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

MINIREVIEW
Therapeutic targeting of molecules involved in
leukocyte–endothelial cell interactions
Nicole C. Kaneider
1
, Andrew J. Leger
1,2
and Athan Kuliopulos
1,2,3
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 leu-
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 corticosteroids, 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:
(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.
4416 FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS
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
Neutrophil Monocyte ⁄ macrophage Lymphocyte
Disease Ischemia-reperfusion injury,
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
Integrin LFA-1, Mac-1 VLA-4 VLA-1, VLA-2, VLA-4, LFA-1
Selectin and ligand
L-selectin, PSGL-1 PSGL-1 L-selectin, PSGL-1, CLA

GPCR CXCR1, CXCR2, LTB4, PAR4 CCR1, CCR2, CXCR2 CCR1, CCR2, CXCR2, CCR5,
CXCR3, CCR4, CCR10, PAFR,
LTB4
N. C. Kaneider et al. Targeting leukocyte–endothelial cell interactions
FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS 4417
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 adhesion 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.
Targeting leukocyte–endothelial cell interactions N. C. Kaneider et al.
4418 FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS
Selectins
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.
N. C. Kaneider et al. Targeting leukocyte–endothelial cell interactions
FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS 4419
Integrins
Integrins constitute a family of 24 heterodimers with a-
and b-subunits whose ligand-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.
Target Drug Disease Mechanism of action Stage of development
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
P-, E- and
L-selectin Efomycine Mouse psoriasis Sialyl-Lewis X
analogue
Preclinical
P- and E-selectin HuEP5C7 Baboon stroke model Blocking antibody Preclinical

E-selectin CDP850 Psoriasis Blocking antibody Phase II
P-, E- and
L-selectin CY1503 Ischemia-reperfusion injury
in lambs
Sialyl-Lewis X
analogue
Preclinical
P- and
L-selectin rPSGL-Ig Myocardial infarction Antibody Phase II (stopped)
P-selectin CY1747 Ischemia-reperfusion injury Blocking antibody Preclinical
CD18 Rovelizumab Myocardial infarction, MS,
stroke
Blocking antibody Phase II in MS
and stroke (stopped,
no effects)
CD18 Erlizumab Myocardial infarction Blocking antibody Phase II (stopped,
no effects)
CD11a Odulimomab Transplant rejection, mouse
model of atopic dermatitis
Blocking antibody Phase III, preclinical
in dermatitis
CD11a Efalizumab Psoriasis Blocking antibody Approved for psoriasis
CD49d Natalizumab MS, Crohn’s disease Blocking antibody Phase III (halted
because of SAEs)
a4b1 TR14035 Asthma Small peptide antagonist Phase II
a4b7 MLN02 Ulcerative colitis Blocking antibody Phase II
ICAM-1 ISIS2302 IBD, rheumatoid Antisense nucleotide Phase III in rheumatoid
Enlimomab arthritis, psoriasis arthritis and Crohn’s disease
Phase II in ulcerative colitis
and in psoriasis

CXCR2 SB 225002 COPD Pyrimidine-based
receptor antagonist
Phase I
CXCR2 SB-332235 COPD Pyrimidine-based
receptor antagonist
Preclinical
CXCR1 and CXCR2 Repertaxin Ischemia-reperfusion primary
graft dysfunction
Small molecule
inhibitor
Phase II for primary graft
dysfunction in lung
transplantation
CXCR1 and CXCR2 x1 ⁄ 2pal-i1 SIRS Pepducin Preclinical
CCL11 CAT-213 Asthma Blocking antibody Phase II
CCR1 BX-471 MS, transplant rejection Nonpeptide antagonist Phase II for MS
CCR3 GW-766994 asthma, allergy Nonpeptide antagonist Preclinical
CCR5 UK-427857 AIDS Nonpeptide antagonist Preclinical
CCR9 Traficet-EN IBD Small molecule drug Phase III
CCR2 INCB3284 Rheumatoid arthritis, obese
insulin-resistant diabetes
Small molecule drug phase II
CXCR4 CTCE-9908 Prostate cancer Small molecule drug Phase II
LTB4 Montelukast Asthma, COPD Small molecule drug Approved for asthma
Zafirlukast
Pranlukast
S1PR1, S1PR3,
S1PR4, S1PR5
FTY720 Transplant rejection S1PR agonist Phase III for kidney
transplantation

Targeting leukocyte–endothelial cell interactions N. C. Kaneider et al.
4420 FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 interve ntion of leukocyte
recruitment by blocking integrins or their counter-recep-
tors (ICAM, VCAMs a nd 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 fo r the firm arrest
of leukocytes to the endothelium [20]. Clinical trials wi th
rovelizumab or erlizumab (mAbs directed aga inst CD18)
(Table 2) failed to show any beneficial effects in ischemia-
reperfusion injury after myocardial infarction or stroke
[22,23]. Blockade of I CAM-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 i n 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 p roven 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. E falizumab has b een 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 ve ry late an tigen-4 to VC AM-1, was
tested in large clinical phase III trials against m ultiple
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 n eed for a dditional s tudies.
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].
N. C. Kaneider et al. Targeting leukocyte–endothelial cell interactions
FEBS Journal 273 (2006) 4416–4424 ª 2006 The Authors Journal compilation ª 2006 FEBS 4421
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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