Title: NSAIDs: learning new tricks from old drugs Federico Díaz-González1 and Francisco Sánchez-Madrid2 1
Department of Internal Medicine, Universidad de La Laguna, Rheumatology Service,
Hospital Universitario de Canarias, Santa Cruz de Tenerife. Spain. 2National Center for Cardiovascular Research, Immunology Service, Instituto Investigaciones Sanitarias Princesa, Universidad Autónoma de Madrid. Spain
Key words: Non-steroidal anti-inflammatory drugs, L-selectin, NADPH oxidase,
Corresponding author:
Federico Díaz-González, Professor of Medicine. Department of
Internal Medicine, Universidad de La Laguna. Staff of Rheumatology, Hospital Universitario de Canárias. C/Ofra s/n, 38320, La Laguna, Santa Cruz de Tenerife. Spain e-mail: [email protected] fax: +34-922646792 List of abbreviations:
ADAM: a disintegrin and metalloproteinase domain, COX:
cyclooxigenase, ICAM-1: Intercellular adhesion molecule 1, IL-1: interleukin-1, iNOS: inducible nitric oxide synthase, LFA-1: lymphocyte function-associated antigen 1, MAPK: Mitogen-activated protein kinases, NADPH-oxidase: nicotinamide adenine dinucleotide phosphate-oxidase, NF-kappa B: nuclear factor kappa-light-chain-enhancer of activated B cells, NSAIDs: non-steroidal anti-inflammatory drugs, PC: phosphatidylcholine, PGE1: prostaglandin E1, PI3K: phosphatidylinositide 3-kinases, PPAR: peroxisome proliferatoractivated receptors, ROS: reactive oxygen species, TNF-: tumor necrosis factor-, VCAM1: Vascular-cell adhesion molecule, VLA-4: very late antigen-4 Received: 29-Oct-2014; Revised: 07-Dec-2014; Accepted: 16-Dec-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/eji.201445222.
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Summary Nonsteroidal anti-inflammatory drugs (NSAIDs) comprise a heterogeneous group of pharmacological agents used for the symptomatic treatment of fever, pain and inflammation. Although the main mechanism of action of NSAIDs consists of inhibiting prostaglandin synthesis by blocking the enzyme cyclooxygenase (COX), clinical and experimental data strongly indicate the existence of additional mechanisms. Some of the COX-independent effects are related to the ability of NSAIDs to penetrate biological membranes and disrupt important molecular interactions necessary for a wide array of cellular functions, including cell adhesion. These effects, in particular those that interfere with L-selectin function in neutrophils during the inflammatory response, may contribute to the anti-inflammatory properties that NSAIDs exert in vivo. Recent contributions in this field have shown that the anti-L-selectin effect of NSAIDs is related to the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane. These findings might represent a novel approach for developing new and effective anti-inflammatory compounds with a better safety profile than the currently available NSAIDs. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of therapeutic agents widely used for the symptomatic treatment of rheumatic disorders. Since the early seventies of last century, it has been widely accepted that the main mechanism of action of these compounds, which is also responsible for the main side effect of gastric mucosal damage, is inhibition of cyclooxygenase (COX), a key enzyme in prostaglandin synthesis [1]. Prostaglandins are group of hormone-like lipid compounds with a wide variety of strong physiological effects, including regulation of inflammation, pain sensitization, and platelet aggregation, among many others. However, a growing body of evidence suggests that NSAIDs have additional anti-inflammatory properties (reviewed in [2]). Some of these effects appear to be related to the ability of NSAIDs to penetrate biological membranes, as evaluated in vitro using membrane mimetic models, cell cultures and molecular dynamic
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simulation systems [3, 4], where they disrupt normal signaling events and modify important processes necessary for cellular function, including cell adhesion [5, 6]. The ability of NSAIDs to interfere with either cell adhesion, for example by cleavage of epithelial cell adhesion molecule protein on tumor cells [6], or with leukocyte adhesion pathways essential for the inflammatory response, such as causing L-selectin shedding on neutrophil [5], has been described. Interestingly, this anti-adhesive effect of NSAIDs has also been shown to influence platelet adhesion, and it has been suggested that coagulation, hemostasis and thrombus formation could be modulated by these compounds independently of the release of pro-inflammatory mediators from platelets [7, 8]. In leukocytes, a group of NSAIDs, including flufenamic, meclofenamic, and mefenamic acids, diclofenac and aceclofenac has been shown to induce the downregulation of L-selectin, whereas another group including phenylbutazone and the oxicams, piroxicam and meloxicam has been shown to modulate the function of the integrin CD11b on neutrophils [5, 9, 10]. Some very recent contributions in this field have shown that the anti-L-selectin effect of NSAIDs also causes a significant anti-inflammatory response in vivo [11], and this anti-inflammatory response has been shown, in vitro in human neutrophils, be related to the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane [12]. In this work we review the “COX-centric” theory of NSAID mode of action, and then dissect the non-prostaglandin-mediated effects of NSAIDs, and how some of these, specifically those that interfere with cell adhesion, might explain the anti-inflammatory effects that such compounds exert in vivo. We also discuss how the effects of NSAIDs that do not rely on prostaglandin inhibition may represent a novel strategy for developing a new family of anti-inflammatory compounds. The therapeutic action of this new compound family would be based on decreasing cell adhesion, rather than on prostaglandin synthesis inhibition, thereby presenting a better safety profile than that of currently available NSAIDs. Recent advances in the understanding of non-prostaglandin-mediated antineoplastic [13] and neuroprotective [14, 15] effects of NSAIDs have also been shown, but fall beyond the scope of this review.
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Challenging the “COX-centric” theory. In the early 1970s, it was proposed that inhibition of prostaglandin synthesis was the mechanism through which aspirin, the first member of the NSAID family, inhibited inflammation [16]. This mechanism later came to be the paradigm view of how NSAIDs exert their action. COX is a key enzyme in prostaglandin synthesis, and most known NSAIDs have been shown to inhibit COX activity. There are two highly related isoforms of COX: COX-1 and COX-2 [17]. COX-1, the “constitutive isoform”, has mainly cytoprotective effects, for instance in the production of gastric mucus and the maintenance of renal blood flow. In contrast, COX-2, the “inducible isoform”, is usually undetectable in most tissues, and its expression increases during the inflammatory response [18]. Based on their chemicals structure, there are now at least 20 different NSAIDs from six major groups available for use in humans (Table 1). All of them are absorbed completely orally, have negligible first-pass hepatic metabolism, and are tightly bound to albumin. In general, NSAIDs are weak organic acids with hydrophobic properties, which facilitate their binding to COX, since it is a membrane protein and the COX active site is located at the end of a hydrophobic channel [17, 19]. Table 1 lists the different members of the NSAID family classified by chemical structure, COX selectivity and proposed mechanism of action. The effect of NSAIDs on prostaglandin synthesis provides an explanation for most of their pharmacological actions, including their anti-pyretic, analgesic and platelet antiaggregant effects, as well as for their deleterious side effects, most notably stomach ulcer and renal insufficiency [20]. However, several lines of evidence suggest that COX blockade may not be the only or even the most important anti-inflammatory mechanism of action of this family of compounds. Evidence challenging COX inhibition as the only mechanism of the anti-inflammatory action of NSAIDs includes the observation that certain prostaglandins, such as PGE1, exert anti-inflammatory activity in vivo [21].
Secondly, non-acetylated
salicylates, a group of poor COX inhibitors [22] have a similar anti-inflammatory
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effectiveness to that of efficient COX-inhibiting NSAIDs in patients with rheumatoid arthritis, pointing to a different mechanism of action [23]. Thirdly, there is a great disparity between the small doses of aspirin required to inhibit prostaglandin synthesis (a few hundred milligrams) and the higher doses required to exert an anti-inflammatory effect in vivo (several grams) [24]. Furthermore, in mice in which the gene encoding COX-2 has been disrupted, the inflammatory response is largely intact [25, 26]. Finally, COX-1-deficient mice show a weak inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate [27]. Surprisingly, these animals did not develop stomach ulcers spontaneously, and even developed less gastric ulceration than wild-type mice when treated with the NSAID indomethacin [27].
COX-independent anti-inflammatory actions of NSAIDs There is a well-known variability in the clinical response of patients to NSAIDs, which suggests that these agents might differ in their mechanism of action. The clinical evidence suggests that the inter-individual variation in the NSAID response cannot be explained by differences in pharmacokinetics, serum concentration or the enantiomeric state of the products [28]. Rather, these data suggest that prostaglandin-independent, NSAIDtriggered responses must play an important role in determining patients’ clinical responsiveness to these agents. During the past twenty years, the description of a number of prostaglandinindependent effects from NSAIDs has contributed to a better understanding of their antiinflammatory activity. Most of the described effects rely on the ability of NSAIDs to insert into the lipid bilayers of biological membranes. The use of a wide range of in vitro experimental techniques has provided significant information about the location of NSAIDs within membranes, and also on their effect on diverse membrane properties, such as fluidity, thickness, or phospholipid packaging, among others. These studies on the interaction between NSAIDs and cell membranes have indeed provided evidence on additional mechanisms to explain both the therapeutic action and toxicity of NSAIDs (reviewed in [3]). For example, a
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recent study in AGS cells showed that NSAIDs have a strong affinity for membrane phospholipids, especially phosphatidylcholine (PC) [29]. This feature may explain the wide range of COX-independent, membrane-associated effects exerted by these compounds, including anion transport, oxidative phosphorylation and cell-cell interactions. Of special interest, in vitro data reported in several studies over two decades indicate that NSAIDs interfere with cell membrane events associated with neutrophil activation. The neutrophil events affected by NSAID insertion at the cell membrane include inhibition of cell aggregation, release of lysosomal enzymes (reviewed in [30]) and chemotaxis [31], activation of the NADPH oxidase complex and the oxidative burst in gastric mucosa cells and neutrophils [12, 32] and upregulation of integrins and conformational changes, and the shedding of L-selectin [5, 9, 33]. Other lines of investigation have postulated that the anti-inflammatory action of some NSAIDs can be related to their ability to modulate transcription factors. Some NSAIDs, including salicylates, aspirin, ibuprofen and some nitric oxide-donating aspirin derivatives, might exert some of their anti-inflammatory actions by inhibiting nuclear factor-kappa B (NF-kappa B), a key transcription factor that controls the inducible expression of many genes involved in inflammation, including those encoding pro-inflammatory cytokines such as TNF- or IL-1, iNOS and adhesion molecules such as ICAM-1 (Intercellular adhesion molecule 1) and VCAM-1 (Vascular-cell adhesion molecule 1) [34]. At high doses, aspirin and sodium salicylate are able to inhibit the activation of NF-kappa B in several cell lines by a mechanism that prevents the degradation of the NF-kappa B inhibitor, I kappa B [35]. This effect has been tested and shown to occur in different cell types, including fibroblasts, epithelial cell, or endothelial cells [36-38]. Other NSAIDs act similarly, but at concentrations comparable to those used in therapy. For example, the NSAID ibuprofen has been shown to inhibit NF-kappa B activation in human T cells, at concentrations similar to those obtained in human plasma (micromolar range) [39]. Although the NF-kappa B inhibitory effect has also been described for other NSAIDs, such as indomethacin, flurbiprofen or sulindac, it is not the
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mechanism of all NSAIDs, since naproxen, a highly effective commonly used NSAID, does not affect NF-kappa B activity [40]. Several NSAIDs have been shown to significantly inhibit neutrophil chemotaxis toward CXCL8 and C5a, an effect that has been linked to the polymerization of F-actin and inhibition of the chemotactic pathway dependent on PI3K/Akt activation [41]. Other reports have claimed that NSAIDs can also regulate other signaling pathways, such as indomethacin, which has been shown to activate the MAPK pathway [42], or diclofenac and celecoxib, which have been shown to function via PPAR [43]; there is the suggestion that these actions may underlie the anti-neoplastic effects of these compounds [44]. However, the implication of these anti-tumor effects in the anti-inflammatory properties of NSAIDs has not yet been determined. All these data suggest that NSAIDs have anti-inflammatory mechanisms of action other than the inhibition of prostaglandin synthesis (Table 1). However, the role of most of these COX-independent processes in clinical inflammation remains to be clarified.
Inhibition of cell adhesion by NSAIDs Clinical experience shows that NSAIDs are more effective in acute rather than chronic inflammatory diseases, suggesting that NSAIDs interfere preferentially with the early steps of the inflammatory response. An early event essential for an effective inflammatory response is the transmigration of leukocytes across the vascular endothelium, and their accumulation in inflamed tissues. This cellular extravasation requires a succession of highly coordinated adhesion events between flowing leukocytes and endothelial cells, a process commonly known as the adhesion cascade. The adhesion cascade can be divided into four successive steps (Figure 1), each of which is essential for leukocyte extravasation, such that if one step is blocked, leukocyte emigration does not occur properly [45, 46].
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A major effort in the field of inflammation is currently aimed at developing antagonists of adhesion receptors, an approach known as anti-adhesive therapy [47-49]. This strategy is based on the understanding that if any of the sequential adhesion events of the adhesion cascade is inhibited, overall inflammation will decrease, ameliorating its deleterious effects. In neutrophils, two groups of NSAIDs have been shown to have anti-adhesive properties, one interfering with the function of L-selectin, the other with CD11b/CD18 [5, 9], two adhesion molecules that play key roles in the adhesion cascade (Figure 1). Remarkably, NSAID-induced shedding of L-selectin has been demonstrated not only in vivo in both humans and mice [5, 11], but also in vitro, where the concentration of NSAIDs required to induce L-selectin shedding in human neutrophils [5, 12] are within the range (micromolars) reached in plasma by oral administration of NSAIDs in humans [50, 51].
L-selectin-based effects of NSAIDs L-selectin is constitutively expressed by most leukocytes, and is enzymatically cleaved and released after cell activation through the processing of its ectodomain by ADAM17 [52] and ADAM8 [53], members of the disintegrin and metalloproteinase domain (ADAM) family of surface metalloproteases (Figure 2). L-selectin plays a major role in the early recruitment of neutrophils to inflammatory foci by mediating their rolling on the endothelial surface [54]. Both in vitro [55] and in vivo [56] experiments have shown that soluble L-selectin is able to reduce intravascular leukocyte rolling and adhesion, and consequently decreases neutrophil extravasation presumably by competing with cell surface L-selectin to binding to endothelial ligands. A number of NSAIDs have been shown to induce rapid cleavage and shedding of Lselectin in human neutrophils in vitro [5, 10]. Analysis of the structure-function relationship of some NSAIDs has shown that the ability to downregulate L-selectin expression is dependent on the presence of diphenylamine or its related compound N-phenylanthranilic acid in the NSAID structural core [10].
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The anti-L-selectin action of NSAIDs involves neither non-specific activation of neutrophils nor prostaglandin inhibition, but has been shown to require the presence of ADAM17 [10]. Shedding of L-selectin in neutrophils can be induced by oxidative attack on the pro-domain thiol group of ADAM17, which masks its functional catalytic domain [57]. A group of NSAIDs, including diclofenac and flufenamic acid, was recently shown to interfere with the ability of human neutrophils to initiate interaction with endothelial cells by triggering L-selectin-shedding through the production of reactive oxygen species (ROS), a highly oxidizing agent generated at the plasma membrane [12]. Data obtained in vitro with neutrophils from patients with chronic granulomatous disease (a hereditary defect in the NADPH oxidase complex that results in the reduction of ROS production) demonstrate that NSAIDs such as diclofenac, require NADPH oxidase-dependent generation of superoxide anion to trigger L-selectin shedding [12] (Figure 2). How NSAIDs activate NADPH oxidase remains unknown. However, given that PC has an inhibitory effect on the NADPH oxidase complex [58], it is conceivable that NSAID–PC interaction at the plasma membrane might reduce this inhibitory effect, facilitating superoxide anion production [29]. These findings are in accordance with the emerging view that ROS have regulatory functions that limit inflammation (reviewed in [59]). Several studies have investigated whether induction of L-selectin shedding by NSAIDs is sufficient to reduce inflammation in vivo. In healthy human volunteers, the basal surface expression of L-selectin on circulating neutrophils is significantly reduced by therapeutic doses of indomethacin [5]. In the zymosan air pouch model of acute inflammation, intramuscular treatment with N-phenylanthranilic acid, a diphenylaminerelated NSAID that promotes L-selectin shedding in neutrophils in vitro [10] and in vivo [11], was shown to interfere with the ability of neutrophils to accumulate at inflammatory foci [11]. At the doses used in that study, N-phenylanthranilic acid did not block COX and did not show any additive anti-inflammatory effect to treatment with Mel-14, a functional blocking monoclonal antibody against murine L-selectin [11]. These observations indicate that the anti-inflammatory effect of NSAIDs in vivo may be mediated by the induction of L-
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selectin shedding. The development of new compounds specifically designed to target Lselectin might therefore be a productive strategy for controlling the pathologic inflammatory response.
NSAIDs and anti-adhesive therapy A major effort in the field of inflammation is currently directed at developing antagonists of adhesion receptors. For more than ten years the field of leukocyte cell adhesion has been considered a potential source of novel and potent targets for the treatment of inflammation. Until now, most advances in this field have been in the development of therapeutic agents that block the function of integrins. Anti-adhesive therapies targeting the major leukocyte integrins, such as LFA-1 and VLA-4, have proved relatively successful in the treatment of human inflammatory disorders, including psoriasis, Crohn’s disease and multiple sclerosis [47, 60]. However, the inhibition of selectins or their ligands has only proved beneficial in certain animal models of inflammation, such as hemorrhagic-traumatic shock in baboons [61] and ischemia/reperfusion injury in rat cells [62], with a limited clinical success in human inflammatory conditions such as experimental endotoxemia and multiple trauma (reviewed in [63]). The anti-inflammatory effect of the anti-L-selectin action of NSAIDs in vivo [11] suggests that a strategy based on inducing L-selectin shedding could offer advantages over L-selectin-ligand blockade using antibodies [64] or synthetic selectin antagonist [65]. This approach would prevent the inflammatory response through a dual action of i) reducing neutrophil L-selectin surface expression and ii) quenching endothelial Lselectin ligands by generating higher concentrations of soluble L-selectin in plasma.
Conclusions In addition to their unquestionable role in the human therapeutic arsenal, NSAIDs have made an immense contribution to our knowledge of prostaglandin physiology, platelet aggregation, cytoprotection and renal blood flow. Currently, NSAIDs continue to surprise us
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with non-prostaglandin-related therapeutic effects of potential benefit in the field of neuroprotection or cancer. In the field of cell adhesion, NSAIDs have effects that not only provide a mechanistic explanation for their anti-inflammatory properties, but also provide new ideas for managing inflammation. In this review we have summarized the most important prostaglandin–independent anti-inflammatory effects of NSAIDs, emphasizing their action on L-selectin expression in neutrophils, a capability that underlies their antiinflammatory action in vivo. The induction of L-selectin shedding, either dependent on NADPH oxidase activation or by other as yet unknown mechanism, could develop into a successful anti-inflammatory treatment by preventing neutrophil rolling and consequently decreasing the intensity of the inflammatory response more effectively than L-selectin-ligand blockade alone, a tested strategy with limited clinical effects. The development of new compounds reproducing the ability of NSAIDs to induce L-selectin shedding might yield new therapeutic tools for the clinical management of inflammatory conditions.
Acknowledgements Funding for this project came from Fondo de Investigaciones Sanitarias, cofinanced by the European Regional Development Fund (FIS PI12/02499), and REUNINVES (Asociación para la Ayuda a la Investigación en Reumatología del Hospital Universitario de Canarias) to F.D-G, also from the Spanish Ministry of Science and Innovation (SAF2011-25834), Comunidad de Madrid (INDISNET-S2011/BMD-2332), Instituto Salud Carlos III (Red Cardiovascular RD 12-0042-0056), and ERC-2011-AdG 294340-GENTRIS to F-S-M. The authors thank S. Bartlett for English editing, M. Vicente-Manzanares for critical reading of the manuscript and artwork, and Mercedes Guerra for the documentation work.
Conflict of Interest Disclosure The authors declare no commercial or financial conflict of interest
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Figure 1. The adhesion cascade. Members of three major cell-surface adhesion receptor families are implicated in leukocyte extravasation: selectins (represented by L-selectin, blue, left), integrins (represented by LFA-1, grey, left), and the immunoglobulin superfamily (not shown). In response to tissue injury, proinflammatory mediators such as IL-1 and TNF- are released that induce a rapid change in the adhesive properties of the endothelial cells (EC) lining the blood vessels near the damaged tissue. Flowing leukocytes (Lk) are immediately captured and begin to roll over the endothelial surface (right). This initial leukocyte– endothelium interaction is mainly mediated by selectins. L-selectin (CD62L) (constitutively expressed by most leukocytes), E-selectin (CD62E) and P-selectin (CD62P) (both expressed by activated endothelial cells) specifically interact with carbohydrate moieties linked to mucin-like molecules expressed by activated endothelial cells and leukocytes (green). The selectin–mucin interaction is responsible for the rolling of leukocytes along the endothelium. During rolling, leukocytes are activated by locally produced chemokines bound to the endothelium, which cause both the shedding of L-selectin, and the activation of the integrin adhesion receptors CD11a/CD18 and CD11b/CD18, as well as the increased expression of CD11b/CD18. The activated leukocyte integrins (open conformation) interact with their endothelial counter-receptor intercellular adhesion molecule (ICAM)-1 (orange), resulting in the firm adhesion of leukocytes to the vessel surface. Finally, leukocytes migrate across the endothelium into the tissues, usually by squeezing between endothelial cells. Several endothelial junctional proteins participate in this final step, including CD31, CD99 and JAMs (junctional adhesion molecules). All of these interactions are required for neutrophil transmigration, but in the case of lymphocytes the rolling and firm adhesion steps are mainly mediated by interaction between the integrin adhesion receptor very late activation antigen (VLA)-4 and its endothelial counter receptor vascular cell adhesion molecule (VCAM)-1 [66]. NSAIDs have been found to interfere with molecules essential for the rolling or firm adhesion steps of the adhesion cascade.
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Figure 2. NSAID activity on L-selectin shedding from neutrophils: a proposed mechanism. Superoxide anion production by the NADPH oxidase complex is proposed to drive NSAID-induced L-selecting shedding from neutrophils. NSAIDs insert into the lipid bilayer of the cell membrane where they interact with phospholipids, such as phosphatidylcholine (PC, green). PC is capable of interfering with the activation of the NADPH oxidase complex (orange), and it is likely that NSAID–PC interaction at the plasma membrane ablates the inhibitory effects of PC, causing activation of NADPH oxidase and consequently the release of superoxide anion to the extracellular milieu. Superoxide generates an oxidative attack on the pro-domain thiol group of ADAM-17 (grey), which masks its catalytic domain. Upon removal of this mask, ADAM17 is activated and able to process the extracellular domain of L-selectin (blue), causing its release from the cell membrane.
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Table 1. NSAID families Drug
Mechanism of action
Reference
Inhibition of PG synthesis L-selectin sheeding Inhibition of NFKappa B Inhibition of iNOS Inhibition of COX-2 gene transcription L-selectin sheeding Inhibition of NFKappa B Inhibition of iNOS
[16]
Inhibition of prostaglandin synthesis Inhibition of NFKappa B Inhibition of neutrophil PI3K/Aktdependent chemotactic pathways L-selectin sheeding Inhibition of NFKappa B L-selectin sheeding Inhibition of neutrophil PI3K/Aktdependent chemotactic pathways
[1]
Inhibition of prostaglandin synthesis L-selectin sheeding
[1]
Non-selective NSAID agents Salicylate Aspirin (acetylated)
Salicylates (non-acetylated)
[5, 10] [35] [67] [68] [69] [35] [67]
Diflunisal Salsalate Propionic acids
Ibuprofen Naproxen
Ketoprofen Flurbiprofen
Oxaprozin
Acetic acids
Diclofenac
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[39] [41]
[5] [70] [10] [41]
[5]
26
Inhibition of VLA-4
[71]
activation Etodolac Tolmetin Sulindac
Inhibition of NF-
[72]
Kappa B Cleavage of epithelial
[6]
cell adhesion molecule Ketorolac Indomethacin
Inhibition of NF-
[73]
Kappa B Induction of L-selectin
[5]
shedding Inhibition of VLA-4
[71]
activation Aceclofenac
Induction of L-selectin
[69, 74]
shedding Inhibition of VLA-4
[71]
activation Inhibition of
Oxicams (enolic acid)
[1]
prostaglandin synthesis Piroxicam
Inhibition of 2
[9]
integrin activation Meloxicam
Inhibition of 2
[9]
integrin activation
Fenamates (anthanilic acid)
Inhibition of
[1]
prostaglandin
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synthesis Meclofenami c acid Mefenamic acid
Non-acidic Nabumetone (naphthylbutanone
Induction of L-selectin
[10]
shedding Induction of L-selectin
[10]
shedding Inhibition of
[1]
prostaglandin
)
synthesis by its active metabolite: 6methoxy-2-naphthyl acetic acid Inhibition of
Selective COX-2 inhibitors
[1]
prostaglandin synthesis Celecoxib Eterocoxib
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28
Department of Internal Medicine, Universidad de La Laguna, Rheumatology Service,
Hospital Universitario de Canarias, Santa Cruz de Tenerife. Spain. 2National Center for Cardiovascular Research, Immunology Service, Instituto Investigaciones Sanitarias Princesa, Universidad Autónoma de Madrid. Spain
Key words: Non-steroidal anti-inflammatory drugs, L-selectin, NADPH oxidase,
Corresponding author:
Federico Díaz-González, Professor of Medicine. Department of
Internal Medicine, Universidad de La Laguna. Staff of Rheumatology, Hospital Universitario de Canárias. C/Ofra s/n, 38320, La Laguna, Santa Cruz de Tenerife. Spain e-mail: [email protected] fax: +34-922646792 List of abbreviations:
ADAM: a disintegrin and metalloproteinase domain, COX:
cyclooxigenase, ICAM-1: Intercellular adhesion molecule 1, IL-1: interleukin-1, iNOS: inducible nitric oxide synthase, LFA-1: lymphocyte function-associated antigen 1, MAPK: Mitogen-activated protein kinases, NADPH-oxidase: nicotinamide adenine dinucleotide phosphate-oxidase, NF-kappa B: nuclear factor kappa-light-chain-enhancer of activated B cells, NSAIDs: non-steroidal anti-inflammatory drugs, PC: phosphatidylcholine, PGE1: prostaglandin E1, PI3K: phosphatidylinositide 3-kinases, PPAR: peroxisome proliferatoractivated receptors, ROS: reactive oxygen species, TNF-: tumor necrosis factor-, VCAM1: Vascular-cell adhesion molecule, VLA-4: very late antigen-4 Received: 29-Oct-2014; Revised: 07-Dec-2014; Accepted: 16-Dec-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/eji.201445222.
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1
Summary Nonsteroidal anti-inflammatory drugs (NSAIDs) comprise a heterogeneous group of pharmacological agents used for the symptomatic treatment of fever, pain and inflammation. Although the main mechanism of action of NSAIDs consists of inhibiting prostaglandin synthesis by blocking the enzyme cyclooxygenase (COX), clinical and experimental data strongly indicate the existence of additional mechanisms. Some of the COX-independent effects are related to the ability of NSAIDs to penetrate biological membranes and disrupt important molecular interactions necessary for a wide array of cellular functions, including cell adhesion. These effects, in particular those that interfere with L-selectin function in neutrophils during the inflammatory response, may contribute to the anti-inflammatory properties that NSAIDs exert in vivo. Recent contributions in this field have shown that the anti-L-selectin effect of NSAIDs is related to the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane. These findings might represent a novel approach for developing new and effective anti-inflammatory compounds with a better safety profile than the currently available NSAIDs. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of therapeutic agents widely used for the symptomatic treatment of rheumatic disorders. Since the early seventies of last century, it has been widely accepted that the main mechanism of action of these compounds, which is also responsible for the main side effect of gastric mucosal damage, is inhibition of cyclooxygenase (COX), a key enzyme in prostaglandin synthesis [1]. Prostaglandins are group of hormone-like lipid compounds with a wide variety of strong physiological effects, including regulation of inflammation, pain sensitization, and platelet aggregation, among many others. However, a growing body of evidence suggests that NSAIDs have additional anti-inflammatory properties (reviewed in [2]). Some of these effects appear to be related to the ability of NSAIDs to penetrate biological membranes, as evaluated in vitro using membrane mimetic models, cell cultures and molecular dynamic
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2
simulation systems [3, 4], where they disrupt normal signaling events and modify important processes necessary for cellular function, including cell adhesion [5, 6]. The ability of NSAIDs to interfere with either cell adhesion, for example by cleavage of epithelial cell adhesion molecule protein on tumor cells [6], or with leukocyte adhesion pathways essential for the inflammatory response, such as causing L-selectin shedding on neutrophil [5], has been described. Interestingly, this anti-adhesive effect of NSAIDs has also been shown to influence platelet adhesion, and it has been suggested that coagulation, hemostasis and thrombus formation could be modulated by these compounds independently of the release of pro-inflammatory mediators from platelets [7, 8]. In leukocytes, a group of NSAIDs, including flufenamic, meclofenamic, and mefenamic acids, diclofenac and aceclofenac has been shown to induce the downregulation of L-selectin, whereas another group including phenylbutazone and the oxicams, piroxicam and meloxicam has been shown to modulate the function of the integrin CD11b on neutrophils [5, 9, 10]. Some very recent contributions in this field have shown that the anti-L-selectin effect of NSAIDs also causes a significant anti-inflammatory response in vivo [11], and this anti-inflammatory response has been shown, in vitro in human neutrophils, be related to the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane [12]. In this work we review the “COX-centric” theory of NSAID mode of action, and then dissect the non-prostaglandin-mediated effects of NSAIDs, and how some of these, specifically those that interfere with cell adhesion, might explain the anti-inflammatory effects that such compounds exert in vivo. We also discuss how the effects of NSAIDs that do not rely on prostaglandin inhibition may represent a novel strategy for developing a new family of anti-inflammatory compounds. The therapeutic action of this new compound family would be based on decreasing cell adhesion, rather than on prostaglandin synthesis inhibition, thereby presenting a better safety profile than that of currently available NSAIDs. Recent advances in the understanding of non-prostaglandin-mediated antineoplastic [13] and neuroprotective [14, 15] effects of NSAIDs have also been shown, but fall beyond the scope of this review.
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Challenging the “COX-centric” theory. In the early 1970s, it was proposed that inhibition of prostaglandin synthesis was the mechanism through which aspirin, the first member of the NSAID family, inhibited inflammation [16]. This mechanism later came to be the paradigm view of how NSAIDs exert their action. COX is a key enzyme in prostaglandin synthesis, and most known NSAIDs have been shown to inhibit COX activity. There are two highly related isoforms of COX: COX-1 and COX-2 [17]. COX-1, the “constitutive isoform”, has mainly cytoprotective effects, for instance in the production of gastric mucus and the maintenance of renal blood flow. In contrast, COX-2, the “inducible isoform”, is usually undetectable in most tissues, and its expression increases during the inflammatory response [18]. Based on their chemicals structure, there are now at least 20 different NSAIDs from six major groups available for use in humans (Table 1). All of them are absorbed completely orally, have negligible first-pass hepatic metabolism, and are tightly bound to albumin. In general, NSAIDs are weak organic acids with hydrophobic properties, which facilitate their binding to COX, since it is a membrane protein and the COX active site is located at the end of a hydrophobic channel [17, 19]. Table 1 lists the different members of the NSAID family classified by chemical structure, COX selectivity and proposed mechanism of action. The effect of NSAIDs on prostaglandin synthesis provides an explanation for most of their pharmacological actions, including their anti-pyretic, analgesic and platelet antiaggregant effects, as well as for their deleterious side effects, most notably stomach ulcer and renal insufficiency [20]. However, several lines of evidence suggest that COX blockade may not be the only or even the most important anti-inflammatory mechanism of action of this family of compounds. Evidence challenging COX inhibition as the only mechanism of the anti-inflammatory action of NSAIDs includes the observation that certain prostaglandins, such as PGE1, exert anti-inflammatory activity in vivo [21].
Secondly, non-acetylated
salicylates, a group of poor COX inhibitors [22] have a similar anti-inflammatory
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effectiveness to that of efficient COX-inhibiting NSAIDs in patients with rheumatoid arthritis, pointing to a different mechanism of action [23]. Thirdly, there is a great disparity between the small doses of aspirin required to inhibit prostaglandin synthesis (a few hundred milligrams) and the higher doses required to exert an anti-inflammatory effect in vivo (several grams) [24]. Furthermore, in mice in which the gene encoding COX-2 has been disrupted, the inflammatory response is largely intact [25, 26]. Finally, COX-1-deficient mice show a weak inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate [27]. Surprisingly, these animals did not develop stomach ulcers spontaneously, and even developed less gastric ulceration than wild-type mice when treated with the NSAID indomethacin [27].
COX-independent anti-inflammatory actions of NSAIDs There is a well-known variability in the clinical response of patients to NSAIDs, which suggests that these agents might differ in their mechanism of action. The clinical evidence suggests that the inter-individual variation in the NSAID response cannot be explained by differences in pharmacokinetics, serum concentration or the enantiomeric state of the products [28]. Rather, these data suggest that prostaglandin-independent, NSAIDtriggered responses must play an important role in determining patients’ clinical responsiveness to these agents. During the past twenty years, the description of a number of prostaglandinindependent effects from NSAIDs has contributed to a better understanding of their antiinflammatory activity. Most of the described effects rely on the ability of NSAIDs to insert into the lipid bilayers of biological membranes. The use of a wide range of in vitro experimental techniques has provided significant information about the location of NSAIDs within membranes, and also on their effect on diverse membrane properties, such as fluidity, thickness, or phospholipid packaging, among others. These studies on the interaction between NSAIDs and cell membranes have indeed provided evidence on additional mechanisms to explain both the therapeutic action and toxicity of NSAIDs (reviewed in [3]). For example, a
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recent study in AGS cells showed that NSAIDs have a strong affinity for membrane phospholipids, especially phosphatidylcholine (PC) [29]. This feature may explain the wide range of COX-independent, membrane-associated effects exerted by these compounds, including anion transport, oxidative phosphorylation and cell-cell interactions. Of special interest, in vitro data reported in several studies over two decades indicate that NSAIDs interfere with cell membrane events associated with neutrophil activation. The neutrophil events affected by NSAID insertion at the cell membrane include inhibition of cell aggregation, release of lysosomal enzymes (reviewed in [30]) and chemotaxis [31], activation of the NADPH oxidase complex and the oxidative burst in gastric mucosa cells and neutrophils [12, 32] and upregulation of integrins and conformational changes, and the shedding of L-selectin [5, 9, 33]. Other lines of investigation have postulated that the anti-inflammatory action of some NSAIDs can be related to their ability to modulate transcription factors. Some NSAIDs, including salicylates, aspirin, ibuprofen and some nitric oxide-donating aspirin derivatives, might exert some of their anti-inflammatory actions by inhibiting nuclear factor-kappa B (NF-kappa B), a key transcription factor that controls the inducible expression of many genes involved in inflammation, including those encoding pro-inflammatory cytokines such as TNF- or IL-1, iNOS and adhesion molecules such as ICAM-1 (Intercellular adhesion molecule 1) and VCAM-1 (Vascular-cell adhesion molecule 1) [34]. At high doses, aspirin and sodium salicylate are able to inhibit the activation of NF-kappa B in several cell lines by a mechanism that prevents the degradation of the NF-kappa B inhibitor, I kappa B [35]. This effect has been tested and shown to occur in different cell types, including fibroblasts, epithelial cell, or endothelial cells [36-38]. Other NSAIDs act similarly, but at concentrations comparable to those used in therapy. For example, the NSAID ibuprofen has been shown to inhibit NF-kappa B activation in human T cells, at concentrations similar to those obtained in human plasma (micromolar range) [39]. Although the NF-kappa B inhibitory effect has also been described for other NSAIDs, such as indomethacin, flurbiprofen or sulindac, it is not the
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mechanism of all NSAIDs, since naproxen, a highly effective commonly used NSAID, does not affect NF-kappa B activity [40]. Several NSAIDs have been shown to significantly inhibit neutrophil chemotaxis toward CXCL8 and C5a, an effect that has been linked to the polymerization of F-actin and inhibition of the chemotactic pathway dependent on PI3K/Akt activation [41]. Other reports have claimed that NSAIDs can also regulate other signaling pathways, such as indomethacin, which has been shown to activate the MAPK pathway [42], or diclofenac and celecoxib, which have been shown to function via PPAR [43]; there is the suggestion that these actions may underlie the anti-neoplastic effects of these compounds [44]. However, the implication of these anti-tumor effects in the anti-inflammatory properties of NSAIDs has not yet been determined. All these data suggest that NSAIDs have anti-inflammatory mechanisms of action other than the inhibition of prostaglandin synthesis (Table 1). However, the role of most of these COX-independent processes in clinical inflammation remains to be clarified.
Inhibition of cell adhesion by NSAIDs Clinical experience shows that NSAIDs are more effective in acute rather than chronic inflammatory diseases, suggesting that NSAIDs interfere preferentially with the early steps of the inflammatory response. An early event essential for an effective inflammatory response is the transmigration of leukocytes across the vascular endothelium, and their accumulation in inflamed tissues. This cellular extravasation requires a succession of highly coordinated adhesion events between flowing leukocytes and endothelial cells, a process commonly known as the adhesion cascade. The adhesion cascade can be divided into four successive steps (Figure 1), each of which is essential for leukocyte extravasation, such that if one step is blocked, leukocyte emigration does not occur properly [45, 46].
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A major effort in the field of inflammation is currently aimed at developing antagonists of adhesion receptors, an approach known as anti-adhesive therapy [47-49]. This strategy is based on the understanding that if any of the sequential adhesion events of the adhesion cascade is inhibited, overall inflammation will decrease, ameliorating its deleterious effects. In neutrophils, two groups of NSAIDs have been shown to have anti-adhesive properties, one interfering with the function of L-selectin, the other with CD11b/CD18 [5, 9], two adhesion molecules that play key roles in the adhesion cascade (Figure 1). Remarkably, NSAID-induced shedding of L-selectin has been demonstrated not only in vivo in both humans and mice [5, 11], but also in vitro, where the concentration of NSAIDs required to induce L-selectin shedding in human neutrophils [5, 12] are within the range (micromolars) reached in plasma by oral administration of NSAIDs in humans [50, 51].
L-selectin-based effects of NSAIDs L-selectin is constitutively expressed by most leukocytes, and is enzymatically cleaved and released after cell activation through the processing of its ectodomain by ADAM17 [52] and ADAM8 [53], members of the disintegrin and metalloproteinase domain (ADAM) family of surface metalloproteases (Figure 2). L-selectin plays a major role in the early recruitment of neutrophils to inflammatory foci by mediating their rolling on the endothelial surface [54]. Both in vitro [55] and in vivo [56] experiments have shown that soluble L-selectin is able to reduce intravascular leukocyte rolling and adhesion, and consequently decreases neutrophil extravasation presumably by competing with cell surface L-selectin to binding to endothelial ligands. A number of NSAIDs have been shown to induce rapid cleavage and shedding of Lselectin in human neutrophils in vitro [5, 10]. Analysis of the structure-function relationship of some NSAIDs has shown that the ability to downregulate L-selectin expression is dependent on the presence of diphenylamine or its related compound N-phenylanthranilic acid in the NSAID structural core [10].
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The anti-L-selectin action of NSAIDs involves neither non-specific activation of neutrophils nor prostaglandin inhibition, but has been shown to require the presence of ADAM17 [10]. Shedding of L-selectin in neutrophils can be induced by oxidative attack on the pro-domain thiol group of ADAM17, which masks its functional catalytic domain [57]. A group of NSAIDs, including diclofenac and flufenamic acid, was recently shown to interfere with the ability of human neutrophils to initiate interaction with endothelial cells by triggering L-selectin-shedding through the production of reactive oxygen species (ROS), a highly oxidizing agent generated at the plasma membrane [12]. Data obtained in vitro with neutrophils from patients with chronic granulomatous disease (a hereditary defect in the NADPH oxidase complex that results in the reduction of ROS production) demonstrate that NSAIDs such as diclofenac, require NADPH oxidase-dependent generation of superoxide anion to trigger L-selectin shedding [12] (Figure 2). How NSAIDs activate NADPH oxidase remains unknown. However, given that PC has an inhibitory effect on the NADPH oxidase complex [58], it is conceivable that NSAID–PC interaction at the plasma membrane might reduce this inhibitory effect, facilitating superoxide anion production [29]. These findings are in accordance with the emerging view that ROS have regulatory functions that limit inflammation (reviewed in [59]). Several studies have investigated whether induction of L-selectin shedding by NSAIDs is sufficient to reduce inflammation in vivo. In healthy human volunteers, the basal surface expression of L-selectin on circulating neutrophils is significantly reduced by therapeutic doses of indomethacin [5]. In the zymosan air pouch model of acute inflammation, intramuscular treatment with N-phenylanthranilic acid, a diphenylaminerelated NSAID that promotes L-selectin shedding in neutrophils in vitro [10] and in vivo [11], was shown to interfere with the ability of neutrophils to accumulate at inflammatory foci [11]. At the doses used in that study, N-phenylanthranilic acid did not block COX and did not show any additive anti-inflammatory effect to treatment with Mel-14, a functional blocking monoclonal antibody against murine L-selectin [11]. These observations indicate that the anti-inflammatory effect of NSAIDs in vivo may be mediated by the induction of L-
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selectin shedding. The development of new compounds specifically designed to target Lselectin might therefore be a productive strategy for controlling the pathologic inflammatory response.
NSAIDs and anti-adhesive therapy A major effort in the field of inflammation is currently directed at developing antagonists of adhesion receptors. For more than ten years the field of leukocyte cell adhesion has been considered a potential source of novel and potent targets for the treatment of inflammation. Until now, most advances in this field have been in the development of therapeutic agents that block the function of integrins. Anti-adhesive therapies targeting the major leukocyte integrins, such as LFA-1 and VLA-4, have proved relatively successful in the treatment of human inflammatory disorders, including psoriasis, Crohn’s disease and multiple sclerosis [47, 60]. However, the inhibition of selectins or their ligands has only proved beneficial in certain animal models of inflammation, such as hemorrhagic-traumatic shock in baboons [61] and ischemia/reperfusion injury in rat cells [62], with a limited clinical success in human inflammatory conditions such as experimental endotoxemia and multiple trauma (reviewed in [63]). The anti-inflammatory effect of the anti-L-selectin action of NSAIDs in vivo [11] suggests that a strategy based on inducing L-selectin shedding could offer advantages over L-selectin-ligand blockade using antibodies [64] or synthetic selectin antagonist [65]. This approach would prevent the inflammatory response through a dual action of i) reducing neutrophil L-selectin surface expression and ii) quenching endothelial Lselectin ligands by generating higher concentrations of soluble L-selectin in plasma.
Conclusions In addition to their unquestionable role in the human therapeutic arsenal, NSAIDs have made an immense contribution to our knowledge of prostaglandin physiology, platelet aggregation, cytoprotection and renal blood flow. Currently, NSAIDs continue to surprise us
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with non-prostaglandin-related therapeutic effects of potential benefit in the field of neuroprotection or cancer. In the field of cell adhesion, NSAIDs have effects that not only provide a mechanistic explanation for their anti-inflammatory properties, but also provide new ideas for managing inflammation. In this review we have summarized the most important prostaglandin–independent anti-inflammatory effects of NSAIDs, emphasizing their action on L-selectin expression in neutrophils, a capability that underlies their antiinflammatory action in vivo. The induction of L-selectin shedding, either dependent on NADPH oxidase activation or by other as yet unknown mechanism, could develop into a successful anti-inflammatory treatment by preventing neutrophil rolling and consequently decreasing the intensity of the inflammatory response more effectively than L-selectin-ligand blockade alone, a tested strategy with limited clinical effects. The development of new compounds reproducing the ability of NSAIDs to induce L-selectin shedding might yield new therapeutic tools for the clinical management of inflammatory conditions.
Acknowledgements Funding for this project came from Fondo de Investigaciones Sanitarias, cofinanced by the European Regional Development Fund (FIS PI12/02499), and REUNINVES (Asociación para la Ayuda a la Investigación en Reumatología del Hospital Universitario de Canarias) to F.D-G, also from the Spanish Ministry of Science and Innovation (SAF2011-25834), Comunidad de Madrid (INDISNET-S2011/BMD-2332), Instituto Salud Carlos III (Red Cardiovascular RD 12-0042-0056), and ERC-2011-AdG 294340-GENTRIS to F-S-M. The authors thank S. Bartlett for English editing, M. Vicente-Manzanares for critical reading of the manuscript and artwork, and Mercedes Guerra for the documentation work.
Conflict of Interest Disclosure The authors declare no commercial or financial conflict of interest
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Figure 1. The adhesion cascade. Members of three major cell-surface adhesion receptor families are implicated in leukocyte extravasation: selectins (represented by L-selectin, blue, left), integrins (represented by LFA-1, grey, left), and the immunoglobulin superfamily (not shown). In response to tissue injury, proinflammatory mediators such as IL-1 and TNF- are released that induce a rapid change in the adhesive properties of the endothelial cells (EC) lining the blood vessels near the damaged tissue. Flowing leukocytes (Lk) are immediately captured and begin to roll over the endothelial surface (right). This initial leukocyte– endothelium interaction is mainly mediated by selectins. L-selectin (CD62L) (constitutively expressed by most leukocytes), E-selectin (CD62E) and P-selectin (CD62P) (both expressed by activated endothelial cells) specifically interact with carbohydrate moieties linked to mucin-like molecules expressed by activated endothelial cells and leukocytes (green). The selectin–mucin interaction is responsible for the rolling of leukocytes along the endothelium. During rolling, leukocytes are activated by locally produced chemokines bound to the endothelium, which cause both the shedding of L-selectin, and the activation of the integrin adhesion receptors CD11a/CD18 and CD11b/CD18, as well as the increased expression of CD11b/CD18. The activated leukocyte integrins (open conformation) interact with their endothelial counter-receptor intercellular adhesion molecule (ICAM)-1 (orange), resulting in the firm adhesion of leukocytes to the vessel surface. Finally, leukocytes migrate across the endothelium into the tissues, usually by squeezing between endothelial cells. Several endothelial junctional proteins participate in this final step, including CD31, CD99 and JAMs (junctional adhesion molecules). All of these interactions are required for neutrophil transmigration, but in the case of lymphocytes the rolling and firm adhesion steps are mainly mediated by interaction between the integrin adhesion receptor very late activation antigen (VLA)-4 and its endothelial counter receptor vascular cell adhesion molecule (VCAM)-1 [66]. NSAIDs have been found to interfere with molecules essential for the rolling or firm adhesion steps of the adhesion cascade.
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Figure 2. NSAID activity on L-selectin shedding from neutrophils: a proposed mechanism. Superoxide anion production by the NADPH oxidase complex is proposed to drive NSAID-induced L-selecting shedding from neutrophils. NSAIDs insert into the lipid bilayer of the cell membrane where they interact with phospholipids, such as phosphatidylcholine (PC, green). PC is capable of interfering with the activation of the NADPH oxidase complex (orange), and it is likely that NSAID–PC interaction at the plasma membrane ablates the inhibitory effects of PC, causing activation of NADPH oxidase and consequently the release of superoxide anion to the extracellular milieu. Superoxide generates an oxidative attack on the pro-domain thiol group of ADAM-17 (grey), which masks its catalytic domain. Upon removal of this mask, ADAM17 is activated and able to process the extracellular domain of L-selectin (blue), causing its release from the cell membrane.
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Table 1. NSAID families Drug
Mechanism of action
Reference
Inhibition of PG synthesis L-selectin sheeding Inhibition of NFKappa B Inhibition of iNOS Inhibition of COX-2 gene transcription L-selectin sheeding Inhibition of NFKappa B Inhibition of iNOS
[16]
Inhibition of prostaglandin synthesis Inhibition of NFKappa B Inhibition of neutrophil PI3K/Aktdependent chemotactic pathways L-selectin sheeding Inhibition of NFKappa B L-selectin sheeding Inhibition of neutrophil PI3K/Aktdependent chemotactic pathways
[1]
Inhibition of prostaglandin synthesis L-selectin sheeding
[1]
Non-selective NSAID agents Salicylate Aspirin (acetylated)
Salicylates (non-acetylated)
[5, 10] [35] [67] [68] [69] [35] [67]
Diflunisal Salsalate Propionic acids
Ibuprofen Naproxen
Ketoprofen Flurbiprofen
Oxaprozin
Acetic acids
Diclofenac
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[39] [41]
[5] [70] [10] [41]
[5]
26
Inhibition of VLA-4
[71]
activation Etodolac Tolmetin Sulindac
Inhibition of NF-
[72]
Kappa B Cleavage of epithelial
[6]
cell adhesion molecule Ketorolac Indomethacin
Inhibition of NF-
[73]
Kappa B Induction of L-selectin
[5]
shedding Inhibition of VLA-4
[71]
activation Aceclofenac
Induction of L-selectin
[69, 74]
shedding Inhibition of VLA-4
[71]
activation Inhibition of
Oxicams (enolic acid)
[1]
prostaglandin synthesis Piroxicam
Inhibition of 2
[9]
integrin activation Meloxicam
Inhibition of 2
[9]
integrin activation
Fenamates (anthanilic acid)
Inhibition of
[1]
prostaglandin
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synthesis Meclofenami c acid Mefenamic acid
Non-acidic Nabumetone (naphthylbutanone
Induction of L-selectin
[10]
shedding Induction of L-selectin
[10]
shedding Inhibition of
[1]
prostaglandin
)
synthesis by its active metabolite: 6methoxy-2-naphthyl acetic acid Inhibition of
Selective COX-2 inhibitors
[1]
prostaglandin synthesis Celecoxib Eterocoxib
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