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Keywords:

  • Inflammation;
  • leukocyte-endothelial cell adhesion;
  • nuclear factor-kappa B;
  • cytokines;
  • lipid mediators;
  • glucocorticoids;
  • antisense oligonucleotides;
  • selectins;
  • β2-integrins

Abbreviations:
AP-1

activation protein-1

CAM

cell adhesion molecule

ESL

E-selectin ligand

ICAM

intercellular adhesion molecule

IL

interleukin

mAb

monoclonal antibody

NF-κB

nuclear factor kappa-B

MAdCAM

mucosal address in cell adhesion molecule

ODN oligodeoxynucleotide; PSGL-1

P-selectin glycoprotein ligand-1

PSL

P-selectin ligand

PECAM

platelet endothelial cell adhesion molecule

TFD

transcription factor decoy

TNF-α

tumour necrosis factor-alpha

VCAM

vascular cell adhesion molecule

VLA

very late antigens

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

Leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of a variety of diseases that affect different organ systems. Examples of such diseases include atherosclerosis, gastric ulcers, haemorrhagic shock, myocardial infarction, stroke, and malaria (Korthuis et al., 1994; Panés & Granger, 1998). The recognition that leukocytes must firmly adhere to vascular endothelial cells in order to mediate the organ dysfunction and tissue injury associated with these diseases has resulted in an intensive effort to define the factors that modulate this cell-cell interaction. A major focal point of this effort has been directed towards identifying and characterizing the adhesion glycoproteins that enable leukocytes to bind to vascular endothelial cells. Data derived from both in vitro (isolated leukocytes binding to monolayers of cultured endothelial cells) and in vivo (intravital microscopic examination of venules) models of leukocyte-endothelial cell adhesion have revealed the relative contributions of different leukocyte and endothelial cell adhesion molecules (CAMs) to the adhesion responses elicited by various inflammatory stimuli. These studies have also led to an appreciation of the potential cellular and molecular loci that can be targeted to interfere with leukocyte-endothelial cell adhesion. As a result, there is a widely held view that several of these loci also represent novel and potentially powerful therapeutic sites for treatment of acute and chronic inflammatory diseases. The major objective of this review is to discuss some of the major potential targets for therapeutic intervention against inflammation that relate to the process of leukocyte-endothelial cell adhesion and more specifically to the regulation of endothelial CAM expression. That discussion is preceded by a brief description of the major CAMs that participate in the recruitment of leukocytes into inflamed tissue and how the expression of these CAMs is coordinated to ensure an orderly sequence of cell-cell interactions.

Adhesion Molecules

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

Both leukocyte and endothelial CAMs participate in slowing the leukocyte as it exits the capillary and enters the postcapillary venule, which is the major site of leukocyte-endothelial cell adhesion. The initial low affinity interaction between leukocytes and venular endothelium is manifested as a rolling behaviour. Rolling leukocytes can then become firmly adherent (stationary) on the vessel wall, where the process of transendothelial leukocyte migration can occur if a chemotactic signal is generated in the perivascular compartment. Each of the three stages of leukocyte recruitment (Figure 1), i.e., rolling, firm adhesion (adherence) and transendothelial migration, involves the participation of different families of adhesion molecules, including the selectins, β-integrins, and supergene immunoglobulins (Table 1).

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Figure 1. Steps in the recruitment of leukocytes in postcapillary venules. (A) illustrates that in the absence of an inflammatory stimulus, leukocytes are largely flowing in the stream of red cells with no adhesive interactions with venular endothelium. (B) illustrates the low affinity interaction between leukocytes and endothelium that is mediated by selectins and manifested as rolling. (C) illustrates that activation of leukocytes and/or endothelial cells can result in stationary adhesion of leukocytes. (D) illustrates that firmly adherent leukocytes can emigrate from venules into the adjacent interstitial compartment, usually along a chemotactic gradient.

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Table 1. Adhesion molecules involved in leukocyte-endothelial cell adhesionThumbnail image of

Selectins

L-selectin

The selectins are a family of lectin-like molecules that mediate leukocyte rolling. L-selectin is normally expressed on most circulating leukocytes while its ligand is only present on activated endothelium. L-selectin is shed from the surface of activated neutrophils, which consequently limits the ability of these cells to roll on endothelial cells (Tedder et al., 1995a). Mutant mice that are genetically deficient in L-selectin exhibit an attenuated leukocyte recruitment after an inflammatory stimulus (Tedder et al., 1995b). L-selectin binds to a number of different CAMs expressed on the endothelial cell (Table 1) including P-selectin, E-selectin and GlyCAM.

P-selectin

This adhesion molecule is expressed on the surface of activated endothelial cells and platelets. It is stored in Weibel-Palade bodies in endothelial cells and in alpha-granules in platelets. When endothelial cells are stimulated (e.g. with thrombin or histamine), P-selectin is mobilized to the surface of activated endothelial cells within minutes, after which it is either recycled back inside the cell membrane or shed into the plasma (Tedder et al., 1995a). During inflammation, endothelial P-selectin acts to recruit leukocytes into postcapillary venules, while platelet-associated P-selectin promotes the aggregation of leukocytes with platelets to form thrombi (Kunkel et al., 1996). Endothelial cells can also synthesize and express P-selectin in response to endotoxin or cytokines. However, important species difference have been observed in the response to these stimuli: whereas tumour necrosis factor-alpha (TNF-α) and endotoxin increase expression of P-selectin in murine endothelial cells (Eppihimer et al., 1996; Pan et al., 1998b), they do not do so in human endothelial cells (Yao et al., 1996). This differential response may be related to differences in the P-selectin promoter among species (Pan et al., 1998a). Leukocytes normally express several ligands for P-selectin, including L-selectin and P-selectin glycoprotein ligand-1 (PGSL-1) (Panés & Granger, 1998).

E-selectin

Unlike P-selectin, the expression of E-selectin on endothelial cells is entirely under transcriptional control. While E-selectin is not constitutively expressed on endothelial cells, its synthesis (and expression) can be induced by cytokines such as interleukin-1 (IL-1) and TNF-α or by endotoxin (Fries et al., 1993). E-selectin expression is detected as early as 2 h after endotoxin stimulation and returns to baseline values by 8 h. This contrasts with P-selectin which has an early peak derived from the preformed pool, a further transcription-dependent elevation that peaks at about 5 h, followed by a sustained level of expression beyond 12 h (Eppihimer et al., 1996). Ligands on the leukocyte for E-selectin include L-selectin and E-selectin ligand (ESL).

Integrins

Integrins are heterodimeric proteins consisting of alpha and beta subunits. These glycoproteins are expressed on the surface of leukocytes, where they can mediate leukocyte-endothelial cell adhesion within a few minutes after an inflammatory stimulus. The specificity of the various integrins depends on their molecular structure. The integrins associated with leukocyte adhesion belong to the beta-1, beta-2 and beta-7 subfamilies. Members of the beta-2 subfamily contain one of four different alpha chains designated CD11a, CD11b, CD11c, and CD11d that are coupled to a common beta chain, CD18 (see Table 1). The heterodimer CD11a/CD18 is expressed on the surface of most leukocytes and interacts with ICAM-1 and ICAM-2 on endothelial cells to cause firm adhesion (Marlin & Springer, 1987). This integrin is not stored to any appreciable degree within the leukocyte so upregulation most likely results from a conformational change that allows the leukocyte to interact with ICAM-1 (Panés & Granger, 1998).

The heterodimers CD11b/CD18 and CD11c/CD18, expressed by granulocytes and monocytes, are stored in granules and on activation of the leukocyte are rapidly mobilized to the cell surface by fusion with the cell membrane (Panés & Granger, 1998). Activation by inflammatory mediators such as PAF or cytokines such as TNF-α results in a 10–30 fold increase in expression of these integrins on the cell surface. CD11b/CD18 interacts with ICAM-1 on endothelium while the ligand for CD11c/CD18 remains uncertain. The role for the recently described CD11d/CD18 (Shelley et al., 1998) in leukocyte recruitment has not been established. Immunoneutralization of these integrins by appropriately directed antibodies will prevent the adhesion of activated leukocytes to activated endothelium. The adhesion of unstimulated leukocytes to endothelial cells is mediated by CD11a/CD18-ICAM-1 interactions while activated leukocytes use both CD11a/CD18 and CD11b/CD18 to bind to ICAM-1 on the endothelium. The biologic importance of the beta-2 integrins was demonstrated dramatically by the identification of its deficiency in humans. Leukocyte adhesion deficiency is an autosomal recessive trait, characterized by recurrent infections, impaired pus formation and would healing, and abnormalities of many adhesion dependent functions of granulocytes, monocytes and lymphoid cells (Anderson et al., 1985). Features of this disorder can be attributed to deficiency of the cell-surface expression of the entire CD11/CD18 complex, which is, in turn, a result of heterogeneous mutations of the CD18 gene (Anderson et al., 1985). A major role for the beta-2 integrins in mediating leukocyte-endothelial cell adhesion is also supported by experimental results obtained in mutant mice that are genetically deficient in CD18 (Eppihimer et al., 1997; Horie et al., 1997a).

Heterodimers of the beta-1 and beta-7 subfamilies also contribute to recruitment of different leukocyte populations. The beta-1 integrin α4β1 (also designated as very late antigen-4 [VLA-4]) mediates the adhesion of lymphocytes, monocytes, eosinophils and natural killer cells to activated endothelial cells, which express the counter-receptor, vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990). The beta-7 integrin α4β7 is found on lymphocytes that colonize the gut and gut-associated lymphoid tissue. This integrin binds to mucosal address in cell adhesion molecule-1 (MAdCAM-1) on the high endothelial venules of lymphoid tissue and mediates the normal homing of lymphocytes to Peyer's patches (Tsuzuki et al., 1996). It also binds to VCAM-1 under conditions of inflammation.

Immunoglobulin superfamily

Five members of the immunoglobulin (Ig) superfamily act as adhesion molecules; ICAM-1, ICAM-2, VCAM-1, platelet endothelial cell adhesion molecule-1 (PECAM-1) and MAdCAM-1. ICAM-1 is normally found on the surface of endothelial cells, but its expression can be significantly increased upon endothelial activation with cytokines or endotoxin. This upregulation varies from one vascular bed to another and, in general, those beds with high constitutive expression like the lung show less upregulation than vascular beds with low constitutive expression (e.g., skeletal muscle or heart). Following endotoxin challenge, peak ICAM-1 expression occurs at around 5 h and remains elevated for 24 h (Panés et al., 1995). ICAM-1 binds to CD11a/CD18 and CD11b/CD18 on leukocytes and peak levels of ICAM-1 expression are associated with maximum leukocyte adherence. A soluble isoform of ICAM-1 (sICAM-1), which represents shed fragments of endothelial ICAM-1, can be detected in plasma during inflammation (Komatsu et al., 1997). While there is some evidence suggesting that sICAM-1 binds to CD11/CD18 on leukocytes, the functional consequences of this binding remains unclear. However, the importance of ICAM-1 in mediating the recruitment of leukocytes has been demonstrated using both ICAM-1 blocking antibodies and ICAM-1 deficient mice. ICAM-2 is constitutively expressed on endothelial cells and this expression is not influenced by the level of activation of the endothelial cell. ICAM-2 binds to CD11a/CD18 but with a lower affinity than ICAM-1 (Panés & Granger, 1998).

VCAM-1, which can bind to both α4β1 and α4β7 on leukocytes, mediates the trafficking of monocytes and lymphocytes. While the constitutive level of VCAM-1 expression is significantly lower than that of ICAM-1, profound increases in VCAM-1 density on endothelial cells are noted 5–9 h after cytokine stimulation (Henninger et al., 1997). PECAM-1, which is constitutively expressed on platelets, leukocytes and endothelial cells, mediates the adhesion of both platelets and leukocytes to endothelial cells (via homophilic interactions) and the migration of leukocytes through endothelial cells (Muller et al., 1993) as well as migration of these cells through the perivascular basement membrane (Wakelin et al., 1996). While cytokine challenge does not result in an increased expression of PECAM-1 on endothelial cells, the adhesion molecule is redistributed to the borders of adjacent endothelial cells where it participates in endothelial cell-cell interactions that affect leukocyte transmigration and microvascular permeability (Romer et al., 1995).

MAdCAM-1 is expressed on high endothelial venules. MAdCAM-1 serves as a ligand for L-selectin and α4β7 integrin and it is involved in lymphocyte homing to Peyer's patches.

Modulation of leukocyte-endothelial cell adhesion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

Data derived from both in vitro and in vivo studies have implicated a number of chemical and physical factors that can influence both the time-course and magnitude of leukocyte-endothelial cell adhesion. The principal physical influence on the adhesion process is shear stress, a force that is generated by the movement of blood in postcapillary venules. Venular wall shear stress determines the level of leukocyte rolling and firm adhesion, and it dictates the contact area between rolling leukocytes and the endothelial cell surface. Reductions in venular blood flow (shear stress) facilitate leukocyte rolling and adhesion, while increases in blood flow tend to oppose leukocyte-endothelial cell adhesion. At low shear rates, the contact time between adhesion molecules on leukocytes and endothelial cells is increased thereby allowing greater opportunity for formation of the strong adhesive bonds that is necessary for a rolling leukocyte to become stationary (Panés & Granger, 1998).

A large number of biological chemicals have been identified that either inhibit or promote leukocyte-endothelial cell adhesion (see Table 2). Most of the chemicals identified as modulators of leukocyte adhesion fall into the category of pro-adhesive agents. Some of these agents, such as histamine, platelet activating factor and IL-8, can rapidly (within 2–3 mins) increase the level of activation and/or expression of adhesion molecules on leukocytes (e.g., CD11b/CD18) and/or endothelial cells (e.g., P-selectin). Other pro-adhesive agents, such as the cytokine TNF-α, act more slowly to promote leukocyte adhesion by enhancing the transcription-dependent expression of endothelial cell adhesion molecules that act to extend and further increase the leukocyte rolling (E-selectin) and adherence/emigration (ICAM-1) responses.

Table 2. Modulation of leukocyte-endothelial cell adhesionThumbnail image of

The list of endogenous anti-adhesive chemicals that have been identified to date is relatively small. These agents tend to exert their inhibitory actions on both the leukocyte and endothelial cell, and the underlying mechanisms of action remain poorly understood. Some of the anti-adhesive compounds (nitric oxide, PGI2, and adenosine) are also potent vasodilators, which raises the possibility that their actions in vivo can be attributed to increases in venular shear rate. However, there is substantial evidence suggesting that increased shear rates account for only a small component of the inhibitory effect on leukocyte-endothelial cell adhesion. Nitric oxide and glucocorticoids appear to exert at least some of their effects by inhibiting the transcription-dependent expression of endothelial cell adhesion molecules (Panés & Granger, 1998).

Targets for therapeutic intervention

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

The cellular and molecular basis for the recruitment of leukocytes to sites of inflammation is highly complex and multifactorial, however there is sufficient experimental evidence in the literature to outline the key elements and sequential nature of this process. As illustrated in Figure 2, the inflammatory response involves the participation of multiple cell types, including circulating leukocytes, vascular endothelial cells, and perivascular cells (e.g., mast cells, macrophages), with the latter cells contributing to the initiation and perpetuation of inflammation through the generation of a variety of inflammatory mediators. Following the primary insult (infection, injury, or hypersensitivity reaction), macrophages and mast cells are stimulated (e.g., by activated complement) to release mediators, such as histamine, oxygen radicals, platelet activating factor, leukotrienes, and cytokines. The engagement of histamine, leukotrienes and certain other mediators with their receptors on endothelial cells results in the rapid mobilization of P-selectin from its preformed pool in Weibel-Palade bodies to the cell surface. Hence, within minutes there is an increased recruitment of rolling leukocytes in postcapillary venules that allows for an enhanced exposure of the previously circulating cells to other mediators liberated from the inflamed tissue. The slowly rolling leukocytes are exposed to PAF, leukotrienes, and other mediators that rapidly activate, and then promote the shedding of, L-selectin on leukocytes. As the L-selectin is shed, there is a corresponding increase in the expression and activation of β2-integrins on leukocytes. The newly expressed and/or activated CD11/CD18 can then bind to its counter-receptor ICAM-1, which is constitutively expressed on endothelial cells. The β2-integrin/ICAM-1 adhesive interactions enable the inflamed tissue to recruit firmly adherent and emigrating leukocytes within a few minutes after the initial insult. This intimate interaction also allows PECAM-1, which is constitutively expressed on both endothelial cells and leukocytes, to promote the homophilic adhesion and emigration of leukocytes.

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Figure 2. Mechanisms underlying the expression of adhesion molecules on leukocytes and endothelial cells at the onset of inflammation. Perivascular cells such as mast cells and macrophages initiate the response by releasing a variety of inflammatory mediators. Engagement of lipid mediators (LTB4 and PAF) with receptors on neutrophils results in the activation of β2-integrins (CD11/CD18). Engagement of histamine with its receptor (H1) on endothelial cells results in the rapid mobilization of preformed P-selectin from its storage site (Weibel-Palade bodies). Engagement of cytokines (e.g., TNFα) to their receptors on endothelial cells lead to the activation of nuclear transcription factors (e.g., NF-κB) that stimulates the synthesis of adhesion glycoproteins, such as VCAM-1, ICAM-1, and E-selectin, which are subsequently expressed on the cell surface.

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While the rapid inducers of leukocyte rolling, adherence and emigration are eliciting their actions, mast cell- and macrophage-derived cytokines engage with their receptors on endothelial cells. This ultimately (via specific signalling pathways) leads to the activation of nuclear transcription factors that modulate the biosynthesis of endothelial cell adhesion molecules that mediate leukocyte rolling (E-selectin) and adherence (ICAM-1, VCAM-1). Consequently, within a few hours (2–4) after the initial inflammatory insult, there is a profound increase in the density of virtually all endothelial cell adhesion molecules that participate in the trafficking of leukocytes during inflammation. As a result of this increased endothelial CAM expression, the recruitment of leukocytes can be sustained at both a higher level and for a longer duration.

The sequence of events described above suggest that there are several potential cellular and molecular loci that can be targeted to interfere with the leukocyte-endothelial cell adhesion associated with inflammation. The following section addresses three potential targets for therapeutic intervention against inflammation that relate to the process of leukocyte-endothelial cell adhesion. These are: (1) inflammatory mediator release and receptor engagement, (2) adhesion molecule synthesis, and (3) adhesion molecule function.

Inflammatory mediators

Experimental findings

A large number of mediators have been implicated in the initiation of leukocyte-endothelial cell adhesion during inflammation (Table 2). Several experimental strategies have been employed to assess the contribution of specific mediators to this facet of the inflammatory response. These include: (1) detection of the mediator at sites of inflammation characterized by leukocyte adhesion, (2) demonstration that leukocyte-endothelial cell adhesion can be induced by exposure of non-inflamed venules to an exogenous source of mediator, and (3) inhibition of leukocyte adhesion by agents known to either antagonize or inhibit the production of the mediator. Several inflammatory mediators, including histamine, PAF, LTB4, cytokines, and chemokines have been shown to promote leukocyte rolling, adherence and/or emigration when applied directly to postcapillary venules (Panés & Granger, 1998). A role for specific leukocyte and/or endothelial cell adhesion molecules in mediating these actions has been demonstrated for most of the mediators using either monoclonal antibodies directed against the CAMs (Zimmerman et al., 1994a) or mice that are genetically deficient in a specific CAM (Kunkel et al., 1996; Xu et al., 1994). In some instances (e.g., histamine and cytokines), corroborative in vivo evidence of CAM involvement has been obtained from quantitative estimates of endothelial CAM expression in different vascular beds after administration of the inflammatory mediator (Eppihimer et al., 1996; Henninger et al., 1997).

Antagonists to histamine (Kurose et al., 1994c), PAF (Kubes et al., 1990b), leukotrienes (Zimmerman et al., 1990), IL-8 (Mulligan et al., 1993) and TNF-α (Appleyard et al., 1996) have been shown to prevent or attenuate the leukocyte-endothelial cell adhesion observed in different models of acute or chronic inflammation. For example, the histamine receptor (H1) antagonist hydroxyzine has been shown to markedly reduce the leukocyte-endothelial cell adhesion and consequent microvascular injury elicited by Clostridium difficile toxin A (Kurose et al., 1994c). Both PAF- (e.g., WEB 2086) and LTB4-receptor (SC41930) receptor antagonists have proven effective in reducing the leukocyte adhesion observed in tissues exposed to either ischaemia and reperfusion (Kubes et al., 1990a; Zimmerman et al., 1990) or to inhibition of nitric oxide biosynthesis (Arndt et al., 1993). Finally, a role for LTB4 in mediating the leukocyte-endothelial cell adhesion induced by non-steroidal anti-inflammatory drugs, such as aspirin and indomethacin, has been demonstrated using both an LTB4-receptor antagonist (SC41930) and a leukotriene biosynthesis inhibitor (L663,536) (Asako et al., 1992).

The involvement of inflammatory mediators in leukocyte-endothelial cell adhesion has also been addressed using agents that are known to stabilize or inhibit the function of perivascular cells that produce and release inflammatory mediators. Mast cell stabilizers have been shown to attenuate the leukocyte-endothelial cell adhesion elicited in postcapillary venules by either ischaemia-reperfusion (Kurose et al., 1997), oxidized low density lipoproteins (Liao & Granger, 1996), Helicobacter pylori (Kurose et al., 1994b), or Clostridium difficile toxin A (Kurose et al., 1994c). Kupffer cells, the resident macrophages of the liver, have also been implicated in the recruitment of adherent leukocytes. It was shown that treatment with gadolinium chloride, which reduces Kupffer cell function, reduces the recruitment of adherent leukocytes in terminal hepatic venules to the same low level as observed after administration of a TNF-α blocking antibody in a model of ischaemia-reperfusion (Horie et al., 1997b).

Therapeutic applications

A number of drugs have been developed to antagonize the actions of inflammatory mediators. While many of these drugs, including antihistamines and mast cell stabilizers are widely used in the treatment of inflammation, there is no evidence that directly implicates inhibition of leukocyte-endothelial cell adhesion as a primary mode of action. Nonetheless, data derived from experimental models of inflammation suggest that these agents are likely to interfere with the expression of either leukocyte or endothelial CAM. It also reasonable to assume that the new generation of mediator-directed therapeutic agents such as soluble IL-1 receptor antagonists or TNF-α antibodies, which have proven anti-inflammatory actions in the clinical setting (Fisher et al., 1994; Targan et al., 1997), exert at least part of their effect through inhibition of endothelial CAM expression.

A major advantage of inflammatory mediator antagonists is their ability to suppress different components of the inflammatory response. For example, PAF and LTB4 antagonists may also act by interfering with the ability of granulocytes to produce oxygen radicals, release proteases, and increase the expression of β2-integrins. Similarly, cytokine-directed inhibitors can blunt the inflammatory responses of macrophages, mast cells, and circulating leukocytes, all of which release mediators that can amplify the expression of endothelial CAMs. However, a major disadvantage of inflammatory mediator-directed therapeutic strategies is their reliance on the assumption that a single mediator can orchestrate all or most of the redundant processes that contribute to leukocyte recruitment. The large number of mediators that can elicit the upregulation of leukocyte rolling receptors alone would argue against the wisdom of this strategy. Nonetheless, it is reasonable to assume that administration of several antagonists should prove more effective in inhibiting the CAM expression associated with inflammation.

Adhesion molecule synthesis

An important target for therapeutic intervention against inflammation that relates to the process of leukocyte-endothelial cell adhesion is endothelial CAM biosynthesis. Targeting this process has the potential to impact the expression of all endothelial CAMs and consequently exert a profound inhibitory effect on leukocyte recruitment. While there are several strategies that can be used to inhibit the biosynthesis of endothelial CAMs, some of these have recently received considerable attention and appear to hold much promise.

Nuclear transcription factors

Inducible gene expression is a key regulatory mechanism that requires transcriptional activator proteins whose DNA binding or transcription activity is induced upon exposure of cells to specific stimuli. Of the many transcription factors that have been described, nuclear factor kappa-B (NF-κB) and activation protein-1 (AP-1) appear to be particularly relevant to the regulation of genes involved in the inflammatory cascade. Both factors represent families of polypeptides with related DNA-binding activity but distinct transactivating potential.

NF-κB is an inducible, multisubunit transcription factor of higher eukaryotes. The DNA-binding forms of NF-κB exist as dimeric complexes composed of various combinations of members of the Rel/NF-κB family of polypeptides. NF-κB dimers (e.g., p50/p65) are normally sequestered in the cytosol of unstimulated cells via noncovalent interactions with a class of inhibitory proteins called IκBs. These inhibitory proteins prevent nuclear transport and DNA binding of NF-κB/Rel proteins. Signals that induce NF-κB activation cause the dissociation and subsequent degradation of IκB proteins, which allows NF-κB dimers to enter the nucleus and induce gene expression (May & Ghosh, 1988).

NF-κB plays an important role in the expression of a large number of inducible genes, many of which contribute to the cellular responses to stress, injury and inflammation. Consequently, NF-κB can be activated by signals that are associated with such states, including cytokines (such as IL-1 and TNF-α), bacterial endotoxins, and pro-apoptotic and necrotic stimuli such as oxygen free radicals, u.v. light and gamma-irradiation. When cells are exposed to these pathogenic stimuli, a cascade of events leads to the phosphorylation and subsequent degradation of IκB, resulting in NF-κB liberation and its entry into the nucleus, where it activates gene expression (Baeuerle, 1998). NF-κB activation is triggered by the phosphorylation and subsequent conjugation of IκB with ubiquitin, which makes IκB a substrate for degradation by the proteasome proteolytic pathway. Peptide aldehyde inhibitors of the proteasome such as calpain inhibitor 1 and MG-132 (Brown et al., 1995) have been shown to block the degradation of IκB and consequent activation of NF-κB that is elicited by TNF-α.

AP-1 is another transcription factor that is composed of homo- and heterodimers of the products of the jun and fos proto-oncogenes. AP-1 subunits include c-jun, jun-B, jun-D, c-Fos, Fos-B, Fra-1 and Fra-2 (Karin & Smeal, 1992). AP-1 activity is induced by many stimuli, including phorbol esters (which activate protein kinase C), polypeptide hormones, cytokines and hydrogen peroxide (Angel & Karin, 1991; Roebuck et al., 1995). Several mechanisms account for the regulation of AP-1 activity, including phosphorylation and postranslational modification (Angel & Karin, 1991).

Transcription factors and adhesion molecule expression

Binding sites for NF-κB have been identified in the promoter regions of the genes for E-selectin, VCAM-1 and ICAM-1, while a binding site for AP-1 has been localized on the promoter region of the ICAM-1 gene. Point mutations which decrease NF-κB binding to κB elements result in diminished cytokine-induced E-selectin expression on cultured endothelial cells, suggesting that NF-κB plays an important role in cytokine induction of the E-selectin gene (Essani et al., 1996). Two closely spaced functional κB elements have also been identified in the MAdCAM-1 promoter (Takeuchi & Baichwal, 1995). The increased ICAM-1 expression on endothelial cells exposed to hydrogen peroxide (Lo et al., 1993) appears to be mediated through AP-1, and independent of κB elements (Roebuck et al., 1995). Therefore, hydrogen peroxide and cytokines appear to activate ICAM-1 gene transcription in endothelial cells through distinct cis-regulatory elements within the ICAM-1 promoter.

It was recently demonstrated that inhibitors of the proteasomal degradation pathway for IκB lead to decreased nuclear accumulation of NF-κB and the subsequent abrogation of TNF-α induced cell-surface expression of E-selectin, VCAM-1, and ICAM-1 in endothelial cells (Read et al., 1995). This response has important functional consequences because proteasome inhibitors also block both the adherence and emigration of leukocytes in human endothelial cell monolayers.

Therapeutic applications

Recent evidence indicates that the transcription factors AP-1 and NF-κB are major targets for some of the commonly used anti-inflammatory drugs including glucocorticoids, aspirin, salicylates, gold salts and D-penicillamine. Glucocorticoids activate the glucocorticoid receptor (GR) in the cytosol, which then form GR-GR homodimers that bind a specific DNA sequence termed the glucocorticoid response element (GRE). Increased transcription of genes bearing a GRE in their promoter region follows. In addition to positive regulation of gene expression, glucocorticoids inhibit the expression of a wide variety of genes involved in the inflammatory process.

An improved understanding of the anti-inflammatory mechanism of glucocorticoids came from the observation that ligand activated GR inhibits AP-1 and NF-κB mediated transcription (reviewed in Cato & Wade, 1996). For example, glucocorticoids inhibit the NF-κB mediated expression of adhesion molecules ICAM-1 (Tailor et al., 1997), VCAM-1 (Tessier et al., 1996), and E-selectin (Brostjan et al., 1997). Glucocorticoids also inhibit the AP-1 expression of collagenases I and IV (Handel, 1997). Several mechanisms for the mutual antagonism between GR and NF-κB, and between GR and AP-1, have been proposed. For example, a direct protein-protein interaction between the GR and NF-κB has been proposed that will prevent the binding of GR and NF-κB to their respective DNA response elements (Ray & Prefontaine, 1994) (Figure 3). Similarly, binding of GR to Jun and Fos has been thought to cause mutual inhibition of GR and AP-1 DNA binding (Sakurai et al., 1997). Glucocorticoids also increase transcription of the gene for IκB, thereby increasing the formation of this protein which binds to activated NF-κB in the nucleus. The IκB protein probably induces the dissociation of NF-κB from κB sites on target genes and causes NF-κB to move to the cytoplasm (Scheinman et al., 1995).

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Figure 3. Mechanisms used by the glucocorticoid receptor to inhibit transactivation by transcription factors. (1) After the glucocorticoid (GC) activates its receptor (GR), a direct protein-protein interaction between GR and transcription factors (TF) will prevent the binding of TF to the respective DNA response elements. (2) Binding of GR to the promoter region of the IκB gene results in transactivation; IκB binding NF-κB will prevent binding or displace NF-κB from κB sites. (3) Binding of GR to glucocorticoid responsive elements modulates TF-induced transactivation.

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Recent evidence shows that in human inflammatory bowel disease (Ardite et al., 1998) and in asthma (Adcock, 1996), cessation of the inflammatory activity in response to steroid treatment is associated with disappearance of NF-κB from nuclear extracts of intestinal or bronchial mucosa, and that failure to abrogate NF-κB activation results in persistence of the inflammatory process. In patients with steroid-resistant asthma, there appears to be an exaggerated activation of AP-1 that binds to and therefore sequesters activated GR inside the nucleus; this would reduce the availability of GR to inhibit NF-κB, which is normally activated in such patients (Adcock et al., 1995).

Gold salts significantly inhibit AP-1 DNA binding in nuclear extracts at concentrations of 5 mM, which is within the range achieved in the serum of rheumatoid arthritis patients under this treatment. Gold salts have also been shown to inhibit IL-1 induced expression of NF-κB and AP-1 dependent transfected reporter genes (Williams et al., 1992) and to inhibit DNA binding activity of NF-κB in vitro (Yang et al., 1995). Consistent with these effects on pro-inflammatory transcription factors is the observation that gold salts inhibit expression of ICAM-1 and VCAM-1 in endothelial cells (Koike et al., 1994). D-penicillamine inhibits AP-1 DNA binding in nuclear extracts in the presence of free radicals, presumably by forming disulphide bonds with the cysteine residues in the DNA binding domains of Jun and Fos (Handel et al., 1996). Aspirin and sodium salicylate also inhibit activation of NF-κB (Kopp & Ghosh, 1994). It has been shown that salicylate inhibits activation of NF-κB by preventing phosphorylation and subsequent degradation of IκB, and this results in blockade of the TNF-induced increase in mRNA levels of ICAM-1, VCAM-1 and E-selectin, and a dose-dependent inhibition of TNF-induced surface expression of these adhesion molecules (Pierce et al., 1996). Indomethacin, a nonsalicylate cyclo-oxygenase inhibitor, has no effect on surface expression of adhesion molecules, suggesting that the effects of salicylate are not due to inhibition of cyclo-oxygenase (Pierce et al., 1996).

Proteasome inhibitors have been tested in experimental models of inflammation with promising results. In a rat model of experimental colitis induced by peptidoglycan/polysaccharide, proteasome inhibition using MG-341 significantly suppressed the upregulation of VCAM-1 and iNOS in the colon and this was associated with a reduction of colonic inflammation (Conner et al., 1997).

Systemic inhibition of NF-κB activation in humans for prolonged periods carries some risk since there is the potential for severe immunosuppression and enhanced cytokine-induced cytotoxicity (Beg & Baltimore, 1996; Wang et al., 1996). Removal of the p65 gene is lethal to the mouse embryo (Beg et al., 1995) and p50 knockout mice breed normally but have increased susceptibility to infections (Sha et al., 1995). However, because both NF-κB and AP-1 are inducible transcription factors that act in response to environmental stimuli, it may be possible to titrate the dose of an NF-κB directed inhibitor within a dynamic range to achieve a therapeutic, and subtoxic, response.

Antisense oligonucleotides

The potential side effects and nonspecific actions of the currently used transcription factor inhibitors have led to a search for drugs that rely on an alternative strategy for regulation of protein synthetic pathways that are specifically related to the inflammatory process. One such approach that has already yielded significant results is the use of antisense oligodeoxynucleotides (ODNs) (Agrawal, 1996).

Antisense ODNs are single stranded DNA sequences complimentary to a specific messenger RNA (mRNA). In theory, antisense ODNs, through base pairing of complementary bases, specifically bind to a mRNA thereby blocking the expression of the gene product (Sharma & Narayanan, 1995) (Figure 4). The mechanism of antisense ODN inhibition may involve multiple modalities, including the induction of RNAase H activity through the formation of an DNA:RNA hybrid, steric hindrance that interferes with translation, and inhibition of mRNA processing and transport from the nucleus (Sharma & Narayanan, 1995). Although ODNs can undeniably hit their intended targets, when an antisense molecule elicits a biological response, it can be difficult to determine whether the response was elicited because the reagent interacted specifically with its target RNA, or because some non-antisense reaction – involving other nucleic acids or proteins – took place. In order to distinguish between antisense and non-antisense effects, ODNs with an altered (scrambled) nucleotide sequence of the antisense are employed. The ability of ODNs to act as sequence-specific inhibitors of gene expression depends on their entry into the cytoplasm and/or nucleus. Since ODNs are negatively charged, they cannot passively diffuse through cellular membranes. Instead, they appear to enter cells via the processes of adsorptive and fluid-phase endocytosis (Yakubov et al., 1989). The identity of cell membrane proteins that bind ODNs is unclear, however a number of heparin binding proteins such as fibroblast growth factors and vascular endothelial growth factors have been implicated in this process (Guvakova et al., 1995). Of particular interest is the observation that ODNs may enter cells via an interaction with the heparin binding protein CD11b/CD18 (Benimetsaya et al., 1997). This suggests that ODNs may be preferentially taken up by activated leukocytes and thereby exert a more profound influence on leukocyte function. Upon entering the cell, in order to be effective, antisense ODNs must be resistant to nucleases, and have sequence-specific effects (Agrawal, 1996). Although questions of ODN specificity remain, there is growing evidence that antisense molecules can be useful pharmacological tools when applied carefully.

image

Figure 4. Target sites for decoy and antisense ODNs. Antisense ODNs are single stranded DNA sequences that specifically bind to mRNA, thereby blocking expression of the gene product. Transcription factor (TF) decoys are double stranded ODNs that compete for protein binding with the authentic binding elements, thereby interfering with gene regulation.

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Therapeutic applications

Antisense molecules have been produced to block the production of specific subunits of NF-κB. In murine embryonic stem cells, antisense ODN raised against p65 elicit a significant reduction in p65 mRNA, and have profound effects on cell adhesion properties (Sokoloski et al., 1993). Treatment of neutrophils with antisense phosphorothioate ODN to the p65 subunit of NF-κB results in a reduction in the expression of p65 and effectively abolishes the upregulation of CD11b normally elicited by either formyl-met-leu-phe and tissue plasminogen activator, indicating that antisense oligomers to p65 can interfere with neutrophil adhesion molecules (Narayanan et al., 1993). Antisense oligonucleotides to p65/p50 also reduce the expression of CD11b/CD18 on stimulated monocytic HL60 cells (Sokoloski et al., 1993), and the expression of E-selectin, ICAM-1, and VCAM-1 on stimulated human umbilical vein endothelial cells (Lee et al., 1995). A likely consequence of these actions of p65 ODNs is a profound diminution of the inflammatory response in mice with TNBS-induced colitis, as well as in IL-10 deficient mice with colitis. In both models of colitis, administration of p65 antisense was more effective in treating the inflammatory disease than glucocorticoids (Neurath et al., 1996).

ISIS 2302, an antisense phosphorothioate ODN to human ICAM-1, appears to selectively inhibit cytokine-induced ICAM-1 expression in a variety of human cells in vitro and in vivo (Bennett et al., 1994; Yacyshyn et al., 1998). Furthermore, a recent pilot study in patients with Crohn's disease showed that administration of this drug reduced ICAM-1 expression in intestinal mucosa and resulted in a significant decrease in corticosteroid usage relative to placebo treated patients (Yacyshyn et al., 1998). A murine analogue, ISIS 3082, has been shown to be active in multiple models of inflammation, including dextran-sulphate induced colitis (Bennett et al., 1997), allograft rejection (Stepkowski et al., 1994), and endotoxin-induced neutrophil recruitment in the lung (Kumasaka et al., 1996). Two studies have shown that treatment of stimulated human umbilical vein endothelial cells with antisense ODNs directed against ICAM-1, E-selectin, or VCAM-1 results in selective inhibition of protein expression and a corresponding reduction of monocyte (or HL-60 cell) adhesion to the cultured endothelial cells (Bennett et al., 1994; Lee et al., 1995).

Decoy oligonucleotides

Another strategy used to inhibit components of the inflammatory response is decoy ODNs that specifically interfere with regulatory proteins (Morishita et al., 1998). Inhibition of sequence-specific DNA-binding proteins can be achieved with double-stranded ODNs containing the specific binding elements. The transcription factor decoy (TFD) competes for protein binding with the authentic binding elements and consequently interfere with gene regulation (Figure 4). The mechanism of TFD action is distinct from the antisense approach in that the production of the target protein is unaffected and only its capacity to bind to the regulatory DNA element is affected. The TFD strategy is particularly attractive for several reasons: (1) the potential drug targets (transcription factors) are plentiful and readily identifiable, (2) synthesis of the sequence-specific decoy is relatively simple and can be targeted to specific tissues, (3) knowledge of the exact molecular structure of the target transcription factor is unnecessary, and (4) decoy ODNs may be more effective than antisense ODNs in suppressing an inflammatory reaction by virtue of their capacity to inhibit transcription of the multiple genes activated by a given transcription factor. Like antisense ODNs, the same critical parameters for optimal function exist, i.e., TFDs must be nuclease resistant, be taken up by cells, and have sequence-specific effects. A major concern regarding the use of TFDs is nonspecific effects, particularly those of phosphorothioate-substituted ODNs. Non-sequence-specific inhibition may occur from blockade of cell surface receptor activity or interference with other proteins (Gibson, 1996). Furthermore, TFDs containing guanine cytosine dinucleotides may result in immune activation (Khaled et al., 1996). To address these concerns, careful controlled experiments must be performed to eliminate the potential nonspecific effects of TFDs-mediated therapy. Furthermore, scrambled ODNs with several mutations in the consensus sequence should be used as controls.

Therapeutic applications

Based on evidence showing NF-κB activation and increased adhesion molecule expression in tissues exposed to ischaemia-reperfusion, the value of treatment with decoy ODN against NF-κB has been evaluated in this model of acute inflammation (Morishita et al., 1997). In rats, intracoronary administration of NF-κB decoy ODNs before or after coronary artery occlusion markedly reduces the size of the myocardial infarct measured at 24 h after reperfusion. The selectivity of the NF-κB decoy ODN effect was confirmed by the finding that the reduction in infarct size was not observed in rats treated with antisense ODN directed against the iNOS gene. The specificity of the NF-κB decoy in inhibiting cytokine and adhesion molecule expression was also confirmed by in vitro experiments using human and rat coronary artery endothelial cells that were transfected with the NF-κB decoy; the decoy ODN inhibited the expression of ICAM-1, VCAM-1 and E-selectin (Morishita et al., 1997). In another study of rats subjected to myocardial ischaemia-reperfusion, the efficacy of a decoy ODN against NF-κB was confirmed by an enhanced recovery of left ventricular function and coronary flow in rats treated with the NF-κB decoy ODN, compared to groups of rats receiving a scrambled decoy or placebo. The protection against ischaemic damage afforded by the NF-κB decoy ODN was associated with less neutrophil adherence to endothelial cells and a lower tissue level of IL-8 (Sawa et al., 1997). The findings of these two studies suggest that in vivo administration of decoy ODNs against NF-κB may be an effective therapeutic strategy for treatment of myocardial ischaemia.

In a recent study, the two approaches to modulate gene expression were compared, i.e., the ability of an antisense that binds to the mRNA for the ReIA subunit of NF-κB to inhibit cytokine production by TNF-stimulated splenocytes was compared to the responses observed in splenocytes receiving a decoy with double-stranded ODNs that bind the NF-κB protein. TNF-α expression was reduced by both treatments, as were the levels of IL-2. However, the antisense effects did not last beyond 24 h, whereas the decoy ODN was shown to inhibit cytokine production even at 72 h after the initial TNF-stimulation (Khaled et al., 1998).

Limitations to the application of decoy or antisense ODN strategies to modulate transcription factor activity are similar to those discussed above in relation to drugs that block NF-κB activation, with potential inhibition of normal physiological responses as the major concern. Therefore, the application of decoy ODN strategies as gene therapy may be limited to the treatment of those acute inflammatory conditions (e.g., ischaemia-reperfusion) in which activation of transcription factors plays a pivotal role.

Adhesion molecule function

The experimental approach for attenuation of leukocyte-endothelial cell adhesion that has received the most attention is inhibition of adhesion molecule function. This strategy has proven to be very effective in limiting both acute and chronic forms of inflammation in animal models and has received limited attention in the clinical setting. In most of these studies, inhibition of adhesion molecule function was achieved through immunoneutralization with monoclonal antibodies that target specific adhesion glycoproteins. Another strategy has involved the administration of soluble isoforms of the adhesion molecules expressed either on leukocytes (e.g., soluble P-selectin glycoprotein ligand-1 [PSGL-1]) or endothelial cells (e.g., sICAM-1).

Monoclonal antibodies (mAbs) have been applied to both in vitro and in vivo models of inflammation in order to define the specific contribution of leukocyte and endothelial cell adhesion glycoproteins to different steps in the recruitment of leukocytes, i.e., rolling, adherence, and emigration. The same mAbs have also been applied to a variety of animal models of inflammation, including arthritis, malaria, meningitis, acute allograft rejection, haemorrhagic shock, and sepsis (Korthuis et al., 1994). In many model systems, the mAbs have been shown to blunt the recruitment of leukocytes and to diminish the tissue injury that usually accompanies the inflammatory response. The magnitude of the inhibitory effect exhibited by the mAb varies with the model studied and the adhesion molecule targeted. Monoclonal antibodies directed against either the common β-subunit of CD11/CD18 or ICAM-1 appear to offer the highest degree of protection in models of ischaemia-reperfusion injury (Kurose et al., 1994a). P-selectin-directed mAbs have also shown efficacy in similar models, but part of this protective action has been attributed to an attenuation of leukocyte-platelet aggregation (Kubes et al., 1994; Weyrich et al., 1993). There are, on the other hand, relatively few reports describing a protective action of E-selectin specific mAbs in experimental models of inflammation. The latter observation may reflect a limited blocking function of the mAbs employed or the absence of a role for E-selectin in these models of inflammation. A limitation in interpreting negative findings with mAbs is the uncertainty regarding whether blocking doses are achieved in vivo. Target levels are generally based on the minimum mAb concentration required to achieve maximal inhibition of leukocyte adhesion in vitro. Experience gained from open-labelled clinical trials (Kavanaugh et al., 1994) indicates that blocking doses are more difficult to achieve in vivo than predicted from in vitro neutrophil binding assays. Another potential limitation of prolonged mAb usage, at least in chronic models of inflammation, is immunogenicity.

Another approach to blocking adhesion molecule function that is gaining attention in the experimental setting is administration of soluble forms of adhesion receptors, such as ICAM-1, sialyl-Lewis X (SLex), and PSGL-1. It has been shown, for example, that administration of soluble SLex (a fucose-containing carbohydrate ligand to P-selectin found on leukocytes) is as effective as a P-selectin mAb in attenuating leukocyte rolling in inflamed mesenteric venules, while a control, fucose-deficient form of the oligosaccharide was without effect (Zimmerman et al., 1994b). Similarly, it has been shown that soluble PGSL-1 administered to rats reduces the renal dysfunction and necrosis normally caused by ischaemia-reperfusion (Takada et al., 1997). A soluble human form of PSGL-1 has also been tested in a feline model of myocardial ischaemia-reperfusion injury (Hayward et al., 1998). The P-selectin antagonist significantly reduced the reperfusion-induced neutrophil accumulation and myocardial necrosis, and it preserved endothelium-dependent vasorelaxation in reperfused coronary arteries. A recombinant form of soluble murine ICAM-1 was recently shown to be effective in reducing the leukocyte-endothelial cell adhesion elicited in mouse mesenteric venules by ischaemia-reperfusion (Kusterer et al., 1998). Hence, the results obtained from all of the experimental studies employing soluble adhesion molecules or their ligands suggest that these agents may be as effective as other anti-adhesion strategies in limiting the accumulation of leukocytes that occurs in ischaemia-reperfusion and other inflammatory conditions.

Therapeutic applications

Clinical trials employing mAbs to leukocyte or endothelial cell adhesion molecules have met with varying success. Monoclonal antibodies directed against the β2-integrins have been used in patients receiving bone marrow grafts. In one study, a mAb against CD18 was unable to prevent bone marrow rejection in adult leukaemic recipients (Baume et al., 1989), while in another clinical series, a CD11a-specific mAb prevented failure of bone marrow grafts in HLA-mismatched young recipients (van Dijken et al., 1990). The latter mAb (CD11a) was shown to be ineffective in reversing acute rejection in renal transplant recipients (Le Mauff et al., 1991).

The outcome of clinical trials employing ICAM-1 specific mAbs appear to be more promising. A phase 1 clinical study using an anti-ICAM-1 mAb as part of the initial immunosuppressive regimen for renal allograft recipients (judged to be at high risk for delayed allograft rejection) demonstrated a lower incidence of both acute graft rejection and delayed graft function (Haug et al., 1993). The same ICAM-1 specific mAb has been shown to produce an improved clinical response in an open-label, dose escalating phase I-II study in patients with severe rheumatoid arthritis (Kavanaugh et al., 1994). For both indications (kidney transplantation, rheumatoid arthritis), the dosing regimen required to achieve the pharmacokinetic target was much higher than that estimated based on in vitro blocking experiments. Furthermore, a high incidence of ICAM-1 mAb anti-idiotype antibodies was found in the kidney transplant patients receiving the murine anti-human ICAM-1 mAb. However, this antigenicity problem should be alleviated with humanized mAbs.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

The therapeutic potential of drugs that target leukocyte-endothelial cell adhesion for treatment of acute and chronic inflammatory diseases seems promising. While several key steps in the inflammatory cascade that result in leukocyte recruitment appear amenable to pharmacologic inhibition, the challenges posed by the potential for disruption of alternate physiological processes as well as immune suppression are significant. However, these limitations may be overcome by research that focuses on the identification and characterization of chemical pathways that uniquely serve the process of leukocyte-endothelial cell adhesion, either at the level of receptor activation, adhesion molecule biosynthesis, and/or adhesion molecule function. The development of safe and effective drugs that target these molecular components of the inflammatory response may yield novel, improved therapies for the debilitating disorders associated with inflammation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References

DN Granger is supported by grants from the National Institutes of Health (HL26441 and DK43785) and Dr J Panés by grant SAF 97/0040 from Comision Interministerial de Ciencia y Tecnologia.

References

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  4. Adhesion Molecules
  5. Modulation of leukocyte-endothelial cell adhesion
  6. Targets for therapeutic intervention
  7. Conclusions
  8. Acknowledgments
  9. References
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