Soluble vascular cell adhesion molecule (VCAM)-Fc ... - CiteSeerX

Soluble vascular cell adhesion molecule (VCAM)-Fc fusion protein induces leukotriene C4 secretion in platelet-activating factor-stimulated eosinophils Ryosuke Tsuruta, Ronald R. Cobb, Marian Mastrangelo, Elias Lazarides, and Pina M. Cardarelli Department of Biology, Tanabe Research Laboratories, San Diego, California

Abstract: Eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1) is important for cellular recruitment into allergic inflammatory sites. To determine whether eosinophil adhesion to VCAM-1 affects cell function, leukotriene C4 (LTC4) was measured. Human eosinophils were incubated with platelet-activating factor (PAF) in the presence or absence of soluble VCAM-Fc fusion protein (sVCAM-Fc) or immobilized VCAM-Fc. sVCAM-Fc induced a concentration-dependent increase in LTC4 secretion, which was dependent on the presence of PAF and not blocked by cyclic peptides shown to inhibit a4b1-dependent adhesion. Likewise, soluble ICAM-Fc induced a concentrationdependent LTC4 secretion. LTC4 secretion was induced by the calcium ionophore, A23187, and the combination of sVCAM-Fc and A23187 had synergistic properties. It is interesting to note that Mn21 or anti-b1 monoclonal antibody, TS2/16, inhibited LTC4 secretion induced by sVCAM-Fc and PAF. Eosinophil adhesion to VCAM-Fc or interleukin-1b-stimulated endothelial cells did not induce LTC4 secretion. These data suggest that sVCAM-Fcinduced LTC4 secretion depends on distinct signals from those of eosinophil adhesion. J. Leukoc. Biol. 65: 71–79; 1999. Key Words: a4b1 integrin · integrin binding · ICAM-1 · fibronectin · allergy

INTRODUCTION Eosinophils remain essential cells in the inflammatory processes of asthma and other allergic diseases. The bronchial mucosa from patients with asthma features a rich eosinophil infiltrate [1] and bronchoalveolar lavage (BAL) fluid in asthmatics has a higher proportion of eosinophils and higher levels of eosinophil cationic protein (ECP) than those in healthy subjects [2]. A positive correlation has been reported between the proportion of eosinophils in sputum and airway hyperresponsiveness [3]. Activated eosinophils release several mediators including toxic basic proteins, oxygen radicals, cytokines, and lipid mediators such as leukotriene C4 (LTC4). LTC4 induces broncho-

constriction [4] and mucus production [5], which contribute to hyperresponsiveness. Leukotriene receptor antagonists and specific inhibitors of the 5-lipoxygenase pathway have demonstrated efficacy in mild-to-moderate asthmatic patients, suggesting an important role for LTC4 in asthma [6, 7]. The sequence of events that induce activation and secretion of LTC4 by eosinophils at sites of inflammation remains unclear. Platelet-activating factor (PAF), a phospholipid mediator, induces eosinophil degranulation [8] and a transient rise in intracellular calcium concentrations [9]. PAF has been shown to enhance eosinophil formation of LTC4 induced by the calcium ionophore, A23187 alone [10, 11]. In contrast to A23187, neither PAF, complement factor 5a (C5a), nor N-formylmethionyl-leucyl-phenylalanine (fMLP) alone can induce LTC4 secretion [12, 13]. Recently, the mechanisms by which eosinophils adhere to and migrate into extracellular matrices have been elucidated [14, 15]. a4b1 Integrin, which is expressed on human eosinophils, binds to vascular cell adhesion molecule-1 (VCAM-1) [16]. Eosinophil interaction with VCAM-1 leads not only to adhesion but also to the generation of superoxide anions [17]. Eosinophil activation and degranulation after adhesion to integrin ligands such as VCAM-1 or intercellular adhesion molecule-1 (ICAM-1) is not fully understood. In this study LTC4 was selected as a measurement of eosinophil activation. To examine the relationship between binding/adhesion and activation, secretion of LTC4 by eosinophils after binding to a soluble form of VCAM-Fc (sVCAM-Fc) and immobilized VCAM-Fc was compared.

Abbreviations: VCAM-1, vascular cell adhesion molecule-1; sVCAM-1, soluble VCAM-1; VCAM-Fc, VCAM-1 Fc fusion protein; sVCAM-Fc, soluble form of VCAM-Fc; ICAM-1, intercellular adhesion molecule-1; ICAM-Fc, ICAM-1 Fc fusion protein; sICAM-Fc, soluble form of ICAM-Fc; LTC4, leukotriene C4; PAF, platelet-activating factor; BAL, bronchoalveolar lavage; ECP, eosinophil cationic protein; C5a, complement factor 5a; fMLP, N-formylmethionyl-leucyl-phenylalanine; RC*D(ThioP)C*, Arg-Cys-Asp-ThioprolineCys peptide cyclized (*) through the side chain sulfhydryls to a disulfide; ThioP, thiazolidine-4-carboxcylic acid; IL, interleukin; mAb, monoclonal antibody; Ig, immunoglobulin; EDTA, ethylenediaminetetraacetate; PBS, phosphatebuffered saline; HBSS, Hanks’ balanced salt solution; DME, Dulbecco’s modified Eagle’s; BSA, bovine serum albumin; HSA, human serum albumin; BCECF-AM, 28,78-bis(2-carboxyethyl)- 5-(and-6)-carboxyfluorescein acetoxymethyl ester; HUVEC, human umbilical vein endothelial cells. Correspondence: Pina M. Cardarelli, Ph.D., Department of Biology, Tanabe Research Laboratories, 4540 Towne Centre Court, San Diego, CA 92121. E-mail: [email protected]

Journal of Leukocyte Biology

Volume 65, January 1999

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MATERIALS AND METHODS

Eosinophil-sVCAM-Fc assay

Reagents and monoclonal antibodies

Eosinophils (2 3 106/mL in HBSS) were exposed to the indicated concentrations of soluble PAF and/or VCAM-Fc and incubated for 15 min at 37°C and 5% CO2. After this incubation, the cells were centrifuged and the supernatant was collected for LTC4 levels.

Fibronectin was generated as described [18]. A 25-amino acid CS-1 peptide (KDELPQLVTLPHPNLHGEILDVPST) and cyclic peptide (RC*D[ThioP]C) (The asterisk denotes disulfide-linked residues and ThioP is an abbreviation for thiazolidine-4-carboxylic acid) were synthesized as described [19]. PAF, C5a , A23187, and anti-IgG Fc antibody were purchased from Sigma Chemical Co. (St. Louis, MO). PAF antagonist (1-O-hexadecyl-2-acetyl-sn-glycerol-3-phospho(N,N,N-trimethyl)-hexanolamine) was purchased from Calbiochem (San Diego, CA). Human IgG Fc-portion was purchased from Accurate Chemical & Scientific Co. (Westbury, NY). Human recombinant interleukin (IL)-1b was purchased from Upstate Biotechnology (Lake Placid, NY). A blocking mouse IgG1 monoclonal antibody (mAb) recognizing a4 (HP2/1) was purchased from Immunotech (Westbrook, ME). Mouse anti-human VCAM-1 mAb (4B9) was purchased from Genzyme (Cambridge, MA). Mouse anti-human CD32 mAb was purchased from PharMingen (San Diego, CA). Anti-b1 mAb, TS2/16 was a kind gift from Dr. Sanchez-Madrid. VCAM-1 (without Fc) was a kind gift from Dr. Heinrikson from Pharmacia and Upjohn.

Cloning of VCAM-1/ICAM-1 and expression of recombinant soluble VCAM-1/ICAM-1 The VCAM-1 used in these studies is a truncated form that contains all seven extracellular immunoglobulin (Ig) domains, including the a4b1 binding sites in domains 1 and 4. The VCAM-1 was expressed using a baculovirus expression vector containing the human Fc sequence and Sf9 cells grown in SF900II SFM medium (Life Technologies, Gaithersburg, MD). Recombinant VCAM-1 protein was purified from the supernatants using Protein A affinity purification membranes using the procedures of the manufacturer (Nygene, Goldens Bridge, NY). The molecular mass of the baculovirus-produced VCAM-Fc is approximately 110 kDa. The ICAM-1 used in these studies contains the entire extracellular Ig domains and was derived from pICAM (kind gift of Dr. Brian Seed). The ICAM-1 was expressed using a baculovirus expression vector containing the human Fc sequence and Sf9 cells similar to that described above. Recombinant protein was purified as described for VCAM-1. The molecular mass of the baculovirus-produced ICAM-1 Fc fusion protein (ICAM-Fc) is approximately 70 kDa.

Human eosinophil isolation Human eosinophils were purified from peripheral blood of normal donors or donors with asymptomatic allergies by using density gradient centrifugation and negative selection with immunomagnetic beads as described by Hansel et al. [20]. One hundred milliliters of ethylenediaminetetraacetate (EDTA) -anticoagulated human peripheral blood were mixed with an equal volume of Ca21/Mg21-free phosphate-buffered saline (PBS) containing 1 mM of EDTA, overlayered onto a Percoll solution (density of 1.087 g/mL, pH 7.4), and centrifuged at 800 g for 20 min at room temperature. The resulting plasma and mononuclear cells were removed. The remaining pellet of granulocytes and erythrocytes was resuspended in 3–4 vol of 3% dextran and allowed to settle at unit gravity for 30 min at room temperature. The granulocyte-rich supernatant was separated from the erythrocyte pellet. The cells were spun at 800 g for 10 min at 4°C. The pellet was exposed to 9 vol of ice-cold sterile water for 30 s and then 1 vol of 103 PBS containing 10 mM of EDTA was added to restore isotonicity. The pellet was washed twice with PBS-BSA (1% bovine serum albumin and 1 mM EDTA in Ca21/Mg21-free PBS) at 4°C. The cell pellet was then incubated with CD16-MACS (Miltenyi Biotech, Auburn, CA) immunomagnetic microbeads on the rocker at 4°C for 25 min. The pellet was resuspended in PBS-BSA and loaded onto a magnetic separation column (Miltenyi Biotech). The immunomagnetically labeled neutrophils were retained in the magnetic field and eosinophils were eluted through the column into a collection tube. The negatively selected eosinophils were washed twice with PBS-BSA. The purity of eosinophils as judged by Luxol-fast-blue stain [21] was .98%. Eosinophils were resuspended (2 3 106/mL) in Hanks’ balanced salt solution (HBSS) or in Dulbecco’s modified Eagle’s (DME) medium containing 0.25% human serum albumin (HSA).

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Eosinophil-immobilized VCAM-Fc assay Ninety-six-well microtiter plates (PRO-BINDy assay plate, Becton Dickinson, Franklin Lakes, NJ) were coated with 50-µL aliquots of VCAM-Fc (0.3125–20 µg/mL) diluted in HBSS overnight at 4°C. The wells were washed twice with HBSS and incubated with 100 µL of HBSS containing 2.5% HSA for 2 h at 37°C. Wells were washed twice with 200 µL of HBSS before use. One hundred-microliter aliquots of eosinophils (2 3 106/mL in HBSS) were added to the wells in the presence of 1 µM PAF and incubated for 15 min at 37°C and 5% CO2. After centrifugation for 10 min at 500 g, the supernatants were collected and LTC4 levels were measured.

Eosinophil-VCAM-Fc cell adhesion assay For direct adhesion assays, eosinophils were incubated with 28,78-bis(2carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM, Molecular Probes, Eugene, OR) for 45 min. After labeling, eosinophils were washed twice with PBS-BSA and resuspended in DME medium containing 0.25% HSA at 2 3 106/mL. One hundred-microliter aliquots of eosinophils were added to the wells and incubated for 20 min at 37°C and 5% CO2. The plates were gently rinsed twice with PBS to remove nonadherent cells. The number of eosinophils adherent to VCAM-Fc was quantitated by using a CytoFluor 2300 Fluorescence Measurement System from Millipore (excitation 485 nm, emission 530 nm, sensitivity 3).

Eosinophil-endothelial cell adhesion assay Human umbilical vein endothelial cells (HUVEC) obtained from Clonetics (Walkersville, MD) were cultured (passage 4–6) for 5 days on gelatin-coated 96-well plates (Costar, Cambridge, MA) and incubated in the presence or absence of IL-1b (5 ng/mL) for 6 h. After washing the wells twice with cold HBSS, eosinophils (2 3 106/mL in HBSS) were added to the wells in the presence or absence of PAF or C5a and incubated for 15 min at 37°C and 5% CO2. After a 10-min centrifugation at 500 g, the supernatants were collected and LTC4 was measured.

LTC4 measurement LTC4 was measured by an enzyme immunoassay according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). The concentration of LTC4 was expressed in picograms per 106 cells.

Statistical analysis Data are presented as the mean 6 SE. Student’s t-test was used for paired comparison and analysis of variance (ANOVA) with repeated measures was used for comparison of more than two variables to determine significance. When significant differences were observed between groups, comparisons were made by Bonferroni’s test or Dunnett’s test. P , 0.05 was considered significant.

RESULTS sVCAM-Fc induces LTC4 secretion in PAF-stimulated eosinophils To measure eosinophil activation, eosinophils were stimulated with 1 µM PAF in the presence or absence of 20 µg/mL of sVCAM-Fc (Fig. 1A). Addition of PAF alone or sVCAM-Fc http://www.jleukbio.org

Fig. 1. (A) Effects of VCAM-Fc and PAF on eosinophil secretion of LTC4. Eosinophils were incubated with 1 µM PAF alone, 20 µg/mL of VCAM-Fc alone, 20 µg/mL of VCAM-Fc and 1 µM PAF, or 20 µg/mL of IgG Fc and 1 µM PAF for 15 min at 37°C. The supernatants were collected and LTC4 secretion was measured as described in Materials and Methods. Data are presented as the mean 6 SE from three experiments. *P , 0.001 vs. PAF, VCAM-Fc, or IgG Fc 1 PAF (by ANOVA and Bonferroni’s post test). (B) Inhibition of LTC4 secretion by a PAF antagonist. Eosinophils were preincubated with the indicated concentration of a PAF antagonist for 15 min at 4°C and incubated for an additional 15 min at 37°C with 20 µg/mL of VCAM-Fc and 1 µM PAF.

alone did not induce LTC4 secretion. In contrast, a combination of PAF and sVCAM-Fc resulted in a significant induction of LTC4 secretion. The magnitude of secretion induced by sVCAM-Fc and PAF was four times higher than that of IgG Fc and PAF. When eosinophils were pretreated with a PAF antagonist, LTC4 secretion induced by sVCAM-Fc and PAF was inhibited in a concentration-dependent manner (Fig. 1B). Because a fusion protein of sVCAM-Fc was used in these studies, the effect of the Fc fragment on secretion was evaluated by several approaches. When sVCAM-Fc (20 µg/mL) was pretreated with anti-IgG Fc antibody (0–40 µg/mL) or when eosinophil Fc receptors were blocked by pretreating eosinophils with IgG Fc (200 µg/mL), no inhibition of LTC4 secretion by sVCAM-Fc and PAF was observed (Fig. 2, A and B). Next, a comparison was made between VCAM-Fc, VCAM (without Fc), and IgG Fc-induced LTC4 secretion. The magnitude of the response was highest with sVCAM-Fc while VCAM lacking Fc gave a response that was 32–44% of sVCAM-Fc (Fig. 2C). This suggests that VCAM in the absence of Fc can indeed induce LTC4 secretion. This, together with data shown in Figure 2, A and B, suggests that the Fc fragment has a minimal effect on secretion. Secretion of LTC4 by PAF-stimulated eosinophils increased in a concentration-dependent manner in response to VCAM-Fc or ICAM-Fc (Fig. 3, A and B). LTC4 secretion appeared to plateau at 20 µg/mL of VCAM-Fc or ICAM-Fc. However, the magnitude of secretion induced by VCAM-Fc was two times higher than that of ICAM-Fc. In addition to VCAM-1, a4b1 has Tsuruta et al.

been shown to bind the CS-1 fragment of fibronectin [22]. Soluble fibronectin or CS-1 peptide had a modest effect on LTC4 secretion (Fig. 3C). This effect, however, was not statistically significant (P . 0.05).

Eosinophils adherent to VCAM-Fc do not secrete LTC4 The data thus far indicated that sVCAM-Fc could induce LTC4 secretion, so the question that was asked next was whether eosinophils adherent to VCAM-Fc could also secrete LTC4. In agreement with previously published reports [14, 17], eosinophils showed significant, spontaneous, and concentrationdependent adhesion to immobilized VCAM-Fc that was maximal at 5 µg/mL of ligand (Fig. 4A). Adhesion to VCAM-Fc was significantly inhibited by a4 integrin-specific blocking mAb or anti-VCAM mAb (Fig. 4B). Although the eosinophils could adhere to VCAM-Fc, immobilized VCAM-Fc had no effect on the secretion of LTC4 from PAF-stimulated eosinophils at any concentration tested. These results were similar for both allergic and non-allergic donors (Fig. 5A). It is interesting to note that there was a difference in basal LTC4 secretion by eosinophils between allergic and non-allergic donors. In addition, once eosinophils had adhered to immobilized VCAM-1, they were no longer competent to respond to a secondary sVCAM-Fc and PAF-induced signal to secrete LTC4 (data not shown). Eosinophil secretion of LTC4 induced by VCAM-Fc and PAF

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Fig. 2. Effects of the Fc fragment on LTC4 secretion. (A) VCAM-Fc (20 µg/mL) was pretreated with the indicated concentration of anti-IgG Fc antibody for 15 min at 4°C. The pretreated VCAM-Fc was added to eosinophils in the presence of 1 µM PAF and the cells were incubated for 15 min at 37°C. (B) Eosinophil Fc binding sites were blocked with 200 µg/mL of IgG Fc for 15 min at 4°C, and the cells were incubated for 15 min at 37°C with 20 µg/mL of VCAM-Fc and 1 µM PAF. Data are presented as the mean 6 SE from three experiments. (C) Eosinophils were incubated with 20 µg/mL of VCAM-Fc and 1 µM PAF, 20 µg/mL of VCAM and 1 µM PAF, 20 µg/mL of IgG Fc and 1 µM PAF, or 1 µM PAF alone for 15 min at 37°C. Data are presented from two experiments.

Eosinophils adherent to endothelial cells do not secrete LTC4

(PAF or C5a) eosinophils incubated on IL-1b stimulated HUVEC or non-stimulated HUVEC (Fig. 5B).

To determine whether VCAM-1 presented on an endothelial cell surface affected the secretion of LTC4, eosinophils were incubated on HUVEC-coated wells that had been pretreated with or without IL-1b for 6 h. IL-1b-treated HUVEC express not only VCAM-1 but also ICAM-1 and E-selectin [23]. No secretion of LTC4 was observed from either resting or activated

Anti-VCAM mAb and VCAM-1 cyclic peptide antagonists do not inhibit LTC4 secretion induced by VCAM-Fc and PAF To determine whether the binding of sVCAM-Fc could be inhibited by mAbs or peptides known to block a4b1-VCAM-1

Fig. 3. Effects of VCAM-Fc, ICAM-Fc, or fibronectin/CS-1 peptide on LTC4 secretion by eosinophils. Eosinophils were incubated with VCAM-Fc (A) or ICAM-Fc (B) in the presence (open circles) or absence (filled circles) of 1 µM PAF or incubated with IgG Fc and 1 µM PAF (squares). (C) Eosinophils were incubated with fibronectin (circles) or KDELPQLVTLPHPNLHGEILDVPST peptide (squares) in the presence of 1 µM PAF. Samples were incubated for 15 min at 37°C. Data are presented as the mean 6 SE from three experiments. *P , 0.01 vs. 0 µg/mL, **P , 0.05 vs. 0 µg/mL, #P , 0.05 vs. 0 µg/mL (by ANOVA and Bonferroni’s post test).

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http://www.jleukbio.org

Fig. 4. (A) Concentration-dependent effect of VCAM-1 on eosinophil adhesion. Wells were coated with various concentrations of VCAM-Fc (circles) or IgG Fc (squares). Eosinophils (2 3 105/well) were added and the plates were incubated for 20 min at 37°C. Adhesion of BCECF-stained eosinophils was quantified as described in Materials and Methods. (B) Effects of mAb on eosinophil adhesion. Eosinophils were pretreated with mAb (10 µg/mL) for 15 min, and added to wells coated with 5 µg/mL of VCAM-Fc (open bars) or IgG Fc (filled bars). Data are presented as the mean 6 SD from one experiment.

interaction [19], the effects of mAbs or cyclic peptides were evaluated. Eosinophil secretion of LTC4 in the presence of sVCAM-Fc was not affected when eosinophils were coincubated with anti-VCAM mAb or 100 µM of cyclic peptide

(RC*D[ThioP]C*), a concentration that had been shown to block a4b1-dependent adhesion of Jurkat cells (Fig. 6A). However, LTC4 secretion was significantly inhibited by an a4-specific blocking mAb. Finally, an antibody to CD32 that

Fig. 5. (A) LTC4 secretion after eosinophil adhesion to VCAM-Fc. Eosinophils (2 3 105) were added to wells coated with various concentrations of VCAM-Fc and incubated with 1 µM PAF for 15 min at 37°C. After this incubation, the plates were centrifuged and supernatant was quantified for LTC4 levels. Data are presented as the mean 6 SE from allergic (squares, n 5 2) or non-allergic donors (circles, n 5 2). (B) Secretion of LTC4 by eosinophils after adhesion to HUVEC. HUVEC were incubated with (filled bars) or without (open bars) IL-1b for 6 h at 37°C. Eosinophils were incubated with 1 µM PAF or 0.1 µM C5a and added to monolayers of HUVEC for 15 min. Data are presented as the mean 6 SE from two experiments.

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Eosinophil secretion of LTC4 induced by VCAM-Fc and PAF

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Fig. 6. (A) Effects of mAb/cyclic peptide on eosinophil secretion of LTC4. Eosinophils were pretreated with mAb (10 µg/mL) or RC*D(ThioP)C* (100 µM) for 15 min at 4°C, and incubated for 15 min at 37°C in the presence of 20 µg/mL of VCAM-Fc (open bars) or IgG Fc (filled bars) and 1 µM PAF. Data are presented as the mean 6 SE from three experiments. *P , 0.05 vs. no treatment (by ANOVA and Dunnet’s post test). (B) Effects of VCAM-1 and A23187 on LTC4 secretion by eosinophils. Eosinophils were incubated with 20 µg/mL of VCAM-Fc and 1 µM PAF, 2.5 µM A23187 alone, 20 µg/mL of IgG Fc and 2.5 µM A23187, or 20 µg/mL of VCAM-Fc and 2.5 µM A23187 for 15 min at 37°C. Data are presented as the mean 6 SE from three experiments. *P , 0.001 vs. VCAM-Fc 1 PAF, **P , 0.01 vs. A23187 or IgG Fc 1 A23187 (by ANOVA and Bonferroni’s post test).

defines the major Fc receptor on eosinophils did not block secretion, further demonstrating that the response is not due to the Fc fragment of VCAM-Fc.

activators LTC4 secretion, further suggesting that the mechanism of secretion is distinct from the classical a4b1-VCAM interactions.

A23187 acts synergistically with VCAM-Fc to induce LTC4 secretion

Human donor variability in LTC4 secretion

As shown previously [11, 12], A23187 alone significantly induced LTC4 secretion. The magnitude of the response was three times greater than that of sVCAM-Fc and PAF (Fig. 6B). The addition of both sVCAM-Fc and A23187 had a synergistic effect on LTC4 secretion and the magnitude of the response was eight times greater than that of sVCAM-Fc and PAF. VCAM-Fcinduced LTC4 secretion is inhibited in eosinophils activated with Mn21 or TS2/16 monoclonal antibody. Mn21 or TS2/16 has been reported to induce higher-affinity binding of soluble VCAM-IgG fusion protein by human T cells [24]. Therefore, we evaluated the response with human eosinophils. It is interesting to note that eosinophil secretion of LTC4 induced by either VCAM-Fc and PAF or A23187 was inhibited by Mn21 (Fig. 7A). In contrast, LTC4 secretion induced by A23187 was enhanced by TS2/16 in a concentration-dependent manner, whereas VCAM-Fc- and PAF-induced secretion was inhibited by TS2/16 (Fig. 7B). This indicates that known activations of a4b1, such as Mn21 or TS2/16, do not induce 76



The magnitude of LTC4 secretion by eosinophils was highly variable among donors. Although LTC4 secretion was generally higher among allergic donors, there was no significant difference in LTC4 secretion between eosinophils from allergic donors and cells from non-allergic donors (P 5 0.391, Table 1).

DISCUSSION The studies presented here show that a combination of PAF and soluble VCAM-Fc or ICAM-Fc leads to secretion of LTC4. Neither VCAM-Fc nor PAF alone induced secretion of LTC4. Our results showed that there were differences between PAFinduced eosinophil activation after the addition of sVCAM-Fc or immobilized VCAM-Fc. With sVCAM-Fc, activated eosinophils secreted LTC4 in a concentration-dependent manner. However, eosinophils added to plates containing VCAM-Fc or ICAM-Fc (data not shown) did not induce secretion of LTC4. Furthermore, cell adhesion to VCAM-Fc blocked subsequent http://www.jleukbio.org

Fig. 7. (A) Effects of Mn21 on LTC4 secretion by eosinophils. Eosinophils were incubated for 15 min at 37°C with 20 µg/mL of VCAM-Fc and 1 µM PAF (circles) or 2.5 µM A23187 (squares) in HBSS containing the indicated concentrations of Mn21. Data are presented as the mean 6 SE from two experiments. *P , 0.01 vs. 0 mM Mn21, **P , 0.001 vs. 0 mM Mn21 (by ANOVA and Bonferroni’s post test). (B) Effects of TS2/16 on eosinophil secretion of LTC4. Eosinophils were pretreated with the indicated dilution of TS2/16 for 15 min at 4°C, and incubated for 15 min at 37°C with 20 µg/mL of VCAM-Fc and 1 µM PAF (circles) or 2.5 µM A23187 (squares). Data are presented as the mean 6 SE from two experiments. *P , 0.001 vs. no antibodies, **P , 0.05 vs. no antibodies, 1:500 (by ANOVA and Bonferroni’s post test).

sVCAM-Fc-induced LTC4 secretion. Addition of PAF-activated eosinophils to IL-1b-stimulated endothelial cells also did not induce LTC4 secretion. Our data are in agreement with those of Nagata et al. who showed that eosinophil adhesion to VCAM-1 did not induce LTC4 secretion [17]. The fact that secretion was lower with monometric VCAM (VCAM without Fc) when compared with dimeric VCAM-Fc is consistent with published reports which show that high-avidity binding results from multivalent interactions [24]. Integrins and their ligands have been shown to provide a co-stimulatory signal leading to cellular activation. For example, neutrophil adhesion to integrin ligands induces a profound respiratory burst [25]. T cells incubated with antiCD3 mAb and VCAM-1 induce cell proliferation, whereas anti-CD3 mAb or VCAM-1 alone does not [26]. Several reports have examined integrin-mediated co-activation using eosinophils. Eosinophil secretion of LTC4 after adhesion to fibronectin was augmented by A23187 [27] or PAF [28]. In our studies, neither soluble fibronectin nor CS-1 peptide induced a statistically significant increase in secretion of LTC4 (P . 0.05, Fig. TABLE 1.

Eosinophil LTC4 Secretion from Allergic or Non-allergic Donorsa

Donor

Allergic (n 5 8) Non-allergic (n 5 13) Total (n 5 21)

LTC4 (pg/106 cells)

800.41 6 340.95 658.06 6 371.91 712.29 6 358.74

Secretion of LTC4 by eosinophils was determined after a 15-min incubation with 20 µg/mL of VCAM-Fc and 1 µM PAF. a Plus-minus values are means 6 SD. There were no significant differences between allergic and non-allergic donors.

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3C). A possible explanation for these differences is that BSA that was used to block nonspecific binding sites affected LTC4 secretion induced by immobilized fibronectin. A23187 has been shown to induce LTC4 secretion and to raise the level of intracellular calcium in human eosinophils [29, 30]. A23187 alone induced a threefold greater increase in LTC4 secretion than did VCAM-Fc and PAF (Fig. 6B). It is interesting to note that the combination of VCAM-Fc and A23187 induced an even greater LTC4 secretion than A23187 alone, suggesting a synergistic effect. Although we did not measure intracellular calcium concentration, VCAM-Fc binding might increase intracellular calcium levels or alternatively, VCAM-Fc binding might induce an intracellular activating signal without further enhancement in calcium concentration [31]. The fact that sVCAM-Fc and not immobilized VCAM-Fc induced LTC4 secretion suggests that different cellular responses occur when integrins bind to ligands in solution versus cell attachment to immobilized ligands. Indeed, recent studies support that binding and adhesion can be dissociated. Yauch et al. found that integrin a4 tail deletion markedly impaired static cell adhesion by a mechanism that did not involve altered binding of sVCAM-1 [32]. Furthermore, mutation of integrin a4 EF-hand sites impaired cell adhesion by a mechanism independent of ligand binding [33]. Miyamoto et al. showed that distinct transmembrane signals were transduced with ligand occupancy as compared to receptor aggregation [34]. Taken together, our data may indicate that eosinophil secretion of LTC4 in response to sVCAM-Fc is not mediated through the common binding site that mediates eosinophil a4b1-VCAM adhesion. This hypothesis is further substantiated Eosinophil secretion of LTC4 induced by VCAM-Fc and PAF

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by the observation that anti-VCAM mAb or the cyclic peptide (RC*D[ThioP]C*) did not block LTC4 secretion and fibronectin or CS-1 peptide induced a modest amount of secretion. Although antibodies to a4 blocked sVCAM-Fc 1 PAFmediated LTC4 secretion, neither antibodies to VCAM nor RCD containing peptides were able to block secretion. Recently, cross-linking studies with a leucine-aspartic acid-valine-based small molecule showed that the site of cross-linking was in the b1 chain mapping within residues 130–146 [35]. Likewise, it is predicted that RCD containing peptides would bind to this site. A mAb, HP2/1 has been mapped near residue 200 in integrin a4 [36]. It is possible that the interaction of a4b1 with sVCAM-Fc in the assay system described is mediated by a similar region on a4 but a distinct site on b1. Current studies are ongoing to define the precise binding site. Both Mn21 and TS2/16, which are reported to increase the affinity between sVCAM-IgG and a4b1 on T cells [24], inhibited LTC4 secretion (Fig. 7). These data imply that ligation of low-affinity state a4b1 by sVCAM-Fc induces secretion but ligation of higher-affinity a4b1 does not. The fact that TS2/16 enhanced LTC4 secretion induced by A23187 but inhibited the secretion induced by sVCAM-Fc and PAF suggests that the combination of sVCAM-Fc and PAF not only induces calcium influx but also has other secondary effects. sVCAM-Fc binding to the low-affinity state a4b1 may be necessary for inducing stimulatory signals early in the inflammatory process. LTC4 secretion may occur at an early stage before the a4b1 integrins have undergone conformational changes. Alternatively, adhesion to immobilized ligands may be the result of post-ligand binding events, such as cytoskeletal changes, which strengthen adhesion and promote cell spreading [37, 38]. LTC4 secretion in the soluble assay was highly variable among different donors. Carlson et al. showed that eosinophils from asthmatics released more ECP and eosinophil protein X than cells from healthy subjects [39]. In contrast to these data, no significant differences of LTC4 secretion were found between eosinophils from allergic donors and cells from non-allergic donors, although there was a difference of LTC4 basal secretion after adhesion to immobilized protein. The biological significance of eosinophil LTC4 secretion in response to sVCAM-Fc and not to endothelial cells expressing VCAM-1 needs to be established in vivo. Our data lead to a hypothesis that eosinophil binding to a soluble integrin ligand induces LTC4 secretion. Endothelial cells stimulated with A23187 [40] or thrombin [41] can release PAF, which can activate eosinophils. One hypothesis is that PAF-stimulated eosinophils will bind to sVCAM-1 both in the circulation and in the BAL fluid triggering LTC4 secretion. The consequence of LTC4 secretion would be an increase in vascular permeability [42]. The transmigrating eosinophils will migrate to the site of inflammation and secrete LTC4, which could potentially induce smooth muscle contraction and enhance mucous secretion [4, 5]. These events would contribute to enhanced inflammatory reactions. Soluble forms of integrin ligands have been found in the circulation or in the BAL fluid. The levels of ICAM-1 in serum from asthmatics were higher than in healthy subjects and 78



likewise, the levels of VCAM-1 in serum obtained during asthmatic episodes were higher than those obtained in stable condition [43, 44]. VCAM-1 and ICAM-1 levels in BAL fluid were also shown to be elevated after allergen challenge [45, 46]. Both endothelium and bronchial smooth muscle [47] are potential sources for sVCAM-1 in BAL fluid. However, it should be noted that the reported serum levels of VCAM-1 were 373–641 ng/mL in healthy subjects [48] and these levels are 30 times lower than what was used in the soluble assay described. We speculate that the local concentration of sVCAM-1 in close proximity to the endothelium or epithelium may be higher than those measured in the circulation or in the BAL fluid. In conclusion, sVCAM-Fc induced secretion of LTC4 in a concentration-dependent manner by PAF-stimulated eosinophils. This observation provides evidence for a potential function of soluble forms of integrin ligands in the blood or in the BAL in inflammatory diseases.

ACKNOWLEDGMENTS We express our gratitude toward Tanabe Seiyaku Co. Ltd., Japan for their continued encouragement and support in this area.

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Eosinophil secretion of LTC4 induced by VCAM-Fc and PAF

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