HYDROGEN PEROXIDE ACTIVATION OF ENDOTHELIAL CELL-ASSOCIATED MMPS DURING VCAM-1-DEPENDENT LEUKOCYTE MIGRATION

Abstract

Leukocyte migration from the blood into tissues is vital for immune surveillance and inflammation. Specificity for the site of leukocyte migration is determined by the combination and concentration of adhesion molecules, cytokines and chemokines in the microenvironment. Leukocytes bound at sites of extravasation migrate within minutes. We have focused on the function of the adhesion molecule VCAM-1 and have reported an active function for the endothelium during VCAM-1-dependent leukocyte migration. VCAM-1 activates endothelial cell NADPH oxidase followed by the generation of 1μM H₂O₂. This stimulates endothelial cell-associated matrix metalloproteinase (MMP) activity in minutes, consistent with the time for lymphocyte migration. The endothelial cell NADPH oxidase and endothelial cell MMP activities are required for VCAM-1-dependent lymphocyte migration as determined by scavenging of ROS, by pharmacologic or antisense inhibition of NADPH oxidase and by pharmacologic inhibition of endothelial cell MMPs. Furthermore, antioxidants block VCAM-1 activation of MMPs. In vivo, administration of the antioxidant bilirubin blocks VCAM-1-dependent leukocyte migration into the lung in experimental asthma. In summary, endothelial cells are not simply a scaffold for leukocyte adhesion. Instead, endothelial cells have an active function during VCAM-1-dependent leukocyte transendothelial migration.

INTRODUCTION

Leukocytes migrate from the blood into tissue in response to inflammatory stimuli. Inflammatory stimuli such as cytokines induce endothelial cells to express receptors for leukocytes. The adhesion molecules on the endothelium and their ligands on leukocytes initially mediate a low affinity adhesion that, with the force of the flow of blood, produces a rolling of the leukocytes along the endothelial surface. This binding stimulates intracellular signals in leukocytes that increase the affinity of several leukocyte ligands that mediate firm adhesion to the endothelium. This high affinity adhesion stops the leukocyte from rolling. Then, the leukocyte migrates across the endothelium. The induction of adhesion molecule expression and the adhesion events have been well defined and reviewed elsewhere (12,19,22,25,40,41). However, the signals within endothelial cells that are required for migration of leukocytes across the endothelium are less defined. The focus of this review is on the function of one of the adhesion molecules on the endothelium, vascular cell adhesion molecule-1 (VCAM-1) (Fig.1).

Fig. 1. Working Model for VCAM-1-dependent Leukocyte Migration.

Leukocyte α□4-integrin adhesion to VCAM-1 on endothelial cells activates calcium channels, intracellular calcium release and Rac-1 that then stimulate endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The 1μM H₂O₂ that is generated stimulates MMPs localized on the endothelial cells at the site of stimulation. These endothelial cell MMPs are required for VCAM-1-dependent leukocyte migration.

VCAM-1 binds to α4β1-integrin on leukocytes α4β1-integrin in its low affinity state participates in leukocyte rolling whereas in its high affinity state, it participates in firm adhesion of the leukocyte to the endothelium (3). VCAM-1 activates endothelial signals that are required for VCAM-1-dependent leukocyte migration (Fig.1). These signals are discussed in this review.

VCAM-1 functions in both normal processes and disease pathogenesis. VCAM-1 in combination with other adhesion molecules regulates lymphocyte recirculation. In disease pathogenesis, VCAM-1 regulates T cell infiltration in inflammatory bowel disease (39), eosinophil infiltration in experimental asthma (7), T cell infiltration in experimental allergic encephalomyelitis (6), and melanoma metastasis to the liver (36,50). It also has a role in atherosclerosis as VCAM-1 is the first adhesion molecule expressed prior to atherosclerotic plaque development (18). The VCAM-1 knockout is an embryonic lethal as it is necessary for heart development (14). Thus, it is important to understand the mechanisms for VCAM-1 signaling so that approaches can be developed to modulate VCAM-1-dependent inflammation during disease.

ENDOTHELIAL CELL MODELS FOR STIMULATION OF VCAM-1 SIGNALS

The endothelial cell models used in our studies for VCAM-1 signaling are depicted in Fig. 2. The endothelial cell lines mHEV have the advantage of providing a model to test the functional outcome of inhibition of VCAM-1 signals in that the endothelial cell lines can be used to examine VCAM-1-dependent lymphocyte migration without complications due to lymphocyte binding to other adhesion molecules. The binding of lymphocytes to the mHEV cells is dependent on VCAM-1 and its ligand α□4-integrin but not other adhesion molecules as determined using blocking antibodies (Table 1) (45). This lymphocyte binding to VCAM-1 is required during lymphocyte transendothelial migration as anti-VCAM-1 antibodies and antiα-□4-integrin antibodies also block migration (27). Lymphocyte migration across the mHEV cells is induced by the chemokine MCP-1 which is constitutively produced by the mHEV cells (31). When examining the migration of spleen cells, the cells that migrate across the mHEV cells are >90% lymphocytes (46). Thus, the mHEV cell lines provide a model to examine signals during VCAM-1-dependent lymphocyte migration as well as stimulation of signals by antibody crosslinking of VCAM-1 (Fig.2). In contrast to the mHEV cells, the primary cultures of human endothelial cells express VCAM-1 and other adhesion molecules such as ICAM-1 and PECAM-1. Therefore, the primary cultures of endothelial cells are a model to examine antibody crosslinking of VCAM-1 but can not be used to examine VCAM-1-induced signals during VCAM-1-dependent lymphocyte migration.

Fig. 2. Models for Activation of VCAM-1 ‘Outside-in’ Signals.

The mHEV cell lines are a model to examine signals that are required for VCAM-1-dependent lymphocyte migration. The lymphocyte adhesion and migration across mHEV cells are dependent on lymphocyte binding to VCAM-1 but not other endothelial cell adhesion molecules. Thus, to examine signals involved in VCAM-1-dependent lymphocyte migration, the endothelial cells are treated with irreversible inhibitors of signaling molecules, washed, and used in lymphocyte migration assays. With this approach, the functional outcome of the endothelial cell signals on VCAM-1-specific migration is tested. It is important to determine whether VCAM-1 can directly induce an endothelial signal under investigation as it is possible that subsequent cell interaction events may trigger the signals being studied. To determine whether VCAM-1 can directly induce a signal, VCAM-1 on the endothelial cell lines is stimulated by crosslinking VCAM-1 with anti-mouse VCAM-1 mAb-coated 10μm beads. The functional effects of the force of laminar flow on VCAM-1 signaling are tested by applying laminar flow during binding of the lymphocytes or anti-VCAM-1-coated beads. To examine whether the VCAM-1 signals are stimulated with the same magnitude and time course as primary cultures of endothelial cells, primary cultures of human umbilical vein endothelial cells (HUVECs) or human microvascular endothelial cells from the lung (HMEC-Ls) are cytokines-treated to induce expression of adhesion molecules, washed and then the endothelial cells are stimulated with anti-human VCAM-1 mAb-coated 10μm beads. To examine VCAM-1 signals in the primary cultures of endothelial cells, anti-VCAM-1-coated beads are used as the stimulant for VCAM-1 rather than lymphocytes as lymphocytes bind to multiple receptors on cytokine-activated primary endothelial cells. Using this combination of approaches, it has been demonstrated that VCAM-1 stimulates endothelial cell production of 1μM H₂O₂. As H₂O₂ diffuses at 100μm/sec across membranes, the exogenous addition of 1μM H₂O₂ bypasses VCAM-1 signals upstream of NADPH oxidase and is used to determine whether 1μM H₂O₂ mimics the signals initiated by VCAM-1 signaling in primary cultures of endothelial cells and endothelial cell lines.

Table 1.

Lymphocyte adhesion to mHEV cells

Adhesion Molecules that mediate cell binding:
	mAb that inhibit adhesion to mHEV cells
α4-integrin	R1-2 + 9C10
	PS/2
VCAM-1	M/k-2
	MVCAM.A
Adhesion Molecules that do not mediate cell binding:
	mAb that no not inhibit adhesion to mHEV
LFA-1	M17/4
ICAM-1	3E2
ICAM-2	3C4
L-selectin	MEL-14
activated β1-rintegrin	9EG7
α4β7-lntegrin	DATK32
mHEV cells do not express*:
PECAM-1
ICAM-1
P-selectin
E-selectin
MAdCAM-1
MECA antigens

^*as determined by cDNA microarray and immunolabeling.(Tudor, K-S. R.S.et al. 2000. Biochem. Cell Biol. 78:99-113.)

For antibody crosslinking, VCAM-1 is stimulated by 10 :m beads that are coated with anti-VCAM-1 (Fig.2). These beads are the approximate size of a leukocyte. Lymphocyte binding to the mHEV cells or antibody crosslinking of VCAM-1 stimulates mHEV cell production of 1μM H₂O₂ indicating that both stimulants activate ‘outside-in’ signals in the endothelial cells (8). Furthermore, H₂O₂ is produced after either antibody binding to VCAM-1 on mHEV cells or to VCAM-1 on primary cultures of endothelial cells (8,27). Thus, H₂O₂ production by VCAM-1-expressing cells is consistent in several endothelial cell models. In addition to direct stimulation of VCAM-1, VCAM-1 signaling upstream of H₂O₂ production can be bypassed by exogenous addition of 1μM H₂O₂ (Fig.2). This 1μM H₂O₂ is relatively low as compared to the 50-200μM H₂O₂ produced by macrophages or neutrophils (9,11). It is also much lower than the exogenous 100-1000μM H₂O₂ added to endothelial cells in many studies on oxidative damage to endothelium (5,16,17,21,47). These differences in H₂O₂ levels are important in understanding functions of oxidation as we have reported that 1μM H₂O₂ and >50μM H₂O₂ have opposing effects on signal transduction (10). The function of 1μM H₂O₂ in activation of MMPs is a focus of this review on VCAM-1 signaling. In summary, using a combination of approaches for stimulating VCAM-1 (Fig.2), VCAM-1 ‘outside-in’ signals can be identified and, importantly, it can be determined whether these signals have a functional consequence in VCAM-1-dependent lymphocyte migration.

VCAM-1 ACTIVATION OF ENDOTHELIAL CELL NADPH OXIDASE

Using the approaches described in fig. 2, it was determined that stimulation of VCAM-1 induces the release of intracellular calcium and calcium channels, both of which are required for VCAM-1-dependent lymphocyte migration in vitro (Fig.1) (8,23). Lymphocyte binding to VCAM-1 also activates a small molecular weight G protein, Rac-1 (8,49). Dominant negative Rac-1 blocks VCAM-1-dependent migration of lymphocytes across endothelial monolayers (8). The VCAM-1 stimulation of calcium fluxes and Rac-1 activate the membrane complex NADPH oxidase (Fig.1) (8,49). NADPH oxidase consists of two transmembrane subunits and three cytoplasmic subunits (2,28). NADPH oxidase catalyzes the production of superoxide that then dismutates to H₂O₂. The catalytic domain gp91 phox is one of the transmembrane subunits. Stimulation of VCAM-1 induces endothelial cell production of 1μM H₂O₂ that is dependent on endothelial cell NADPH oxidase as determined by gp91 phox antisense and pharmacological approaches (8). The VCAM-1-dependent lymphocyte migration across the mHEV cells requires endothelial cell NADPH oxidase as it is blocked by pharmacological inhibition of NADPH oxidase with apocynin, blocked by inhibition of flavoproteins with diphenyliodonium, and blocked by scavenging ROS with superoxide dismutase or catalase (Table 2) (27).

Table 2.

Lymphocyte migration across mHEV cells

Lymphocyte Migration across mHEV cells requires:	Inhibitors
α4-integrin binding to VCAM-1	anti-α4 integrin or anti-VCAM-1 mAbs
Endothelial cell NADPH oxidase	gp91 phox antisense, diphenyliodonium, apocynin, Superoxide dismutase, catalase,or bilirubin
Lymphocyte Migration is NOT Mediated by:	Inhibitors
Endothelial cell nitric oxide synthase	L-NMMA or NIO
Endothelial cell xanthine oxidase	allopurinol
Endothelial cell cytochrome P450	methoxsalen or troleandomycin
Endothelial cell PI3 kinase	wortmanin
Endothelial cell tyrosine kinases	herbimycin

References:

Matheny. H., et. 2000. J. Immunol. 164:6550-6559.

Cook-Mills, J.M., et al. 2004. Biochem. J. 378:539-547

Keshavan, P. 2005. J. Immunol. 174:3709-3718

In contrast, the inhibition of flavoproteins including NADPH oxidase in lymphocytes does not block VCAM-1-dependent lymphocyte migration (27). Therefore, NADPH oxidase in endothelial cells but not lymphocytes is required for VCAM-1-dependent lymphocyte migration (Fig.1).

The VCAM-1-dependent lymphocyte migration across mHEV cells is not blocked by pharmacological inhibition of other ROS generating enzymes in endothelial cells such as xanthine oxidase, nitric oxide synthase or cytochrome P450 (Table 2) (27). Although VCAM-1 is a member of the immunoglobulin superfamily and other members of this family can signal through PI3 kinase and tyrosine kinases, inhibition of these enzymes in endothelial cells does not block VCAM-1-dependent lymphocyte migration (Table 2) (27). In summary, pharmacologic and antisense inhibition of NADPH oxidase or scavenging of ROS blocks VCAM-1-dependent ROS generation and VCAM-1-dependent lymphocyte migration (Table 2). Inhibition of NADPH oxidase also blocks VCAM-1-stimulated ROS production by primary cultures of human lung microvascular cells (27). As ROS are required for VCAM-1-dependent lymphocyte migration, it was determined whether they mediate changes in endothelial cell structure. VCAM-1 activates changes in endothelial cell actin structure in endothelial cell lines or TNFα□-activated HUVECs (27). VCAM-1-stimulated ROS also induce a loss of the cytoskeletal proteinβ-catenin at the cell periphery indicating that VCAM-1 induces changes in endothelial cell structure (49). Taken together, VCAM-1 stimulates NADPH oxidase production of H₂O₂ that is required for VCAM-1-dependent lymphocyte migration.

HYDROGEN PEROXIDE ACTIVATION OF MMPS DURING VCAM-1 SIGNALING

It has been reported that the activity of some enzymes is modulated by oxidation. For example, high levels of reactive oxygen species (ROS) inhibit matrix metalloproteinases. Rajagopalan et al. (32) examined the activity of purified MMPs using a broad concentration curve for H₂O₂. Greater than 50 :M H₂O₂ inhibited MMP activity whereas lμM H₂O₂ stimulated activity of purified MMPs (32). However, the effect of low concentrations of H₂O₂ on cell-associated MMPs was not examined in their studies. Therefore we examined whether endothelial cell-associated MMPs were activated by VCAM-1-induced ROS production. To examine VCAM-1-dependent activation of endothelial cell-associated MMPs, endothelial cell lines that constitutively express VCAM-1 as well as primary cultures of endothelial cells were used (Fig.2). The endothelial cells were stimulated with anti-VCAM-1-coated beads as in Fig. 2, washed and endothelial cell-associated MMPs were examined for activity by zymography using gelatin-impregnated SDS-PAGE gels. In addition, these studies focused on examination of endothelial cell-associated MMPs rather than released MMPs as, in vivo, the flow of blood would wash away released MMPs. Thus released MMPs would not have a function at the site of transendothelial lymphocyte migration. The endothelial cells express the gelatinases MMP2 and MMP9 as determined by western blot and gelatin zymography (10). In addition, the endothelial cells express the receptors that hold these MMPs at their cell surface as they express transmembrane MMP14 which binds MMP2 and they express CD44 which binds MMP9 (10).

Anti-VCAM-1-coated beads activate both endothelial cells-associated MMP2 and MMP9 within minutes as determined by gelatin zymography (Table 3) (10).

Table 3.

Activation of MMPs by VCAM-1 signaling

Activation of Endothelial Cell-associated MMP2 & MMP9:
Stimulant:	Time of activation:	Block Activation with:
anti-VCAM-1-beads	15 minutes	GM6001, gp91phox antisense, apocynin.catalase, or bilirubin
exogenous 1 μM H2O2	15 minutes
Activation of Lymphocyte-associated MMP9:
Stimulant:	Time of activation	Block Activation with:
lymphocyte binding to VCAM-1	2 hours	pretreat mHEV cells with dlphenyliodonium or apocynin
exogenous 1 μM H2O2	2 hours
VCAM-1-dependent Lymphocyte Migration Requires Endothelial Cell MMPs:
Inhibition of migration:		MMP Inhibitor:
pretreat endothelial cells with inhibitor		GM6001
No inhibition of migration:
pretreat lymphocytes with inhibitor		GM6001

References:

Deem, TL et al. 2004 Blood. 104:2385-2393.

Keshavan, P et al. 2005. J. Immunol. 174:3709-3718.

The VCAM-1-induced activation of endothelial cell-associated MMPs is blocked by antisense to gp91 phox, blocked by pharmacologic inhibition of NADPH oxidase and blocked by scavenging of H₂O₂ with catalase (Table 3) (10). This demonstrates that VCAM-1 induces signals through endothelial cell NADPH oxidase for the ROS-dependent activation of endothelial cell-associated MMPs. The anti-VCAM-1 activation of endothelial cell-associated MMPs is not different under static versus laminar flow conditions at 2dynes/cm² (10). This is the flow rate at postcapillary venules (24). Thus, the physiological flow rate at postcapillary venules does not alter VCAM-1 activation of MMPs, even though laminar flow places a force on the anti-VCAM-1-coated beads causing a ‘tug’ on VCAM-1. Exogenous addition of 1μM H₂O₂ also activates endothelial cells associated MMP2 and MMP9 (Table 3) (10). Moreover, similar levels of MMP activity were induced by the anti-VCAM-1-coated beads and H₂O₂ stimulants, indicating that they had similar functional effects on the cells. In contrast to the stimulatory effects of 1μM H₂O₂, high concentrations of exogenous H₂O₂ (>50μM H₂O₂) inhibits basal endothelial cell-associated MMP activity (10). This emphasizes the opposing effects of low versus high concentrations of H₂O₂ in that low concentrations of H₂O₂ can activate signal transduction whereas high levels of H₂O₂ inhibit enzymatic activities and induce oxidative damage (5,21,47).

MMP FUNCTION DURING VCAM-1-DEPENDENT LYMPHOCYTE MIGRATION

The time courses for stimulation of the endothelial cell MMPs demonstrates that the endothelial cell-associated MMPs are activated within a few minutes whether stimulated by anti-VCAM-1 or by 1μM H₂O₂ (10). This is in contrast to the reports that T cell-associated MMPs are activated 5-12hrs after interaction with endothelial cells (13,33,51). However, once a leukocyte reaches a site for migration, the process of leukocyte transendothelial migration occurs in less than 2min. It was not shown by Romanic et al (33) whether lymphocyte MMPs or endothelial cell MMPs were important for the transendothelial lymphocyte migration. To determine whether migration is modulated by endothelial cell-associated MMPs or alternatively lymphocyte-associated MMPs, the endothelial cells or lymphocytes were pretreated with the MMP inhibitors, GM6001 (10μM) (Table 3) or BB3103 (0.5mM)( 10). These inhibitors do not affect cell viability. The cells were washed and lymphocyte migration was examined using mHEV cell monolayers on 12μm pore transwell polycarbonate membrane. GM6001 pretreatment of mHEV cells blocks VCAM-1-dependent lymphocyte migration in a dose-dependent manner whereas GM6001 pretreatment of lymphocytes does not affect lymphocyte migration (Table 3) (10). The last wash, from the cells that had been pretreated with GM6001, does not affect migration of untreated cells, indicating that the GM6001-treated cells were sufficiently washed (10). In addition, as expected, the MMP inhibitor GM6001 blocks anti-VCAM-1 stimulated endothelial cell-associated MMP activity (Table 3) (10). In summary, endothelial cell MMP activity but not lymphocyte MMP activity is necessary for VCAM-1-dependent lymphocyte transendothelial migration.

The specific function of MMP2 versus MMP9 in migration has not been examined as it is likely that MMPs located at the sites of ROS generation are not differentially activated by 1μM H₂O₂ since the mechanism for H₂O₂ oxidation of MMPs is conserved among the MMPs. Briefly, MMPs are synthesized in a nonactive form with a proarm that contains a cysteine that is bound to a zinc atom in the active site of the MMPs. Hydrogen peroxide oxidizes this cysteine in the MMP prodomain resulting in the release of the cysteine from the zinc atom (29). This is followed by autocatalytic cleavage of the proarm, generating the active MMP (48). Thus H₂O₂ would not have specificity for MMP isozymes given the conserved cysteine-zinc bond in proMMPs (29) and the rate of diffusion of H₂O₂ at 100μm/sec (26).

THE ANTIOXIDANT BILIRUBIN BLOCKS VCAM-1 SIGNALING

As VCAM-1 signals through generation of H₂O₂ and activation of MMPs, it was determined whether the antioxidant bilirubin blocked VCAM-1-dependent lymphocyte migration in vitro and blocked VCAM-1 activation of MMPs (Table 3). Bilirubin is generated from heme by hemoxygenase-1 (20,34). After generation of bilirubin, it can undergo redox cycling such that oxidation of bilirubin converts it to biliverdin (34,37,42,43). Bilirubin and biliverdin are membrane permeable (30,43). Biliverdin is recycled back to bilirubin by biliverdin reductase and the cofactor NADPH (20). Hemoxgenase-1 and biliverdin reductase are expressed by endothelial cells and endothelial cell lines (20). It has been reported that bilirubin acts as an antioxidant in that it reduces oxidized phospholipids with the approximate rate of antioxidant vitamins (44). We reported that concentrations of bilirubin in the upper physiological range blocks anti-VCAM-1 activation of endothelial cell-associated MMP2 and MMP9 (Table 3) without affecting cell viability (20). In addition, bilirubin blocks VCAM-1-dependent migration of lymphocytes across endothelial cells in vitro (20). In contrast, VCAM-1-dependent lymphocyte migration is not blocked by the stable bilirubin conjugate ditaurobilirubin which can not scavenge ROS (20). The bilirubin inhibition of lymphocyte migration results from an inhibition of migration rather than inhibition of lymphocytes available for migration as the number of lymphocytes bound to the endothelial cell monolayer is unaffected by bilirubin (20). Therefore, the antioxidant bilirubin blocks VCAM-1-dependent lymphocyte migration across endothelial cells and this migration is requires endothelial cell ROS generation.

ENDOTHELIAL CELL-DERIVED ROS MEDIATE A DELAYED ACTIVATION OF LYMPHOCYTE MMPS

VCAM-1 stimulates endothelial cell generation of 1μM H₂O₂ and activates endothelial cell surface-associated MMPs within minutes (10) and it is known that H₂O₂ diffuses rapidly (4). Thus, the endothelial cell-generated H₂O₂ might be expected to activate the MMPs on the surface of leukocytes within minutes as the leukocytes are bound at the site of VCAM-1 stimulation. However, it is reported that lymphocyte MMPs are activated 5-12 hours after interaction with VCAM-1 on endothelial cells (13,33,51). Therefore, we determined whether the VCAM-1-stimulated ROS generation by endothelial cells activates lymphocyte-associated MMPs and whether this occurs in minutes or hours (Table 3). For these studies, splenic lymphocytes were incubated with monolayers of mHEV cells, nonbound lymphocytes were removed by washing, and bound lymphocytes were released from the monolayers by reversing lymphocyte binding with anti-VCAM-1 at 15min, 2hrs, or 5hrs. The MMP activity associated with the lymphocytes that had bound to the endothelial cells was determined by gelatin zymography. The MMPs on these lymphocytes are activated at 2hrs but not at 15min which is in contrast to the activation of endothelial cell-associated MMPs within 15min (10). This delayed activation of lymphocyte MMPs is consistent with the requirement for endothelial cell MMPs but not lymphocyte MMPs during VCAM-1-dependent lymphocyte migration. It is also consistent with the rate of transendothelial migration of leukocytes which occurs within minutes once a lymphocyte reaches a site of migration.

The mechanism for this delayed activation of lymphocyte MMPs was determined. The activation of lymphocyte MMPs is blocked by pharmacological inhibition of endothelial cell ROS generation with diphenyliodonium or apocynin but not by pharmacologic inhibition of ROS-generating enzymes in lymphocytes (10). Thus, endothelial cell-derived ROS during VCAM-1 signaling activates a delayed induction of MMP activity on lymphocytes bound to VCAM-1. In addition, exogenous addition of 1μM H₂O₂ to a suspension of lymphocytes induces activation of lymphocyte-associated MMPs that is also delayed for 2hrs (10). To identify a mechanism for this delayed ROS-mediated activation of lymphocyte MMPs, the expression of MMPs and the expression of the endogenous tissue inhibitors of MMPs (TIMPs) was determined after stimulation of lymphocytes by exogenous addition of 1μM H₂O₂. After a 5hrs treatment with 1μM H₂O₂, the expression of lymphocyte MMP9 is not altered but the expression of TIMP1 and TIMP2 by lymphocytes is reduced by 60-80% as determined by western blot (10). This results in a three fold increase in the MMP9/TIMP ratio reflecting the increased MMP activity at 5hrs. In summary, lymphocyte binding to VCAM-1 activates endothelial cell generation of ROS which induces a delayed activation of lymphocyte MMPs. This delay in ROS-induced MMP activity in lymphocytes is a result of the time required for a reduction in TIMP expression.

ANTIOXIDANT INHIBITION OF VCAM-1-DEPENDENT LEUKOCYTE MIGRATION IN VIVO

The studies on VCAM-1 activation of ROS demonstrate that endothelial cell production of ROS rapidly activates endothelial cell-associated MMPs that are required for VCAM-1-dependent lymphocyte migration in vitro. Therefore, it was determined whether an antioxidant blocks VCAM-1-dependent leukocyte migration in vivo. The in vivo inflammation examined in these studies was VCAM-1-dependent leukocyte infiltration into the lung in experimental asthma (Table 4).

Table 4.

Bilirubin blocks VCAM-1-dependent leukocyte migration into the lungin experimental asthma

OVA challenge stimulates infiltration of;

Eosinophils, Lymphocytes, Neutrophils, Monocytes

Bilirubin blocks OVA-induced infiltration of:

Eosinophils, Lymphocytes

Bilirubin does not alter OVA-induced expression of:

VCAM-1 on endothelium

Th2 cytokines(IL-4,IL-5,IL-6,IL-10)in lung and lymph nodes

Chemokines (MCP-1,eotaxin) in lung lavage fluid

Bilirubin does not alter cytokines not induced by OVA:

IL-2, IL-12,IFNγ, TNFα

References:

Keshavan, P et al. 2005. J. Immunol. 174:3709-3718.

In experimental asthma, leukocyte migration into the lung is induced by sensitization and challenge with the antigen ovalbumin (OVA). It is well established that OVA-stimulated eosinophilia in the lung as well as OVA-stimulated eosinophilia in the skin requires adhesion to VCAM-1 as antibodies to VCAM-1 block the eosinophilia (7,15,35). Therefore, it was determined whether treatment with the antioxidant bilirubin, which blocks VCAM-1-dependent signals in vitro, also blocks VCAM-1-dependent eosinophilia in experimental asthma. Mice were sensitized by intraperitoneal injection of OVA in alum and then challenged by intranasal inhalation of OVA using a standard protocol for experimental asthma (20). At the time of intranasal OVA challenge, a group of mice also received either intraperitoneal injections of bilirubin at upper physiological concentrations or vehicle control (20). The treatment with bilirubin inhibits eosinophil infiltration into the bronchoalveolar lavage by >90% and inhibits lymphocyte infiltration by 60% (Table 4) (20). The migration of eosinophils into the tissue is also reduced by 90% as determined by immunohistochemistry for the eosinophil granule component, major basic protein (20). The reduction in eosinophil and lymphocyte infiltration is consistent with the VCAM-1 dependence of eosinophil migration and the partial VCAM-1 dependence of lymphocyte migration in this lung response to OVA (7). As anticipated, there is no effect of bilirubin administration on the OVA-induced infiltration of monocytes or neutrophils which are independent of binding to VCAM-1 (20). Although there is reduced eosinophilia with the administration of bilirubin, there are sufficient numbers of eosinophils available for migration as there is not a reduction in blood eosinophils in the bilirubin-treated group compared to the nontreated group (20). In fact, there is a 3 fold increase in blood eosinophil numbers with bilirubin administration (20). In summary, the antioxidant bilirubin blocks VCAM-1 signaling in vitro, blocks VCAM-1-dependent lymphocyte migration in vitro and blocks VCAM-1-dependent infiltration of leukocytes in vivo.

The infiltration of eosinophils in response to OVA inhalation is regulated by cytokines, chemokines, and binding to VCAM-1. Bilirubin treatment does not alter the induction of VCAM-1 expression in OVA-treated mice (20). Bilirubin treatment also does not alter the OVA-induced increase in Th2 cytokines (IL-4, IL-5, IL-6 or IL-10) in lung lavage fluid or OVA-restimulated draining lymph node cells (20). Bilirubin does not alter the OVA-induced increase in the chemokines MCP-1 or eotaxin (20). In addition, bilirubin does not increase expression of Th1 cytokines (IL-2, IL-12, IFN□γ or TNFα□) which are not upregulated by OVA stimulation (20). In summary, VCAM-1-dependent eosinophil and lymphocyte infiltration into the lung is reduced by the antioxidant bilirubin without altering the expression of the adhesion molecules, cytokines, or chemokines that regulate leukocyte infiltration in response to OVA. These data are consistent with bilirubin scavenging of endothelial-cell derived ROS generated during VCAM-1 signaling.

SUMMARY

In summary, VCAM-1 is a scaffold on which leukocytes migrate but VCAM-1 also activates signals in endothelial cells that are required for VCAM-1-dependent leukocyte migration. Thus, the endothelium plays an active role in the regulation of VCAM-1-dependent leukocyte migration. The working model (Fig.1) for VCAM-1 function is as follows: leukocyte binding to VCAM-1 stimulates endothelial cell NADPH oxidase (nox2) for the production of superoxide, resulting in the generation of 1μM H₂O₂. This H₂O₂ diffuses rapidly activating enzymes such as the matrix metalloproteinase family of enzymes. These MMPs degrade matrix and endothelial cell surface receptors in cell junctions (1,38). Although H₂O₂ diffuses rapidly, it mediates a rapid localized function as the H₂O₂ is produced in such low concentrations as to oxidize targets at the site of leukocyte transendothelial migration. At distant sites after diffusion of H₂O₂, the H₂O₂ concentration becomes too low for functional consequences. The rapid activation of MMPs that are associated with the endothelial cells is consistent with the rapid process of transendothelial cell leukocyte migration. Furthermore, endothelial cell-associated MMPs but not lymphocyte-associated MMPs are required for VCAM-1-dependent transendothelial migration. The identification of mechanisms for VCAM-1-dependent infiltration of leukocytes provides a basis for designing approaches to modulate VCAM-1-dependent processes during disease.

Abbreviations

ICAM-1

Intercellular Adhesion Molecule-1

mAb

monoclonal Antibody

MMPs

Matrix Metalloproteinases

OVA

Ovalbumin

PECAM-1

Platelet-Endothelial Cell Adhesion Molecule-1

ROS

Reactive Oxygen Species

TIMPs

Tissue Inhibitors of Matrix Metalloproteinases

VCAM-1

Vascular Cell Adhesion Molecule-1