Báo cáo khoa học: Therapeutic targeting of molecules involved in leukocyte–endothelial cell interactions potx

However, the overzeal-ous accumulation of leukocytes in tissues also contributes to a wide variety of diseases, such as atherosclerosis, chronic inflammatory bowel disease, rheumatoid art

M I N I R E V I E W

Therapeutic targeting of molecules involved in

leukocyte–endothelial cell interactions

Nicole C Kaneider1, Andrew J Leger1,2and Athan Kuliopulos1,2,3

1 Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA, USA

2 Department of Medicine, Tufts University School of Medicine, Boston, MA, USA

3 Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA

One of the key characteristics of inflammation is the

recruitment of leukocytes to the site of tissue injury

There are three major subsets of leukocytes with

migratory capacity that are involved in inflammation:

neutrophils, monocytes⁄ macrophages and lymphocytes

Quiescent endothelium acts as a barrier between the

circulating white blood cells and the underlying

sub-endothelial tissue In response to inflammatory stimuli,

endothelial cells undergo a phenotypic change and

act-ively facilitate the recruitment and transmigration of

leukocytes to the site of inflammation

The neutrophil is a short-lived phagocyte that plays

an essential role in the defense against microorganisms,

as witnessed by the life-threatening infections that

occur in patients with neutropenia or in those with

kocyte defects Neutrophils are the most abundant

leu-kocyte type in humans, and accumulate, within hours,

at sites of acute inflammation Once at the site of injury, neutrophils secrete a variety of destructive enzymes, such as myeloperoxidase, elastase, matrix metalloproteases and cathepsins In the absence of proper feedback mechanisms, the destructive power of neutrophils contributes significantly to the pathogene-sis of numerous diseases Neutrophils have been impli-cated in the progression of many inflammatory diseases, including sepsis, the systemic inflammatory response syndrome (Fig 1), the acute respiratory dis-tress syndrome, chronic obstructive pulmonary disease and others (Table 1) Few currently available therapeu-tic agents, including cortherapeu-ticosteroids, effectively down-regulate the pro-inflammatory activity of neutrophils Monocytes are long-lived leukocytes and play a crit-ical role in the orchestration of the inflammatory response Monocytes migrate from the blood into

Keywords

endothelium; inflammatory diseases;

leukocytes; therapeutic targets

Correspondence

A Kuliopulos, Tufts-NEMC, 750 Washington

St., Box 7510, Boston, MA 02111, USA

Fax: +1 617 636 7855

Tel: +1 617 636 8482

E-mail: athan.kuliopulos@tufts.edu

(Received 15 May 2006, accepted 12 July

2006)

doi:10.1111/j.1742-4658.2006.05441.x

Inflammation is traditionally viewed as a physiological reaction to tissue injury Leukocytes contribute to the inflammatory response by the secretion

of cytotoxic and pro-inflammatory compounds, by phagocytotic activity and by targeted attack of foreign antigens Leukocyte accumulation in tis-sues is important for the initial response to injury However, the overzeal-ous accumulation of leukocytes in tissues also contributes to a wide variety

of diseases, such as atherosclerosis, chronic inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, vasculitis, systemic inflammatory response syndrome, juvenile diabetes and psoriasis Many therapeutic inter-ventions target immune cells after they have already migrated to the site of inflammation This review addresses different therapeutic strategies, used to reduce or prevent leukocyte–endothelial cell interactions and communica-tion, in order to limit the progression of inflammatory diseases

Abbreviations

GPCR, G protein-coupled receptor; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; LFA-1, lymphocyte function-associated antigen-1; PAR, protease activated receptor; PSGL-1, P-selectin glycoprotein ligand-1; S1P, sphingosine-1-phosphate; VCAM-1, vascular cell adhesion molecule-1.

various tissues where they transform into

macrophag-es Cells of the mononuclear phagocytotic system have

been linked to a variety of inflammatory diseases, in

particular to atherosclerosis, where macrophages

trans-form into foam cells and mediate atherosclerotic

pla-que formation (Fig 2) Because macrophages produce

a wide range of biologically active molecules involved

in both beneficial and detrimental outcomes in inflam-mation, therapeutic interventions that target macro-phages and their products may be a fruitful avenue to control chronic inflammatory conditions

Lymphocytes provide acquired immunity and repre-sent the collective memory of the immune system Naı¨ve lymphocytes reside mainly in lymphoid organs,

Fig 1 Cell surface molecules as potential targets in systemic inflammatory response syndrome as a neutrophil-driven disease.

Table 1 Targeting cell surface molecules of predominant cell types in inflammatory diseases ARDS, acute respiratory distress syndrome; CLA, cutaneous lymphocyte-associated antigen; COPD, chronic obstructive pulmonary disease; GPCR, G protein-coupled receptor; LFA-1, lymphocyte function-associated antigen-1; LTB4, leukotriene B4; PAFR, platelet activating factor receptor; PSGL-1, P-selectin glycoprotein ligand-1; SIRS, systemic inflammatory response syndrome; VLA-4, very late antigen-4.

Predominant cell type

SIRS, COPD, ARDS, cystic fibrosis, osteomyelitis, Goodpasture syndrome, immune complex-mediated vasculitides, pyelonephritis.

glomerulonephritis, gout

Atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis,

COPD, asthma

Multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, type-1 diabetes, allograft rejection, lupus, asthma, atopic dermatitis

CXCR3, CCR4, CCR10, PAFR, LTB4

whereas effector and memory lymphocytes move into

inflamed tissue when attracted by an array of

chemo-kines [1,2] T lymphocytes play central roles in

adap-tive immune responses against protein antigens Two

major B-cell subsets have been described to date [3,4]

B1 cells produce low affinity IgM that is reactive to a

limited number of highly conserved microbial and host

antigens B2 cells are the most numerous type of B

cells found in tissues and lymphoid organs, and their

major role is to produce antibodies for the defense

against extracellular bacteria [5] B2 cells undergo

clonal expansion, isotype switching and develop into

memory cells, and can process and present antigens to

T cells to amplify or regulate adaptive immune

responses

A central feature of inflammation is the ingress of

circulating leukocytes across the endothelium and

underlying basement membranes into the affected

tis-sue Excessive, unregulated and sustained activation of

the endothelium that occurs during severe

inflamma-tory processes leads to endothelial dysfunction and

damage Exposure of endothelial cells to

pro-inflam-matory mediators results in an up-regulation of E- and

P-selectin, vascular cell adhesion molecule-1 (VCAM-1),

intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules which mediate leukocyte rolling and firm adhesion Local chemokines secreted by the endothelium or subendothelial components direct leukocyte chemotaxis across the vascular intima Emerging therapeutic strategies aimed at controlling inflammation interfere at various stages of the multi-step recruitment cascade of leukocytes The function

of inflammatory adhesion molecules can be modula-ted by competitive blockade, altered surface expres-sion of ligands and adheexpres-sion molecules on the cell surface, or by inhibition of chemokine G protein-coupled receptor (GPCR) signaling [6] Several anti-inflammatory drugs indirectly inhibit components involved in leukocyte–endothelial cell interactions For example, compounds that block interleukin (IL)-1

or tumor necrosis factor-a have potent effects on the expression of E-selectin, VCAM and other cell adhe-sion molecules on endothelial cells [7,8] Corticoster-oids, nonsteroidal anti-inflammatory drugs or statins have also been shown to decrease the expression of adhesion molecules and pro-inflammatory chemo-kines, by nuclear factor-jB dependent gene transcrip-tion [9–11] (Fig 3)

Fig 2 Cell surface molecules as potential targets in atherosclerosis as a macrophage-driven disease.

Selectins consist of three members of C-type lectins

that bind sialyl-Lewis X carbohydrate ligands, such as

P-selectin glycoprotein ligand-1 (PSGL-1) [12]

P-selec-tin is stored in granules of endothelial cells and

plate-lets and it translocates to the cell surface following

exposure to inflammatory stimuli E-selectin is

exclu-sively expressed by endothelial cells, and L-selectin is

expressed on many subclasses of leukocytes [13] The

interaction of P- and E-selectin with leukocyte PSGL-1

and other sialyl Lewis-X glycoconjugates initiates the

attachment, rolling and homing of leukocytes on

endo-thelium Conversely, L-selectin on leukocytes binds to

endothelial ligands containing sulfated sialyl-Lewis X

like molecules

Inhibiting leukocyte rolling by blocking selectins

affects the accumulation of leukocytes in many

experi-mental settings [14,15] Blocking selectin activity with

humanized antibodies has been studied extensively in several clinical disorders Initial preclinical studies

in asthma, psoriasis, ischemia-reperfusion injury, or myocardial infarction were promising; however, the results of clinical trials with mAbs against E-, P- and L-selectins were disappointing [6] Attention was switched to the common ligand of all selectins, namely sialyl-Lewis X, as a broad-based therapeutic target [15] Outcomes from human trials using mimet-ics of sialyl-Lewis X or small molecule inhibitors of selectins have been more promising than those using selectin-directed antibodies [16] The synthetic inhib-itor bimosiamose (Table 2), a sialyl-Lewis X mimetic, improved psoriasis manifestations and allergen-indu-ced asthma in humans [17,18] Moreover, a new class

of selectin inhibitors, called efomycines, found as

a fermentation by-product of Streptomyces BS1261, have shown promising results in mouse models of skin inflammation [19]

Fig 3 Cell surface molecules as potential targets in inflammatory bowel disease as a lymphocyte-driven disease.

Integrins constitute a family of 24 heterodimers with

a-and b-subunits whose liga-and-binding activity is regulated

by conformational changes, transcriptional induction and redistribution from intracellular pools [20] Integrins mediate cell–cell, cell–extracellular matrix and cell–patho-gen interactions by binding to distinct, but overlapping,

Table 2 Selectin, integrin and GPCR antagonists in clinical and preclinical studies COPD, chronic obstructive pulmonary disease; IBD, inflammatory bowel disease; MS, multiple sclerosis; S1PR, sphingosine-1-phosphate receptor; SAE, severe adverse effect; SIRS, systemic inflammatory response syndrome.

P-, E- and L -selectin Bimosiamose Asthma, psoriasis Sialyl-Lewis X

analogue

Phase II P-, E- and L -selectin OC229648 Mouse model of peritonitis Sialyl-Lewis X

analogue

Preclinical

analogue

Preclinical

P-, E- and L -selectin CY1503 Ischemia-reperfusion injury

in lambs

Sialyl-Lewis X analogue

Preclinical

stroke

Blocking antibody Phase II in MS

and stroke (stopped,

no effects)

no effects)

model of atopic dermatitis

Blocking antibody Phase III, preclinical

in dermatitis

because of SAEs)

Phase II in ulcerative colitis and in psoriasis

receptor antagonist

Phase I

receptor antagonist

Preclinical CXCR1 and CXCR2 Repertaxin Ischemia-reperfusion primary

graft dysfunction

Small molecule inhibitor

Phase II for primary graft dysfunction in lung transplantation

insulin-resistant diabetes

Small molecule drug phase II

Zafirlukast Pranlukast S1PR1, S1PR3,

S1PR4, S1PR5

transplantation

combinations of ligands [20] Their structural and

func-tional diversity allows the integrins to play pivotal roles in

many biological processes, including inflammation,

he-mostasis and wound healing [21] Dysregulation of

inte-grins, however, contributes to the pathogenesis of many

diseases Therefore, therapeutic intervention of leukocyte

recruitment by blocking integrins or their

counter-recep-tors (ICAM, VCAMs and mucosal addressin cell

adhe-sion molecule-1) is likely to exert anti-inflammatory

effects in several diseases Extensive efforts have been

focused on the discovery and development of integrin

antagonists for clinical applications b2 (CD11⁄ CD18)

and a4 (CD49d) integrins are essential for the firm arrest

of leukocytes to the endothelium [20] Clinical trials with

rovelizumab or erlizumab (mAbs directed against CD18)

(Table 2) failed to show any beneficial effects in

ischemia-reperfusion injury after myocardial infarction or stroke

[22,23] Blockade of ICAM-1, the counter-receptor for

CD18 on endothelial cells, with a mAb (enlimomab),

showed negative effects in a phase II clinical trial in stroke

patients [24] These results dampened the enthusiasm for

targeting integrin function in ischemic settings However,

in inflammatory diseases, the inhibition of CD11a, which,

together with CD18 forms the lymphocyte

function-asso-ciated antigen-1 (LFA-1) complex, has been proven to be

beneficial For example, odulimomab, which interferes

with leukocyte migration by inhibiting CD11a, is used for

the treatment of graft-versus-host disease and suppresses

atopic dermatitis in animal models [6,25] Efalizumab is a

humanized IgG1 mAb that also targets the CD11a chain

of LFA-1 and prevents LFA-1 from interacting with

ICAM-1 Efalizumab has been successfully used in phase

III clinical trials in patients with psoriasis [26]

Natal-izumab (tysabri), a mAb to the a4 integrin chain that

blocks the binding of very late antigen-4 to VCAM-1, was

tested in large clinical phase III trials against multiple

sclerosis [27] and Crohn’s disease [28] The outcome in

these studies was very promising; however, the occurrence

of progressive multifocal leukoencephalopathy in

natal-izumab-treated patients has required further risk–benefit

analysis of this promising therapy In a clinical trial of

ulcerative colitis, MLN02, an antibody against the a4b2

heterodimer, was tested in 181 patients and found to

induce complete clinical and endoscopical remission in

33% (14% in the placebo group) [29] However, the

long-term beneficial effects of MLN02 in clinical practice are

not known, suggesting the need for additional studies

GPCR

GPCRs play a vital role in the signaling processes

that control cell motility, growth, blood coagulation

and inflammation GPCRs are the largest known

family of cell-surface receptors and are activated by chemokines, proteases, lipids and a wide variety of other molecules involved in inflammation Multiple chemokines play critical roles in the initiation and perpetuation of inflammatory diseases Activation of chemokine receptors by their ligands leads to the acti-vation of integrins, resulting in firm adhesion to the endothelium Therefore, for many years, chemokine receptors and their ligands have been an attractive hunting ground for pharmaceutical companies (Table 2) There are several possible approaches to inhibit specific chemokines These range from block-ing antibodies against chemokines or their receptors, small molecule receptor antagonists, or compounds that interdict components of downstream signal trans-duction pathways

In disease states such as systemic inflammatory response syndrome and, more specifically, severe sep-sis, an inability to down-regulate the inflammatory response leads to a hyperactivated state To reduce neutrophil migration along chemotactic gradients, early efforts targeted receptor–ligand interactions with peptido-mimetics or utilized blocking antibodies The first small molecule chemokine receptor antagonist was SB225002, which exhibited nanomolar inhibition against IL-8 binding to CXCR2, but not CXCR1 [30]

In chronic obstructive pulmonary disease, CXCR2 and IL-8 are up-regulated in the airways, and therefore blocking CXCR2 with SB225002 or other CXCR2 inhibitors may be particularly beneficial and studies are now entering the first clinical trials (Table 2) Fur-thermore, several preclinical studies with other CXCR1 and CXCR2 blocking agents have been shown to be efficacious in ischemia-reperfusion and sepsis models and are now being evaluated in the clinical situation [31–33]

The discovery that the chemokine receptors, CCR5 and CXCR4, are the coreceptors for CD4 in human immunodeficiency virus infection, provided a strong impetus for the rapid development of CCR5 and CXCR4 antagonists In addition, the activation of CCR5 by regulated on activation, normal, T-cell expressed, and secreted (RANTES) has also been linked to the development of atherosclerosis, asthma, atopic dermatitis and other inflammatory diseases CCR1 antagonists have been tested in multiple scler-osis and transplant rejection [34–36] Small molecule CCR3 inhibitors have shown beneficial effects in aller-gen models of asthma [37] Targeting CCR2 might be

a potential strategy for preventing macrophage activa-tion in asthma, multiple sclerosis, rheumatoid arthritis and atherosclerosis [38], and this is being evaluated in clinical studies [38]

Sphingosine-1-phosphate (S1P) receptors were

iden-tified in the context of defining the ligand for

endothel-ial differentiation gene-1 Four S1P receptors (S1PR2,

S1PR3, S1PR4 and S1PR5) were subsequently

identi-fied and found to be expressed by many cell types

Recently, studies with a small molecule –

2-amino-2-[2-(4-octylphenyl) ethyl] propane-1,3-diol hydrochloride

(FTY720) – identified during a screen for

immunosup-pressant agents, have shown that FTY720 is an agonist

for S1PR1, S1PR3, S1PR4 and S1PR5 FTY720 is a

prodrug that requires activation by endogenous

sphingosine-1-kinase The active metabolite traps T

cells in lymph nodes and initiates their homing into

lymphoid organs [39] FTY720 has been shown to be

efficacious in the prevention of kidney transplant

rejec-tion and might exert beneficial effects in other

inflam-matory diseases [40,41]

Another family of GPCRs, namely the protease

acti-vated receptors (PARs), has been shown to trigger

inflammatory responses following tissue injury PARs

are tethered-ligand receptors that are activated by

pro-teolytic cleavage of their extracellular domains [42]

Four different PARs have been identified: PAR1,

PAR2, PAR3 and PAR4 Activation of endothelial

and leukocyte PARs by proteases of the blood

coagu-lation cascade has a profound impact on inflammation

Thus, PARs are considered to be promising

therapeu-tic targets, and development of selective antagonists

for the PARs might provide an alternative strategy for

the treatment of inflammatory diseases [43,44]

Covic et al discovered a novel class of compounds,

termed pepducins, that inhibit receptor–G protein

signaling [43] These cell-penetrating lipopeptides are

derived from the intracellular loops of PARs and other

GPCRs The hydrophobic lipid moiety is used to

transport the peptide across the cell membrane and

tethers the pepducin to the inner leaflet of the lipid

bilayer in molecular proximity to the intracellular

loops of the receptor Pepducins were first designed to

block PAR1 and PAR4 signaling in human platelets

and required their cognate receptors for activity

[43,44] P1pal-7, a PAR1 antagonist pepducin, has

been shown to inhibit tumor growth and angiogenesis

in a breast cancer mouse model [45] Second

genera-tion pepducins derived from the first intracellular loop

of GPCRs have proven to be highly selective against

chemokine receptors and PARs [33,46] Pepducins

tar-geted against CXCR1 and CXCR2 chemokine

recep-tors completely blocked IL-8 induced neutrophil

migration without suppressing the response to bacterial

fMLP Moreover, even delayed treatment with

CXCR1⁄ 2 pepducins conferred nearly 100% survival

in a mouse model of sepsis in the absence of

antibiot-ics [33] These findings are of particular importance because the current treatment options for sepsis are primarily supportive

Future directions The challenge of the future will be to identify the key leukocyte subsets that initiate the pathologic processes

of a certain disease and specifically inhibit leukocyte migration and activation without compromising the normal function of the immune system The concept of immuno-modulation, rather than immuno-suppression, will probably be the optimal treatment for many inflammatory diseases such as the systemic inflam-matory response syndrome, atherosclerosis, asthma, chronic obstructive pulmonary disease, auto-immune disease and transplant rejection

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