PERSPECTIVES
Fast Flowing Eosinophils
Signals for Stopping and Stepping Out of Blood Vessels

David Broide

Department of Medicine, University of California San Diego, La Jolla, California

	Article

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The recruitment of eosinophils to sites of allergic inflammation in vivo is a multistep process characterized by initial eosinophil intravascular tethering and firm adhesion to endothelium, followed by sequential eosinophil diapedesis between endothelial cells and chemotaxis into tissues (1). The selective tissue recruitment of eosinophils is controlled by multiple factors, including the profile of eosinophil adhesion receptors, the density of their ligands expressed on endothelial cells, as well as the tissue- and disease-induced expression patterns of chemokines. The importance of eosinophil and endothelial cell adhesion receptor interactions to eosinophil recruitment in vivo is suggested from studies using animal models of allergic inflammation in which inhibition of either eosinophil or endothelial adhesion molecules significantly reduces eosinophil tissue recruitment (4). Studies using intravital videomicroscopy to visualize eosinophil endothelial interactions under conditions of blood flow in vivo have confirmed that eosinophil adhesion to endothelium occurs via a series of discrete steps, which include initial eosinophil tethering to endothelium, activation-dependent adhesion, and transendothelial migration (7). The eosinophil adhesion receptors subserving these functions in vivo have been identified in mice treated with neutralizing antibodies to adhesion molecules, or in adhesion molecule-deficient mice. These eosinophil adhesion receptors comprise receptors allowing eosinophils to tether to endothelium (L-selectin, PSGL-1, 41, 47) (7) and receptors mediating eosinophil firm adhesion to endothelium (2 integrin, 41, 47) (10). Although in vitro and in vivo studies have provided important insight into the receptors used by eosinophils to tether to, and to firmly adhere to, endothelium, more recent studies are providing insight into the signals that mediate the ability of eosinophils to rapidly change from rolling along the endothelial surface of a blood vessel to firmly adhering to the interior of the blood vessel and subsequently transmigrating into tissues.

Progression of circulating leukocytes, including eosinophils, from the bloodstream into inflamed tissues, occurs by a defined sequential series of activation and adhesion steps to endothelium. A variety of different chemotactic mediators (eotaxin, PAF, IL-8, LTB4, C5a, FMLP) can induce activation-dependent adhesion of leukocytes to endothelium under flow conditions in vitro (11). These chemotactic mediators probably act through a common leukocyte cellular signaling mechanism, as all the aforementioned chemotactic mediators bind to seven transmembrane receptors coupled to the pertussis toxin-sensitive  subunit of the G protein Gi (Gi a) (14, 15). Chemokines can potentiate VLA-4-mediated tethering to endothelium within < 0.1 s of contact through Gi protein signaling (16). Interestingly, in the absence of G(q) signaling, eosinophils failed to accumulate in the lungs following allergen challenge, which is attributed to the failure of hemopoietically-derived cells to elaborate GM-CSF in the airways (17). In studies using a single cell adhesion assay, GM-CSF has also been shown to upregulate the biophysical strength of eosinophil adhesion to VCAM-1, suggesting another pathway for G(q) signaling to influence eosinophil adhesion to endothelium (18). Although activation-dependent arrest of rolling leukocytes has been a part of the paradigm of leukocyte recruitment for many years, in vivo evidence to support the hypothesis that endothelial proteoglycans present chemokines to rolling leukocytes in vivo is not yet available. The current paradigm suggests that chemokines released locally at sites of inflammation bind to endothelial proteoglycans (i.e., CD44, heparan, etc.) on the lumenal surface of blood vessels to promote the conversion of a leukocyte from a rolling interaction with endothelium to a leukocyte firmly adherent to endothelium (19). The chemokine would be retained on the lumenal surface of the endothelium by binding to endothelial proteoglycans, and through this interaction it would not be washed away by blood flow. Leukocytes rolling along the endothelium would thus bind chemokines presented on endothelial proteoglycans. Chemokines would then rapidly upregulate leukocyte  integrin function and induce activation-dependent firm adhesion of the circulating leukocyte to endothelium. Firm arrest of leukocytes occurs very rapidly in vitro, often within seconds, and is dependent upon the binding of leukocyte integrins (2 and/or 1) to endothelial immunoglobulin superfamily adhesion molecules (ICAM-1, and/or VCAM-1). The in vitro evidence supporting this hypothesis includes the demonstration that chemokines bind to proteoglycans present on the lumenal surface of endothelium (i.e., RANTES, MIP-1 binds to CD44) (19), and flow chamber studies demonstrating that chemoattractants (IL-8, PAF, MIP-1) can induce integrin-mediated leukocyte adhesion to ICAM-1 or VCAM-1 (11). However, in those studies, the chemoattractants were added exogenously to endothelium, and not endogenously generated to be presented by endothelium, raising concerns that the concentration of chemoattractants that flowing or rolling leukocytes might actually encounter in vivo may be much lower than when the chemokine is added exogenously in vitro. Therefore, it is still unclear whether allergen-stimulated expression of chemoattractants presented by endothelial proteoglycans actually stimulates eosinophil integrin avidity changes under conditions of blood flow in vivo.

Several studies have focused on the role of eotaxin in activation-dependent adhesion of eosinophils to endothelium (13, 23, 24). The eotaxins are a family of CC chemokines that coordinate the recruitment of eosinophils to sites of allergic inflammation. The three known members of the eotaxin family (eotaxin-1, eotaxin-2, and eotaxin-3) have low sequence homology, with eotaxin-1 being encoded on a different chromosome (17q) from that of eotaxin-2 and eotaxin-3 (7q). All three eotaxins signal through cell surface CCR-3 receptors and are potent eosinophil chemoattractants in vitro. The three eotaxins have also been studied in terms of their effect on eosinophil adhesion to endothelium and on transmigration across endothelium. Some, but not all, studies suggest that eotaxin promotes activation-dependent adhesion of eosinophils to endothelium under conditions of flow (13, 23, 24). In vitro flow chamber studies have demonstrated that eosinophils pretreated with anti-CCR3 antibodies have an approximately 22% inhibition of eosinophil activation-dependent adhesion to endothelium stimulated with TNF (13). On the one hand, this study supports the concept of cytokine-activated endothelial cells inducing CCR-3-dependent eosinophil adhesion under conditions of flow. However, the relatively modest contribution of the CCR-3 receptor to eosinophil activation-dependent adhesion suggests that alternate CCR-3-independent pathways are also operative. Some studies have also demonstrated a role for eotaxin in de-adhesion from endothelium as opposed to adhesion to endothelium. For example, flow chamber studies have demonstrated that eotaxin-2 induces de-adhesion of eosinophils bound to VCAM-1 (23), whereas static adhesion studies of eosinophils bound to the CS-1 region of fibronectin have demonstrated that eotaxin induces a transient increase in the strength of adhesion followed by a sustained reduction in eosinophil adhesion to CS-1 (25). Therefore, the relative contribution of CCR-3 versus non- CCR-3 pathways to eosinophil activation-dependent adhesion or de-adhesion from endothelium in vivo is not yet known. Eosinophil activation-dependent adhesion to endothelium will likely share some common pathways with neutrophils (i.e., the PAF receptor is expressed by both neutrophils and eosinophils) as well as have unique pathways (i.e., CCR-3 is expressed by eosinophils but not by neutrophils).

In this issue of the AJRCMB, Tachimoto and coworkers provide interesting evidence that eotaxin-2, acting via MAP kinases, may facilitate eosinophil recruitment at sites of allergic inflammation by shifting their adhesion molecule usage away from VCAM-1-dominated to ICAM-1-dominated pathways (26). Using a flow chamber, they demonstrate that eosinophils accumulate well on VCAM-1, but poorly on ICAM-1. When eotaxin-2 was co-immobilized with each of these endothelial adhesion molecules, fewer cells adhered to VCAM-1 and more adhered to ICAM-1. As CCR-3 activation by eotaxin induces downregulation of eosinophil 1 integrin function and upregulation of 2 integrin function, eotaxin-2 is likely to be shifting adhesion molecule usage away from VCAM-1-dominated to ICAM-1-dominated pathways by regulating changes in eosinophil  integrin function. Eotaxin-2 is constituitively expressed and can be induced by allergen challenge (27), suggesting a potential role for eotaxin-2 in eosinophil adhesion receptor usage in vivo. The differences in levels of eosinophil adhesion to VCAM-1 and ICAM-1 in the absence of eotaxin-2 (26) is likely due to the difference in tethering and firm adhesive functions of eosinophil 1 and 2 integrins. Eosinophils express 41 (the counterligand for VCAM-1), which is able to subserve both the initial tethering function required to slow down eosinophils under conditions of flow, and the subsequent firm adhesion step required for the eosinophils to firmly adhere to VCAM-1 (7). In contrast, under conditions of flow, eosinophils accumulate poorly on ICAM-1, even though they express 2 integrin counterreceptors which subserve firm adhesion to ICAM-1. As eosinophil 2 integrins are not able to subserve the initial tethering step to ICAM-1 to slow eosinophils down before firm adhesion, eosinophils do not accumulate on ICAM-1 (26). These studies underscore the sequential nature of eosinophil tethering and firm adhesion to endothelium, which is also noted in vivo, where fast-flowing eosinophils always first tether to endothelium before becoming firmly adherent.

In contrast to in vitro studies, in vivo studies demonstrating that chemoattractants induce eosinophil activation-dependent adhesion are limited. One chemoattractant that can induce activation-dependent adhesion of eosinophils in vivo is the complement fragment C3a (28). Superfusion of the IL-1-stimulated rabbit mesentery with C3a in vivo results in rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a does not evoke subsequent transmigration of the adherent eosinophils into tissues. In contrast to C3a, C5a induces both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium (28). Both C3a- and C5a-dependent adhesion to venular endothelium is blocked by pretreatment of eosinophils with anti-4 and anti-2 integrin mAbs. In vitro, both C3a- and C5a-dependent transmigration of eosinophils across IL-1-stimulated endothelial monolayer is mediated by 41 and M2 integrins. Overall, these studies of eosinophils and neutrophils suggest that C3a is an eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion, but not transmigration in vivo. C5a, in contrast, is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils. The potential importance of complement to eosinophilic inflammation and asthma is suggested from studies of allergen-challenged mice deficient in C3, which exhibit diminished airway hyperresponsiveness and lung eosinophilia (29). In addition, increased levels of C3a and C5a have been observed 24 h following allergen challenge in the airways of subjects with allergic asthma, but not control subjects (30). The increased airway levels of C3a and C5a correlated with airway eosinophil levels. Thus, complement fragments may also play an important role in eosinophil activation-dependent adhesion and tissue recruitment.

Following activation-dependent adhesion of eosinophils to endothelium, eosinophils transmigrate across endothelium into tissues. The role of adhesion molecules (platelet endothelial cell adhesion molecule [PECAM]), chemokines (eotaxin-3), and shear stress, have all been investigated to determine their role in facilitating this step of eosinophil transmigration across endothelium. PECAM (or CD31) is a cell adhesion molecule which belongs to the Ig superfamily and is expressed on endothelial cells as well as circulating leukocytes, including eosinophils (31). The homophilic PECAM interaction of neutrophil or monocyte PECAM with endothelial PECAM is very important to neutrophil and monocyte transendothelial migration, as neutralizing antibodies to PECAM inhibit neutrophil and monocyte transendothelial migration by ~ 80% in vitro and in vivo (32, 33). In contrast, studies with eosinophils suggest that eosinophil transendothelial migration is PECAM-independent in vivo in both a mouse model of asthma (34) and in a parasite model of ocular onchocerciasis (35). Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis, and RT-PCR studies demonstrate that eosinophils, like neutrophils, express the six extracellular domains of PECAM (34). Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM (34). The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM, and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs do not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, or airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo (34). PECAM- independent eosinophil recruitment has also been demonstrated in a murine model of ocular onchocerciasis in which Ags from the parasitic worm Onchocerca volvulus are injected into the corneal stroma (35). The presence of a PECAM-independent pathway for leukocyte recruitment has been suggested from studies of PECAM-deficient mice (36). PECAM-deficient neutrophils exhibit no defect in migration in vitro (36), and PECAM-deficient mice showed similar levels of neutrophil migration compared with wild-type mice in several different models of inflammation in vivo (36). Leukocyte recruitment in PECAM-deficient mice may therefore be mediated by compensatory induction of normally redundant PECAM-independent pathways in these mutant mice. Similar and/or different PECAM-independent pathways may be mediating eosinophil tissue recruitment in vivo.

In vitro flow chamber studies suggest that shear stress (present under conditions of blood flow in vivo) plays a more important role in eosinophil and lymphocyte, compared with neutrophil, transmigration across endothelium (37). Eosinophils exhibit a basal level of transmigration across endothelium under static conditions in vitro, but require shear stress to reach maximal levels of transmigration (37). Shear stress is also required for T lymphocyte transmigration across cytokine-stimulated human umbilical vein endothelial cells (HUVECs) in vitro, as no transmigration occurs under static conditions (38). In contrast, neutrophil transmigration across cytokine-stimulated HUVECs is accelerated by shear stress, but is not dependent on shear stress (39). Under shear stress conditions, eosinophil transmigration across IL-4-stimulated HUVECs is rapid, with 50% of eosinophils transmigrating within 7 min (37). Eotaxin-3, which binds to CCR3 on eosinophils, appears to play an important role in eosinophil transmigration across IL-4-stimulated endothelium under conditions of shear stress (37). Endothelial surface-associated expression of eotaxin-3 is induced by IL-4, and greater than 65% of shear-dependent eosinophil transmigration across IL-4- stimulated HUVECs can be blocked by either pertussis toxin or by an anti-CCR3 monoclonal antibody (37).

Thus, endothelial surface-associated expression of eotaxin-1, eotaxin-2, and eotaxin-3 may play important roles in eosinophil activation-dependent adhesion (13, 24), de-adhesion (23), shift in eosinophil adhesion molecule usage away from VCAM-1-dominated to ICAM-1-dominated pathways (26), and transmigration of eosinophils across endothelium under conditions of flow (37). As the kinetics of expression of eotaxin-1 (peaks early at 6 h) differs from that of eotaxin-2 and eotaxin-3 (peaks at 24 h), in vivo studies will need to be performed to determine whether and at what time points individual eotaxin family member, alone and in combination with other chemoattractants, play a role in eosinophil adhesion, de-adhesion, or endothelial transmigration.

	Footnotes

Address correspondence to: David Broide, M.D., University of California-San Diego, Basic Science Bldg., Rm. 5090, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail: dbroide{at}ucsd.edu

(Received in original form April 22, 2002).

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