor H89 reduced the binding of EC3bi to monocytes adhering to a surface coated with mAb against VLA-5 or a surface without mAb coating. These results show that tyrosine kinases are involved in the VLA-5-induced activation of CR3 on monocytes.

Figure 1.

Figure 1

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Effect of protein kinase inhibitors on the binding of EC3bi to adherent monocytes. Monocytes, preincubated with protein kinase inhibitors tyrphostin-1 (tyr1), tyrphostin-A47 (tyrA47), staurosporine (stauro), H7 or H89 were plated on surfaces pretreated with HAP medium (a) or precoated with anti-VLA-5 mAb (b). Next, EC3bi were allowed to adhere to these cells. Values are the mean±SD of at least five separate experiments. Difference in adherence of B. pertussis to monocytes and indicated control was determined by paired two-tailed t-test: *P<0·05, **P<0·01 versus indicated control.

Effect of tyrphostin on activation of CR3 on monocytes at different time-points

The kinetics of tyrosine phosphorylation by protein tyrosine kinases after cross-linking of VLA-5 on monocytes adhering to a surface coated with the mAb against VLA-5, was assessed by adding tyrphostin-A47 at various time-points. Incubation of monocytes with tyrphostin-A47 for 10 min before the adherence assay or addition of this compound at the start of this assay resulted in 60–70% inhibition of the binding of EC3bi to monocytes (Fig. 2). Addition of tyrphostin-A47 5 min after the start of the adherence assay, inhibited the binding by only 30%; addition at 10 min or later had almost no effect. Addition of the inactive analogue tyrphostin-1 at the start of this assay did not inhibit the binding of EC3bi to monocytes (data not shown). Thus, protein tyrosine kinases have only an effect during a limited period after cross-linking of VLA-5 on monocytes, indicating that activation via these kinases is a transient process.

Figure 2.

Figure 2

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Effect of tyrphostin-A47 on activation of CR3 on monocytes at different time-points. Tyrphostin-A47 was added before or during adherence of monocytes on surfaces coated with mAb against VLA-5 and the binding of EC3bi was assessed. Results of a representative experiment are expressed as the percentage inhibition of binding of EC3bi to monocytes relative to control (no tyrphostin added).

Tyrosine phosphorylation of proteins in monocytes after cross-linking VLA-5

Since tyrphostin-A47 was only effective in inhibiting CR3 activation on monocytes for a limited period after cross-linking of VLA-5, the role of protein tyrosine kinases was further assessed by phosphorylation of proteins on their tyrosine residues.

Monocytes in suspension were incubated with anti-VLA-5 mAb. Thereafter, at various intervals cross-linking was induced by adding goat anti-mouse F(ab′)2. The tyrosine-phosphorylated protein content of total monocyte-lysates was determined after their separation on a polyacrylamide gel and transfer to nitrocellulose paper, using an anti-phosphotyrosine mAb. The results showed that cross-linking of VLA-5 for 1 min led to tyrosine phosphorylation of several proteins with a molecular weight between 55 000–86 000 and 100 000–120 000, which was maximal between 2 and 10 min after cross-linking of VLA-5 (Fig. 3). Dephosphorylation of tyrosine on other proteins, for instance a 90 000 MW protein, was also observed. One of the signals which can occur upon activation of monocytes is the increase of the intracellular calcium (Ca2+) concentration. Cross-linking of VLA-5 on monocytes in suspension with mAb against VLA-5 and goat anti-mouse F(ab′)2 did not result in an increase in intracellular Ca2+ for at least 20 min (data not shown).

Figure 3.

Figure 3

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Phosphorylation of proteins on tyrosine residues after cross-linking of VLA-5 on monocytes. Cross-linking of VLA-5 receptors on monocytes in suspension was assessed by incubation of the cells with anti-VLA-5 mAb before the addition of goat anti-mouse F(ab′)2. At the indicated time-points cells were lysed and tyrosine phosphorylation in total monocyte-lysates was determined (representative experiment).

Effect of protein tyrosine kinase inhibitor in the adherence of B. pertussis to monocytes

Adherence of B. pertussis fimbriae to VLA-5 on monocytes leads to cross-linking of these receptors which results in the activation of CR3 and firm binding of bacteria via FHA to their surface.5 Cross-linking of VLA-5 on monocytes with anti-VLA-5 mAb leads also to CR3 activation which involves protein tyrosine kinases. These findings raised the question whether protein tyrosine kinases are involved in the adherence of B. pertussis to monocytes. When monocytes were preincubated with tyrphostin-A47 before allowing adherence on HAP medium pretreated surfaces, binding of B. pertussis was significantly inhibited (P<0·01) compared to monocytes incubated with the inactive analogue tyrphostin-1 or HAP medium (Fig. 4).

Figure 4.

Figure 4

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Effect of a protein tyrosine kinase inhibitor on binding of B. pertussis to adherent monocytes. Monocytes were preincubated with tyrphostin-A47 (tyrA47), tyrphostin-1 (tyr1), or HAP medium before being allowed to adhere on HAP medium-treated surfaces. Next, B. pertussis was allowed to adhere to these cells. Values are the mean±SD of at least six separate experiments. Difference in adherence of B. pertussis to monocytes and indicated control was determined by paired two-tailed t-test: **P<0·01 versus indicated control.

DISCUSSION

From the present study we conclude that protein tyrosine kinases are involved in VLA-5-mediated activation of CR3 on human monocytes. Furthermore, these kinases mediate the adherence of B. pertussis to human monocytes. These conclusions are based on the inhibition of adherence of B. pertussis and EC3bi to activated CR3 in the presence of staurosporin, a non-specific protein kinase, and tyrphostin-A47, a specific protein kinase inhibitor. Protein kinase A and C inhibitor (H7) and protein kinase A inhibitor (H89) had no effect on EC3bi binding.

Tyrphostin-A47 was most effective when added before, or at the start of, assays with monocytes adhering to a surface coated with anti-VLA-5 mAb. This indicates that tyrosine kinase-mediated activation of CR3 after cross-linking of VLA-5 is a rapid, but transient, process. This was supported by the rapid tyrosine phosphorylation of several proteins with a molecular weight between 55 000–86 000 and 100 000–120 000, which was maximal between 2 and 10 min after cross-linking of VLA-5. It remains to be established, which proteins phosphorylated on their tyrosine residues are involved in CR3 activation.

Monocytes cultured on a surface not coated with anti-VLA-5 mAb bind EC3bi via CR3, which could be inhibited by staurosporin and tyrphostin-A47. This binding activity might be due to monocyte activation as a result of the isolation procedure of these cells. For neutrophils it has been demonstrated that the Arg-Gly-Asp amino acid-containing region of C3bi binds to the β3-integrin leucocyte response integrin on these cells, followed by activation of the β2-integrin receptor CR3, which then binds another region of C3bi.25 It might be that such auto-activation also occurs in monocytes.

Besides the observed up-regulation of the expression of CR3 on neutrophils after clustering of VLA-5 and the laminin receptor VLA-6 on these cells,26 recent reports indicate also that cross-linking of various β1-integrins on several cell types induces intracellular signals which are mediated by tyrosine phosphorylation. For instance, the interaction of VLA-4 with fibronectin in combination with VLA-5 activates natural killer cells via protein tyrosine kinases,27,28 whereas cross-linking of VLA-3 on human epidermal carcinoma cells29 and cross-linking of VLA-4 on B lymphocytes as well as T lymphocytes30,31 resulted in an enhanced tyrosine kinase activity.

Our results showed that B. pertussis did not bind to two other fibronectin-binding receptors, VLA-4 and VNR, and cross-linking of these receptors on monocytes did not enhance the binding of B. pertussis. Thus, although these three integrin receptors can bind the same ligand, activation of the cell via these integrin receptors can still be specific.

Motivation for the present study was the activation of CR3 on monocytes after interaction of B. pertussis with the β1-integrin receptor VLA-5.4,5 This interaction of B. pertussis with VLA-5 on monocytes is not unique for these bacteria. Yersinia pseudotuberculosis32 and Y. enterocolitica33 can also bind to various β1-integrins on several mammalian cells via their virulence factor invasin and consequently promote their ingestion. Furthermore, Trypanosoma cruzi,34,35Leishmania sp.,36Staphylococcus aureus37,38 and various mycobacterial species39,40 can bind fibronectin and micro-organisms with bound fibronectin interact with VLA-5 and other fibronectin receptors. In this regard it is interesting that fibronectin and fimbriae of B. pertussis have similar binding specificities.41 Whether binding of micro-organisms to VLA-5 results in activation of CR3 is not known as yet.

Based on our previous studies4–7 together with the present results, we conclude that cross-linking of VLA-5 on the monocyte by B. pertussis resulting in activation of CR3 is mediated via protein tyrosine kinases.

Acknowledgments

The authors wish to express their gratitude to Peter N. Nibbering and Henry Beekhuizen for helpful suggestions. This work was supported by the National Institute of Public Health and the Environment, Bilthoven, the Netherlands.

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