Published online before print October 2, 2003
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4/ß1 integrin (very late antigen-4) on stimulated human neutrophils
Departments of
* Internal Medicine,
Biomedical Engineering, and
Pharmacology, University of Virginia, Charlottesville
1Correspondence: Cardiovascular Research Center, MR-5 Room 1314, 415 Lane Rd. (Box 801394), University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail: gws3u{at}virginia.edu
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4/ß1 integrin very late antigen-4 (CD49d/CD29) is up-regulated on circulating neutrophils of septic patients. Although no individual agent mimics this effect of sepsis, we now report that following priming of human neutrophils with lipopolysaccharide or tumor necrosis factor (TNF-), addition of formyl-Met-Leu-Phe (fMLP) results in a "stimulated", sepsis-like, four- to fivefold rise in CD49d expression. TNF/fMLP stimulation also produced a similar increase in CD49d-mediated adhesion of neutrophils to a vascular cell adhesion molecule-1 (VCAM-1)-coated surface. Adenosine is a naturally occurring, anti-inflammatory mediator released from injured or inflamed tissues. We observed that stimulated neutrophil CD49d expression was decreased by activation of A2A adenosine receptors (A2AAR) with the selective agonist 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylicacid methyl ester (ATL146e; EC50=6.4 nM). ATL146e (100 nM) also reduced the fraction of stimulated neutrophils that adhered to VCAM-1 from 38 ± 6% to 27 ± 5%. Inhibition of CD49d expression was equally inhibited by ATL146e, added before or after TNF priming, and was reversed by incubation with the A2AAR-selective antagonist 4-{2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl}-phenol (ZM241385; 100 nM). A suboptimal ATL146e concentration (1 nM) combined with the type IV phosphodiesterase inhibitor rolipram (100 nM) synergistically decreased stimulated CD49d expression by >50%. The cyclic adenosine monophosphate (cAMP)-dependent kinase [protein kinase A (PKA)] inhibitor H-89 (10 µM) reversed the effect of ATL146e on stimulated CD49d expression. Other means of increasing cAMP in neutrophils also decreased stimulated CD49d expression. We conclude that adenosine binding to A2AAR counteracts stimulation of neutrophil CD49d integrin expression and neutrophil binding to VCAM-1 via a cAMP/PKA-mediated pathway.
Key Words: inflammation • adhesion molecules • tumor necrosis factor • lipopolysaccharide • cytokines
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4/ß1; CD49d/CD29 integrin). Expression is stimulated, and VLA-4-dependent adhesion occurs when human neutrophils are exposed to a chemoattractant [e.g., formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or interleukin (IL)-8] in the presence of the microfilament-disrupting agent dihydrocytochalasin (DHCB). Increased VLA-4 expression is also observed on neutrophils following their emigration through endothelium [1
2
3
]. Expression of VLA-4 on neutrophils facilitates adhesion and migration through fibroblast monolayers [4
] and up-regulates neutrophil ß2 integrin expression [5
]. VLA-4 expression contributes to joint infiltration by neutrophils in a rat model of adjuvant-induced arthritis [6
] and sequestration of neutrophils within rat lung in a model of endotoxemia [7
, 8
]. Recently, it has been noted that peripheral blood neutrophils from septic patients (but not from postoperative patients or patients with local infections) have increased CD49d (the -subunit of VLA-4) expression and display increased adhesion to the VLA-4 ligand vascular cell adhesion molecule-1 (VCAM-1). In addition, exposure of neutrophils derived from healthy volunteers to plasma from septic patients increases their adhesion to VCAM-1 [9
]. No single cytokine (or other factor) has been isolated from septic patient plasma that increases neutrophil expression of CD49d. This suggests the possibility that multiple factors acting together within the blood may be required to increase CD49d expression. We and others [10
, 11
] have shown that fMLP stimulation of oxidative activity in purified human neutrophils is greatly enhanced if the cells are first primed by pre-exposure to tumor necrosis factor (TNF). We now show that this mode of neutrophil activation, i.e., lipopolysaccharide (LPS) or TNF priming followed by exposure to fMLP, mimics the effect of sepsis to stimulate cell-surface expression of CD49d and enhances adhesion of neutrophils to a VCAM-1-coated surface. Below, we refer to neutrophils that have been primed by exposure to TNF and then treated with fMLP as "stimulated."
Adenosine is an endogenous-purine nucleoside formed as the result of the breakdown of adenine nucleotides that are released from ischemic tissues, activated platelets, mast cells, and dying cells. Several recent studies show that slowing the metabolism of endogenous adenosine or treatment with exogenous adenosine receptor (AR) agonists can decrease sepsis-induced inflammatory cascades with a concomitant reduction in morbidity and mortality [12
13
14
15
16
17
18
]. Human neutrophils contain 
1000 A2AARs/cell that bind 125I-4-{2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl}-phenol (125I-ZM241385) with a dissociation constant of 0.12 nM [19
]. In vitro, A2AAR agonists act, at least in part, through cyclic adenosine monophosphate (cAMP)-mediated pathways to decrease the oxidative burst and the release of primary granule contents from stimulated neutrophils [11
, 20
21
22
]. In this study, we found that a cAMP-dependent pathway is also responsible for A2AAR-mediated inhibition of stimulated neutrophil VLA-4 expression.
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was a gift from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan; specific activity=600 pg U-1). Stock solutions were made in Hanks’ balanced salt solution (HBSS)–0.1% human serum albumin (HSA) at 2 x 105 U ml-1, aliquoted into single-day samples, and frozen at -70°C. Soluble recombinant human VCAM-1 and neutralizing antibody to human CD49d (HP 1/2) were gifts from Roy Lobb (Biogen, Inc., Cambridge, MA). 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e) was supplied by Adenosine Therapeutics, LLC (Charlottesville, VA). ZM241385 [23
] was a gift from Simon Poucher (AstraZeneca Pharmaceuticals, Cheshire, UK). 4-(3'-Cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone (rolipram) was a gift from Berlex Laboratories (Cedar Knolls, NJ). Neutrophil isolation medium (Ficoll-Hypaque) was purchased from ICN Biomedicals (Aurora, OH) and from Accurate Chemicals and Scientific (Westbury, NY). RD1-conjugated antibodies (Ab) to human CD29 (4B4), fluorescein isothiocyanate (FITC)-conjugated Ab to human CD49d (HP 2/1), FITC, and RD1-conjugated isotype control antibodies and nonconjugated mouse immunoglobulin G (IgG)2a were from Coulter/Immunotech (Miami, FL). N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was purchased from Calbiochem (La Jolla, CA). Human recombinant IL-8 was purchased from Peprotech (Rocky Hill, NJ). The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): DHCB, dibutyryl (db)-cAMP, dimethyl sulfoxide, fMLP, forskolin, LPS from Escherichia coli strain 026:B6, and Tween-20. Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Indianapolis, IN).
Neutrophil purification
Human neutrophils were purified from normal heparinized (10 U/ml) venous blood by a one-step Ficoll-Hypaque separation procedure [24
] yielding 95% neutrophil cells, >95% viable, as determined by trypan blue exclusion and containing <50 pg/ml endotoxin. Following separation, the granulocytes were washed with HBSS three times.
Neutrophil VLA-4 (CD49d/CD29) expression
Neutrophils (1x106/ml) were incubated with or without ATL146e, ZM241385, rolipram, db-cAMP, forskolin, H-89, phorbol 12-myrsitate 13-acetate (PMA), LPS [+1% pooled human serum (PHS)], TNF, DHCB, IL-8, or fMLP at 37°C in HBSS containing 0.1% HSA as stated in Results. Samples were then placed on ice, labeled with FITC-conjugated antibody to CD49d and RD1-conjugated antibody to CD29 for 30 min at 4°C in the dark or FITC- and RD1-conjugated, isotype-matched control antibodies. The samples were washed with 1 ml phosphate-buffered saline (PBS) and were then resuspended in PBS containing 0.5% paraformaldehyde. The neutrophil population was selected for acquisition and analysis by gating on forward- and side-light scatter. The fluorescence intensity was measured with a FACSCalibur flow cytometer (BDIS, San Jose, CA), and a minimum of 10,000 events were collected. Analysis was performed with CellQuest software (BDIS) at an excitation wavelength of 488 nm and emission of BP 530/30 for FITC-stained cells and an emission of BP 585/42 for RD1-stained cells. Unless otherwise stated, neutrophil CD49d/CD29 expression data are reported as neutrophil mean fluorescent intensity (MFI) as a percentage of stimulated neutrophil MFI (TNF/fMLP or PMA) corrected for nonspecific staining (by subtraction of the isotype-control MFI).
The numbers of CD49d-binding sites per neutrophil were calculated with Simply Cellular microbeads (Bangs Laboratories, Fishers, IN) according to the manufacturer’s directions. As it has been reported that many FITC-tagged HP 2/1 have been observed to contain contaminating, nonspecific FITC–IgG2a [25 ], the samples for receptor enumeration were blocked (15 min; 4°C) with untagged mouse IgG2a (10 µg/ml) before staining.
Neutrophil static firm adhesion to a VCAM-1-coated surface
Recombinant human VCAM-1 was diluted to 5 µg/ml in PBS and applied to polystyrene slides and was allowed to adsorb for 2 h at room temperature. Plates were rinsed and blocked to prevent nonspecific adhesion with 0.5% Tween 20 in PBS overnight at 4°C. The plates were incorporated as the bottom section of a parallel plate-flow chamber. Human neutrophils (2x106/ml HBSS) were incubated (37°C) with or without an antibody to CD49d (HP 1/2), isotype-matched control antibody, ATL146e, TNF, and fMLP, as stated in Results. The cells were then perfused into the chamber for 10 s and incubated without flow for 5 min. Nonadherent cells were removed by initiating a short (5-s) flow of 1 dyne/cm2 wall shear stress. The results are reported as the neutrophils adhered as a percent of those present before washing.
Statistical analysis
One-way ANOVA and the Dunnett’s or Bonferroni’s post-test analyzed data, using GraphPad PRISM software (San Diego, CA). Single comparisons were done by the paired Student’s t-test, nonlinear regression sigmoid curves were fitted to the dose-response data, and EC50 were determined using GraphPad PRISM software. Data are displayed as means ± SEM unless otherwise stated. Differences were considered significant at P < 0.05.
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Figure 1. Stimulation of CD49d expression on human neutrophils. Neutrophil CD49d expression was assayed by fluorescence-activated cell sorter (FACS) as described in Materials and Methods. Neutrophils (1x106 ml-1) were incubated ± ATL146e (100 nM; 37°C for 10 min), primed ± TNF- (10 U ml-1; 0–60 min; 37°C), or ±LPS (100 ng/ml+1% PHS; 30 min; 37°C) and were then stimulated with ±fMLP (1 µM 10 min; 37°C) or ±IL-8 (100 ng/ml; 10 min; 37°C) in the presence of ADA (1 U ml-1). Sequential addition of one compound followed by another is depicted by ">". (A) LPS and TNF prime human polymorphonuclear neutrophils for increased CD49d expression in response to fMLP stimulation. *, TNF stimulates CD49d expression (P<0.01, cf., unstimulated neutrophils); **, LPS > fMLP stimulates more neutrophil CD49d expression compared with LPS priming alone (P<0 0.001); ***, TNF > fMLP stimulates more neutrophil CD49d expression compared with TNF priming or fMLP stimulation alone (P<0.001 and P<.001, respectively; n=six–18 samples from at least three separate experiments). (B) Effect of length of priming time with TNF on fMLP-stimulated neutrophil CD49d expression (n=six–18 samples from at least three separate experiments). (C) TNF priming enhances fMLP-stimulated neutrophil CD49d expression but not CD29 expression. ATL146e inhibits TNF > fMLP-stimulated CD49d expression but not CD29 expression. Data are reported as the percent of cells in the four FACS dot-plot quadrants that are CD29+/CD49d-, CD29+/CD49d+, CD29-/CD49d-, and CD29-/CD49d+ (n=five to six samples from three separate experiments).
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Although unstimulated human neutrophils express little 4 (CD49d), they do express ß1 (CD29) integrins, and CD29 expression is not affected by stimulation with PMA or the complement component C5a [28
]. In the present experiments, we confirmed that unstimulated neutrophils (unlike the very low CD49d expression) do express CD29 (Fig. 2
). We observed a small (20%), nonsignificant increment in increased expression of CD29 upon stimulation with TNF/fMLP (P>0.05). Consequently, upon stimulation with TNF/fMLP, there were markedly fewer CD29+/CD49d- cells with a corresponding increase in double-positive cells (CD29+/CD49d+; Figs. 1C
and 2 ).
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Figure 2. ATL146e reduces increased expression of 4 (CD49d) integrin (but not ß1; CD29) on neutrophils primed with TNF and then stimulated with fMLP. Representative dot plots from one experiment are shown (n=five to six). The methods are the same as in Figure 1C
. The quadrant lines delimit the isotype-control fluorescence (lower left quadrants). Mean percent of cells within each quadrant (for all experiments) is indicated. (A) Unstimulated. (B) TNF-primed > fMLP-stimulated. (C) ATL146e (100 nM)-treated > TNF-primed > fMLP-stimulated.
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Figure 3. ATL146e treatment decreases CD49d-mediated neutrophil adhesion to a VCAM-1-coated surface. Neutrophils (1–2x106/ml) were incubated ± a neutralizing antibody to CD49d (HP 1/2; 2 µg/ml; 37°C; 10 min) or nonspecific IgG1 (2 µg/ml; 37°C; 10 min) and were then primed ±TNF (10 U ml-1; 20 min; 37°C) and stimulated ±fMLP (1 µM 10 min; 37°C) in the presence of ADA (1 U ml-1). Sequential addition of one compound followed by another is depicted by ">". (A) HP 1/2 decreased stimulated neutrophil adhesion (*, P=0.038, compared with/without HP 1/2). (B) Incubation with ATL146e (100 nM; 37°C; 10 min) before TNF priming decreased stimulated neutrophil adhesion to the VCAM-1-coated surface (**, P=0.024, compared with/without ATL146e). Each point is the mean ± SEM (n=three to six).
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Figure 4. A2AAR-mediated inhibition of CD49d expression on neutrophils. Neutrophils were incubated as in Figure 1
. Sequential addition of one compound followed by another is depicted by ">". (A) ATL146e (100 nM) inhibited TNF-primed and fMLP-stimulated CD49d expression but not PMA (100 nM)-stimulated expression (*, P<0.05, compared with/without ATL146e). (B) ATL146e (100 nM) inhibited stimulated neutrophil CD49d expression if added before or after TNF priming (**, P<0.05, compared with/without ATL146e). (C) Incubation with the selective A2AAR antagonist ZM241385 (100 nM; 10 min; 37°C) before treatment with ATL146e (100 nM) prevented ATL146e-mediated inhibition of TNF/fMLP-stimulated neutrophil CD49d expression (***, P<0.05, compared with/without ZM241385). Each point is the mean ± SEM (n=four–40).
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Figure 5. Dose-dependence of ATL146e to block neutrophil expression of CD49d. Neutrophils were incubated ±ATL146e (0–1 µM) and TNF (10 U/ml; 20 min; 37°C) followed by fMLP stimulation (1 µM; 10 min; 37°C). EC50 = 6.36 nM. Each point is the mean ± SEM (n=six–16).
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Figure 6. Effects of agents to modify protein kinase A (PKA) activity on neutrophil CD49d expression. Neutrophils were incubated as in Fig. 1
.). Sequential addition of one compound followed by another is depicted by ">". (A) A suboptimal concentration of ATL146e (1 nM) with rolipram (100 nM) synergistically decreased TNF/fMLP-stimulated neutrophil CD49d expression (*, P<0.05, compared with/without rolipram; **, P<0 0.001, compared with/without rolipram). (B) db-cAMP (1 mM) or forskolin (50 µM) decreased TNF/fMLP-stimulated neutrophil CD49d expression (*, P<0.05, compared with/without rolipram; **, P<0.001, compared with/without db-cAMP or forskolin). (C) The PKA inhibitor H-89 (10 µM; 37°C; 10 min) added before ATL146e addition reversed ATL146e inhibition of TNF/fMLP-stimulated CD49d expression (**, P<0.001, compared with/without ATL146e; ***, P<.001, compared with/without H-89). Each point is the mean ± SEM (n=six–40).
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30 min) facilitates reactive oxygen species (ROS) release and degranulation from cells subsequently excited with a second stimulus such as the chemotactic peptide fMLP [11
]. Similarly, we found in this study that although TNF and fMLP alone induced little expression of neutrophil CD49d, TNF priming followed by fMLP stimulation resulted in a four- to fivefold increase in CD49d expression. A comparable effect was observed with LPS priming (Fig. 1A)
. Hence, more than a single factor is required to stimulate CD49d expression. The relatively rapid (30 min) up-regulation of CD49d suggests that CD49d is preformed within human neutrophils. By itself, fMLP does not stimulate expression of CD49d, although it does stimulate degranulation of secondary granules [20
]. These data suggest that CD49d molecules may be sequestered within primary granules, rather than in the more labile secondary granules. Our data are in agreement with the observation that unstimulated and stimulated human neutrophils do not contain mRNA for CD49d [25
]. That is, the neutrophils are not synthesizing CD49d during stimulation. We found that stimulation of CD49d expression correlates with increased neutrophil adhesion to a VCAM-1-coated surface. This is consistent with a functional effect of VLA-4 to mediate VCAM-1-dependent adhesion (Fig. 3A) . In addition, it has been shown that VLA-4-mediated adhesion of neutrophils to VCAM-1 can initiate release of ROS and proteases with the potential to damage the underlying endothelium [31 ].
The endogenous autocoid adenosine and various synthetic A2AAR agonists reduce inflammation and decrease stimulation of neutrophil oxidative activity and primary granule release [20 21 22 , 32 , 33 ]. The present experiments extend these studies. The very potent and selective A2AAR agonist, ATL146e, by binding selectively to A2AARs, inhibited the expression of functionally active VLA-4 in neutrophils stimulated by TNF/fMLP (Figs. 3B 4C and 5) . The difference in potency of ATL146e to affect VLA-4 expression and adhesion may indicate that only a minimum number of VLA-4 receptors on the neutrophil surface are required for adhesion. Analogous to the effects of A2AAR agonists on the oxidative burst [34 , 35 ], ATL146e did not affect increased CD49d expression stimulated by PMA (Fig. 4A) nor did it matter if the A2AAR agonist was added before or after priming by TNF (Fig. 4B) . These data suggest that ATL146e inhibits the fMLP-stimulated response rather than priming by TNF.
Elevated neutrophil [cAMP]i can inhibit neutrophil activation as measured by Mac-1 expression, release of ROS, or degranulation [20 , 36 , 37 ]. By binding to A2AARs, agonists increase neutrophil [cAMP]i and inhibit neutrophil stimulation by TNF/fMLP [20 , 22 , 38 ].Three findings of the present study support the conclusion that ATL146e inhibits stimulated CD49d expression via a [cAMP]i-dependent mechanism: The effect of a suboptimal concentration of ATL146e (1 nM) was markedly enhanced by addition of the type IV phosphodiesterase inhibitor rolipram (Fig. 6A) ; treatment with the cAMP analog, db-cAMP, or with the adenylyl cyclase activator, forskolin, mimicked the effect of ATL146e (Fig. 6B) ; and the PKA inhibitor H-89 blocked ATL146e activity (Fig. 6C) .
Data from a recent clinical trial with natalizumab, a CD49d-neutralizing monoclonal Ab (mAb), demonstrate an increased rate of clinical remission and a decreased presence of various other indicators of Crohn’s disease severity [39 ]. In addition, treatment of multiple sclerosis patients with natalizumab in short-term trials results in fewer brain lesions and fewer relapses [40 ]. As natalizumab can neutralize CD49d independently of cell type, it is possible that neutralization of neutrophil VLA-4 contributes to the favorable clinical effects of natalizumab in these diseases.
As mAb treatment carries the clinical disadvantages of high cost, lack of oral availability, and the potential for the host to develop immunity to the mAb, it has been proposed that other therapies should be considered [41 ]. Stable and orally available, small molecules are attractive candidates for therapy. In addition, unlike antibodies to single antigens, synthetic, small molecules can potentially have multiple effects by inhibiting the inflammatory cascade at several levels. For example, A2AAR agonists and PKA activation decrease not only CD49d expression but also reduce neutrophil release of proteases, production of ROS, and spreading on biological surfaces [20 21 22 , 42 , 43 ]. For the purpose of treating inflammatory diseases, the anti-inflammatory effects of A2AAR activation on cells other than neutrophils may contribute therapeutic benefit(s). By binding to A2AARs, agonists decrease TNF release from monocytes/macrophages and enhance release of the anti-inflammatory cytokine IL-10 [14 , 16 , 19 , 44 45 46 47 ]. In addition, AR agonist treatment protects animals challenged with endotoxin or live bacteria [13 14 15 16 17 18 ]. It will be of interest in future experiments to determine if A2AAR agonist-mediated inhibition of neutrophil VLA-4 expression contributes to the observed beneficial effects of these compounds in sepsis.
Received June 30, 2003; revised August 28, 2003; accepted September 2, 2003.
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4-integrin: a newly identified pathway of neutrophil recruitment in critically ill septic patients Nat. Med. 7,465-470[CrossRef][Medline] production and protect mice against endotoxin challenge J. Immunol. 151,389-396[Abstract]
production and prevents lethality in endotoxemic mice Eur. J. Pharmacol. 358,261-268[CrossRef][Medline]4-integrins on human neutrophils J. Leukoc. Biol. 68,243-2504 Integrins as therapeutic targets in autoimmune disease N. Engl. J. Med. 348,68-72 production via adenosine A2a receptor-dependent and independent mechanisms FASEB J. 14,2065-2074This article has been cited by other articles:
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