REVIEWS

PI3K and PI3K: partners in crime in

inflammation in rheumatoid arthritis
and beyond?
Christian Rommel, Montserrat Camps and Hong Ji

Abstract | Dysregulated signal transduction in innate and adaptive immune cells is known
to be associated with the development of various autoimmune and inflammatory diseases.
Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network
heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases
(PI3Ks) generate lipid-based second messengers that control an array of intracellular
signalling pathways that are known to have important roles in leukocytes. In light of the
recent progress in the development of selective PI3K inhibitors, and the beneficial effects
of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the
therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis
and other immune-mediated diseases.

Merck Serono International

S.A., 9 Chemin des Mines,
1211 Geneva, Switzerland.
Correspondence to C.R.
e-mail: christian.rommel@
merckserono.net
doi:10.1038/nri2036
Published online
9 February 2007

The high degree of complexity and plasticity of the

intracellular signal-transduction network is often considered to be an a priori impediment to the success of
targeted therapies. Nevertheless, signal-transduction
pathways that underlie disease processes are gradually
being unravelled, and the deciphering of the role and
hierarchy of signalling components and their interactions has encouraged the development of effective
and safe drugs that specifically target crucial elements
of intracellular signalling1.
The aetiology of chronic inflammatory diseases,
such as rheumatoid arthritis, is ill-defined. Fortunately,
our understanding of their pathogenesis at the cellular
and molecular level is more advanced. Indeed, recent
progress in the analysis of the cytokine and chemokine
network and its signal-transduction machinery has led to
a more rational selection of targets for therapeutic intervention in inflammation. The phosphoinositide 3-kinase
(PI3K) isoforms PI3K and PI3K are key enzymes in
leukocyte signalling and therefore represent promising
targets for the intervention of the signalling pathways
that are involved in inflammatory and autoimmune
diseases. In this Review, we describe the PI3K signalling pathways that are involved in immune responses
relevant to the pathogenesis of rheumatoid arthritis and
other inflammatory diseases, and highlight the multiple
roles of PI3K isoforms in the complex interplay between
innate and adaptive immunity.

NATURE REVIEWS | IMMUNOLOGY

PI3K families
PI3Ks belong to a large family of lipid signalling kinases
that phosphorylate phosphoinositides at the d3 position
of the inositol ring, creating d3 phosphorylated inositol
lipids. PI3Ks are evolutionary conserved from yeast to
high mammals and are divided into class I, II and III
PI3Ks, according to their molecular structure, cellular regulation and in vivo substrate specificities24. Class I PI3Ks,
which include class IA (consisting of PI3K, PI3K and
PI3K isoforms) and class IB (consisting of PI3K only),
are a family of dual-specificity lipid and protein kinases
that control many cellular functions, such as growth and
proliferation, survival and apoptosis, as well as adhesion
and migration (FIG. 1). This class of enzymes catalyse the
phosphorylation of phosphatidylinositol-4,5-bisphosphate
(PtdIns(4,5)P2) giving rise to the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)24. In
contrast to phosphatidylinositol-3-phosphate (PtdIns3P),
the levels of PtdIns(3,4,5)P3 are undetectable in resting
cells and following stimulation increase substantially
albeit transiently. All four class I PI3Ks are heterodimers
composed of a catalytic subunit of 110 kDa and a tightly
associated regulatory subunit that controls their expression, activation and subcellular localization57. The
110-kDa catalytic subunits of class IA PI3K isoforms
form heterodimers with a regulatory subunit known as
p85, of which there are five distinct isoforms. Class IA
PI3K isoforms are often activated by growth factor and
VOLUME 7 | MARCH 2007 | 191

2007 Nature Publishing Group

REVIEWS
Class IA PI3K

Class IB PI3K

Receptor
Receptor
tyrosine
kinase

GPCR
Plasma membrane

PtdIns(4,5)P2

PtdIns(3,4,5)P3

PtdIns(4,5)P2
G protein

Y
PTK

RAS

p110,
Y P p85 p110,
p110
GAB2

p85 p110,
p110,
p110

p110

Class IB PI3K

Growth

P p85 p110,
p110,
p110

p101/p84

Metabolism Survival
Proliferation Differentiation Development

Class IA PI3Ks

Apoptosis

Adhesion

Motility

Migration

Phagocytosis

Figure 1 | Class I PI3Ks: linking cell-surface receptors to signalling networks and cell functions. After cell stimulation,
all class I phosphoinositide 3-kinases (PI3Ks) are recruited to the inner face of the plasma membrane, where they generate
phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) by direct phosphorylation of phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2)2,117. Class IA PI3K isoforms (p110, p110 and p110) bind directly or indirectly to receptors
through the interaction of their regulatory subunits (p85) with tyrosine-phosphorylated recognition motifs on the
receptor cytoplasmic domains or soluble adaptor proteins such as GAB2 (GRB2-associated binding protein 2)3. The only
class IB PI3K isoform, PI3K, is recruited to G-protein-coupled receptors (GPCRs) by direct interaction with G-protein
subunits, through both the catalytic p110 and the regulatory subunits10,14,118. RAS, in its active GTP-bound state, also binds
directly to the p110 subunit of PI3K further regulating its catalytic activity119. Activation of all class I PI3Ks leads to the
generation of the lipid second messenger PtdIns(3,4,5)P3 at the cell membrane, which serves as a docking platform for
pleckstrin-homology-domain-containing proteins (such as AKT, also known as PKB), as well as other homology-domaincontaining proteins. This leads to a cascade of phosphorylating events and proteinprotein interactions of downstream
targets to control multiple biological processes2. PTK, protein tyrosine kinase.

G-protein-coupled receptors
(GPCRs). One of a large group
of seven-transmembranespanning receptors that bind
a diverse set of molecules,
including chemokines,
complement components,
bioactive amines, hormones
and neurotransmitters. GPCRs
are coupled to heterotrimeric,
GTP-binding proteins
composed of and
subunits, which, after
receptor activation by ligand
binding, split into monomers
and dimers, which interact
and activate or inhibit
downstream components of
various signalling pathways.

Mammalian target of
rapamycin
(MTOR). MTOR is a conserved
serine/threonine protein kinase
that regulates cell growth and
metabolism, as well as cytokine
and growth-factor expression,
in response to environmental
cues. MTOR receives
stimulatory signals from RAS
and phosphoinositide 3-kinase
(PI3K) downstream of growth
factors, as well as nutrients,
such as amino acids, glucose
and oxygen.

cytokine receptors through a tyrosine-kinase-dependent

mechanism6,7. Besides PI3K, which is mainly expressed
by leukocytes, the expression of the class IA PI3K isoforms
is ubiquitous3,6,8.
The 110-kDa catalytic subunit of PI3K, the only
class IB PI3K isoform described so far, associates with
one of two regulatory subunits, p101 and p84 (also
known as p87PIKAP), that are distinct from any of the
p85 subunits911. Notably, PI3K activation is driven by
the activation of G-protein-coupled receptors (GPCRs),
such as chemokine receptors. This activation is mediated by the direct binding of both the regulatory and
catalytic subunits of PI3K to G-protein subunits,
by subsequent membrane translocation of PI3K and by
further direct catalytic activation of PI3K by G-protein
subunits and RASGTP1014 (FIG. 1). Similar to PI3K,
PI3K is mainly expressed by white blood cells, although
its expression pattern might be broader than originally
reported1518.
Our understanding of the physiological function and
importance of individual PI3K isoforms has recently
improved owing not only to the development of genemodified mice but also to the recently developed pharmacological tools. Wortmannin and LY294002 are potent
PI3K inhibitors and they have been extensively used for
more than a decade to analyse PI3K-driven pathways,
mostly in ex vivo studies19. However, these molecules
do not show specificity for a particular class I PI3K isoform and, moreover, they have been shown to also block

192 | MARCH 2007 | VOLUME 7

class II and class III PI3Ks, as well as other closely related

enzymes, such as mammalian target of rapamycin (MTOR),
and unrelated enzymes, such as casein kinase 2 (CK2),
myosin light chain kinase (MLCK) and polo-like kinase
(PLK)2022. Consequently, some of the phenotypes that
were originally attributed to class I PI3Ks, using these
inhibitors, could also be due to the inhibition of other
related pathways. Concomitantly, some of the phenotypes
observed in gene-modified mice that lack the entire PI3K
protein (gene deletion; knockout mice), as opposed to
mice that only lack PI3K enzyme activity (site-directed
kinase-inactive mutant; knock-in mice), could also be
caused by the loss of protein functions that are independent of class I PI3K enzymatic activity16,2325. Studies involving the knockout of genes that encode the regulatory
subunits have helped to identify the functions of PI3K
isoforms in B cells2629,30. However, in some cases, deletion
of the regulatory subunits also impaired the expression
level of the corresponding catalytic subunits. Similarly, the
knockout of genes that encode the class IA p110 catalytic
subunits leads to an excess of the regulatory subunits,
which might block signalling that is independent of p110
activity. In other words, the phenotypes that result from
the knockout of either the p85 or p110 subunits might
be more complex than originally anticipated5,6,8. Because
genetic and pharmacological experimental approaches
have both advantages and disadvantages31, it is best, whenever possible, to combine both strategies, in particular
with respect to validating drug targets.

www.nature.com/reviews/immunol
2007 Nature Publishing Group

REVIEWS

Anaphylaxis
Severe and rapid allergic
reaction triggered by the
activation of high-affinity Fc
receptors for IgE in sensitized
individuals. An anaphylactic
shock is the most severe type
of anaphylaxis and will usually
lead to death in minutes if left
untreated.

MRLlpr mouse
A mouse strain that
spontaneously develops
glomerulonephritis and other
symptoms of systemic lupus
erythematosus (SLE). The lpr
mutation causes a defect in
FAS (also known as CD95),
preventing the apoptosis of
activated lymphocytes; the
MRL strain contributes
disease-associated mutations
that have yet to be identified.

Pannus
A sheet of inflammatory
granulation tissue, composed
of immune cells, blood vessels
and fibrous cells, that spreads
from the synovial membrane
and ultimately invades the joint
in rheumatoid arthritis.

Roles of class I PI3Ks in vivo

The differential tissue distribution of PI3K isoforms is a
key factor in the distinct biological functions of PI3Ks.
Not surprisingly, genetic ablation of the omnipresent
PI3K or PI3K isoforms results in embryonic lethality,
which indicates that, at least during development, PI3K
and PI3K have essential and non-redundant roles8,32,33.
The fact that heterozygous mice are viable indicates that
low levels of PI3K and PI3K activity are sufficient
for normal development. However, even if the involvement of these molecules in diseases, such as cancer
and metabolic disorders, makes them attractive candidate drug targets, the adverse effects that might result
from pharmacological inhibition of PI3K and PI3K
activities remain unclear17. By contrast, mice deprived of
PI3K or PI3K expression or function are viable, fertile
and apparently healthy with a normal life span, even in
conventional animal facilities. Nevertheless, PI3K- and
PI3K-mutant mice, generated either by deletion of the
whole gene (p110d- and p110g-knockout mice) or by
mutation of the kinase domain (PI3K (Asp910Ala) or
PI3K (Lys833Arg) kinase-inactive knock-in mice), show
severely altered phenotypes when their immune system
is acutely stressed8,2325,3436.
PI3K deficiency leads to impaired in vitro and in vivo
recruitment of neutrophils and macrophages to the sites of
inflammation and, in response to inflammatory stimuli34,
it leads to impaired neutrophil oxidative burst35,36 and
dendritic-cell migration37, as well as impaired T-cell activation35,36. PI3K-mutant mice, both knockout and kinaseinactive knock-in mice, show specific defects in B-cell
signalling that lead to impaired B-cell development and
generation of T-cell-dependent and T-cell-independent
antibodies after antigen stimulation in vivo 2325.
Interestingly, only the PI3K kinase-inactive knock-in
mice, and not the p110d-knockout mice, exhibit impaired
T-cell activity in vitro and in vivo. By contrast, both p110gknockout mice and PI3K kinase-inactive knock-in mice
show impaired mast-cell-mediated allergic responses and
consequently attenuated passive anaphylaxis in vivo38,39.
Although the precise roles of PI3K and PI3K in
disease remain undefined, the basic phenotypes of the
PI3K- and PI3K-mutant mice gained interest from pharmaceutical companies to pursue the development of orally
active and selective small-molecule inhibitors of PI3K for
the treatment of disorders that are triggered by uncontrolled innate and/or adaptive immune responses17,18,40.
As a result, the use of newly developed isoform-selective
small-molecule inhibitors has confirmed some of the phenotypes shown by genetic-based studies. Recently, treatment with a new, orally active, small-molecule inhibitor of
PI3K has been shown to reduce glomerulonephritis and
prolong survival in the MRLlpr mouse model of systemic
lupus erythematosus (SLE)41. Moreover, p110g-knockout
mice are largely protected in mouse models of effectorphase arthritis, and oral treatment of wild-type mice with
a selective PI3K inhibitor suppressed the progression
of joint inflammation and damage in both lymphocyteindependent and lymphocyte-dependent mouse models
of rheumatoid arthritis, reproducing the protective effects
shown in p110g-knockout mice42,43.

NATURE REVIEWS | IMMUNOLOGY

Another study has addressed the complexity of

chemokine-induced neutrophilia and class I PI3K signalling in lung tissue, and defines the PI3K isoform as
a promising target for airway inflammatory diseases44.
Interestingly, a similar study, using specific inhibitors of
PI3K, showed attenuated allergic airway inflammation
and hyper-responsiveness in a mouse model of asthma45.
Furthermore, a selective PI3K inhibitor has recently
been shown to protect mice against anaphylactic allergic
responses, reproducing the protection that was originally
observed in PI3K kinase-inactive knock-in mice38. So,
PI3K and PI3K have non-redundant important functions in mast cells, neutrophils, dendritic cells, B cells
and T cells, and the activities of these immune cells are
crucial during the onset, progression and maintenance
of chronic inflammatory diseases, such as rheumatoid
arthritis. The fact that some in vivo models of inflammation have revealed similar roles for PI3K and PI3K
in immune-cell signalling indicates a potential partnership between these class I PI3Ks in the regulation
and function of immune responses. Despite all the
experimental evidence collected so far, whether pharmacological inhibition of PI3K and/or PI3K effectively
ameliorates chronic inflammatory diseases in humans
remains largely unknown. In the following sections, we
describe expected sites and modes of action, as well
as the potential therapeutic benefits, of selective PI3K
inhibitors in the pathology of rheumatoid arthritis and
other inflammatory diseases.

Roles of PI3K and PI3K in rheumatoid arthritis

Rheumatoid arthritis is a chronic progressive, debilitating inflammatory disease that affects approximately 1%
of the worlds population. Rheumatoid arthritis is generally considered to be an immune-mediated disease.
Microscopically, rheumatoid joints are characterized
by inflammatory infiltrates in the synovium and the
synovial fluid, by pannus formation and, ultimately, by
cartilage and bone erosion. Detailed studies in animal
models that recapitulate many of the features of rheumatoid arthritis in humans have proposed the involvement
of multiple steps in the pathogenesis of the disease, with
an interplay between the adaptive and innate immune
systems43,4648. The activities of T cells47, B cells (including the production of autoantibodies and immune complexes49,50, and cytokine secretion51,52), dendritic cells53,
mast cells5456, macrophages63,64 and neutrophils57,58 have
all been shown to have important roles and to affect at
least one or several aspects of the disease (FIG. 2).
PI3K and PI3K function in T cells. T cells are implicated in most, if not all, of the immune-mediated
inflam matory diseases. In rheumatoid arthritis,
expression of the MHC class II molecule HLA-DR
confers strong susceptibility to the disease, suggesting a role for CD4+ T cells5961. Memory T cells, as well
as T-cell-generated cytokines, have been detected in
the synovium from rheumatoid joints6265. Recently, a
co-stimulatory modulator, consisting of a fusion protein of cytotoxic T-lymphocyte antigen 4 (CTLA4) and
immunoglobulin Fc (abatacept (Orencia; Bristol-Myers

VOLUME 7 | MARCH 2007 | 193

2007 Nature Publishing Group

REVIEWS
a Initiation (periphery)

b Propagation (synovium)

IL-4, IL-10, IL-13

CD40L

Immune complex

Mast cell
Synovial
fibroblast

CD40

MMP, PGE2,
CCL5, IL-18

Autoantibody

T cell
B cell

TCR
MHC
Dendritic
cell

c Tissue damage (bone and cartilage)

BCR

Monocyte

FcR

CCLs

TNF, IL-1, CCL2, CCL3,

IL-6, CXCL8, LTB4, VEGF
and others

H+, cathepsin K
Osteoclast

ROS, NO

Macrophage
CCR
Neutrophil

Pannus formation

Figure 2 | PI3K acts at multiple steps and various stages in the pathogenesis of rheumatoid arthritis. This figure
illustrates one scenario depicting the stepwise progression of the development of rheumatoid arthritis43,4648,52.
Disease progression can be divided into three separate, albeit interrelated, phases. a | Disease initiation takes place in
peripheral lymphoid organs. Disease is probably initiated by dendritic cells that present self-antigens to autoreactive
T cells, which in turn activate autoreactive B cells through cytokines and co-stimulatory molecules, resulting in the
production of autoantibody and the deposition of immune complexes in the joint. After the migration of immune cells
into the joint, antigen presentation can also occur at the synovium. Direct T-cell contact can also activate other cells in
an antigen-independent manner. b | Disease propagation is mediated by immune complexes binding to Fc receptors
(FcRs) on macrophages, neutrophils and mast cells. This leads to the release of pro-inflammatory cytokines and
chemokines by these activated cells, as well as to marked leukocyte infiltration and consequent synovial pannus
formation. c | High levels of cytokines and chemokines activate and recruit synovial fibroblasts, macrophages,
osteoclasts and neutrophils, which release proteases, reactive oxygen species (ROS), nitric oxide and prostaglandin E2
(PGE2). Synoviocytes are also activated and recruited, and these cells invade cartilage. All steps collectively contribute
to irreversible bone and cartilage destruction. Phosphoinositide 3-kinases (PI3Ks), most notably PI3K and PI3K, have
crucial and specific roles at all stages of disease progression: in antigen signalling in B and T cells, and in signalling
downstream of FcRs, cytokine receptors and chemokine receptors in mast cells, macrophages, neutrophils and
synoviocytes. BCR, B-cell receptor; CCL, CC-chemokine ligand; CCR, CC-chemokine receptor; CD40L, CD40 ligand;
CXCL, CXC-chemokine ligand; IL, interleukin; LTB4, leukotriene B4; MMP, matrix metalloproteinase; TCR, T-cell receptor;
TNF, tumour-necrosis factor; VEGF, vascular endothelial growth factor.

Squibb)), has shown convincing therapeutic benefit for

individuals with rheumatoid arthritis66,67. So, consistent
with results from animal studies48, T cells have important roles, systemically and/or locally, in rheumatoid
arthritis48,68,69.
In T cells, class I PI3Ks can be activated by crosslinking
the T-cell receptor (TCR), with or without co-stimulation
by CD28, or by activating the interleukin-2 (IL-2) receptor or chemokine receptors. Sustained elevated levels
of PtdIns(3,4,5)P3 cause lymphoproliferation and, conversely, prevention of PtdIns(3,4,5)P3 production potently
blocks T-cell proliferation6. p110g-knockout T cells
show normal TCR-mediated early signalling events but,
compared with wild-type cells, they proliferate less after
stimulation with CD3-specific antibody and produce
fewer cytokines in response to TCR-dependent and TCRindependent stimuli in vitro. This indicates that PI3K
does not act downstream of the TCR but regulates a second signal, probably through GPCRs36. T-cell-restricted
activation of class IA PI3K isoforms, by transgenic expression of a mutated form of p85 regulatory subunit that
lacks the C-terminal NH2 domain (p65PI3K-transgenic
mice), which results in constitutive activation of PI3K70,71,

194 | MARCH 2007 | VOLUME 7

increases the generation of CD4+ T cells in the thymus72

and enhances peripheral T-cell survival. This results in
the triggering of an invasive lymphoproliferative disease
with symptoms similar to SLE73. Interestingly, deletion
of the negative regulator of the class I PI3K pathway, the
tumour suppressor phosphatase and tensin homologue
(PTEN), also induces SLE-like disease, confirming the
capacity of the PI3K pathway to trigger this pathology74,75.
It is not clear which of the class IA PI3K isoforms is implicated in this disease, however, deletion of the class IB
isoform PI3K blocks SLE-like symptoms and extends
the life span of p65PI3K-transgenic mice76. Long-term
administration of a PI3K-selective inhibitor also results
in amelioration of disease in the MRLlpr mouse model
of SLE41. Although PI3K is important for mature T cells
to migrate towards lymphoid chemokines77,78, it has been
found that PI3K does not reduce CD4+ T-cell invasion
or lymphoproliferation but rather inhibits pathogenic
CD4+ T-cell survival in p65PI3K-transgenic mice76.
As an increase in pathogenic CD4+ memory T cells
is a hallmark of many autoimmune disorders, including rheumatoid arthritis, PI3K might be a valuable
target for the treatment of this pathology. It will be of

www.nature.com/reviews/immunol
2007 Nature Publishing Group

REVIEWS
interest to investigate whether long-term blockade of
PI3K inhibits arthritogenic T-cell survival or invasion
besides reducing neutrophil and macrophage chemotaxis in inflammatory arthritis42. Nonetheless, PI3K
kinase-inactive knock-in mice also show reduced
T-cell proliferation and activation following in vitro
stimulation with CD3-specific antibodies, as well as
impaired antigen-specific T-cell responses in vivo25. In
addition, PI3K has an important role in the differentiation and expansion of both T helper 1 (TH1) cells and
TH2 cells in vitro and in vivo79, and, interestingly, it is
also required for CD4+CD25+FOXP3+ (forkhead box
P3) regulatory T-cell activity80. Therefore, it will also
be interesting to assess PI3K kinase-inactive knock-in
mice, or selective PI3K small-molecule inhibitors,
in different autoimmune disease models.
Although PI3K has been successfully validated as a
target for the treatment of rheumatoid arthritis, at least
in animal models (both lymphocyte-dependent and
lymphocyte-independent models), it is not clear to what
extent the inhibition of PI3K in T cells contributes to
the effects seen in the lymphocyte-dependent models of
rheumatoid arthritis42. Notably, inhibition of PI3K also
showed therapeutic efficacy in collagen-induced arthritis
when the inhibitor was administered to mice with established joint inflammation, a stage of the disease that had
passed at least the early requirement of T-cell activity.
Both p110g-knockout mice and PI3K kinaseinactive knock-in mice showed a defective capacity
to mount contact hypersensitivity reactions37 (H.J. and
C.R., unpublished observations). The defect in p110gknockout mice is attributed to impaired migration
towards draining lymph nodes but not to the antigenpresenting function of antigen-loaded dendritic cells
from the periphery 37, although the intrinsic defect
in T-cell function of PI3K- or PI3K-mutant mice in
this model has not been addressed. The fact that PI3K
mediates the migration of antigen-presenting cells to
the draining lymph nodes suggests a role for PI3K in
connecting innate and adaptive immune systems at the
early stage of immune reactions.

Collagen-induced arthritis
An animal model of
rheumatoid arthritis. Collageninduced arthritis develops
in susceptible rodents and
primates after immunization
with cartilage-derived type II
collagen.

Contact hypersensitivity
A T-cell-mediated immune
response that is evoked
following antigen
administration in the skin. It is
marked by monocyte and/or
macrophage infiltration and
activation, and depends on the
production of T-helper-1-type
cytokines.

PI3K and PI3K function in B cells. In rheumatoid

arthritis, B cells and plasma cells constitute about 5%
of the infiltrate in the pannus and are the main source
of autoantibody81. Although a direct link with disease
pathogenesis is not yet clear, autoantibodies can form
immune complexes or directly activate complement
pathways and/or other immune cells, leading to tissue
damage. An unequivocal role for B cells in the pathology
of rheumatoid arthritis has been shown by the impressive efficacy of the B-cell-depleting CD20-specific
monoclonal antibody rituximab (Rituxan; Genentech
and Biogen Idec; and MabThera; F. HoffmanLaRoche)
in a recent clinical trial82,83. In addition, B cells have been
directly or indirectly implicated in many animal models
of arthritis48.
A prominent role for PI3K in B-cell function has not
been reported so far. PI3K seems to mediate the production of T-cell-dependent IgG1 after immunization
in vivo, probably through its role in T-cell function36.

NATURE REVIEWS | IMMUNOLOGY

By contrast, PI3K is essential for B-cell development

and function. IgM-specific antibody-induced B-cell
proliferation is abolished in cells isolated from PI3K
kinase-inactive knock-in mice, whereas B-cell proliferation induced by IL-4, CD40 or lipopolysaccharide
(LPS) is only partially affected25. Accordingly, both
T-cell-dependent and T-cell-independent antibody
responses in these mice are strongly reduced in vivo following antigen challenge25. In addition, PI3K activity
is indispensable for B-cell-receptor-induced DNA synthesis and proliferation, and IL-4-induced survival84,85.
These observations indicate that PI3K has a crucial and
non-redundant role in B-cell function that is not compensated by other class I PI3Ks, and they propose that
a specific inhibitor of PI3K might effectively suppress
B-cell- and T-cell-mediated functions in diseases, such
as rheumatoid arthritis.
PI3K and PI3K function in neutrophils. Neutrophils
not only represent the first line of defence against bacterial infection but they are also involved in many diseases in which the functions of adaptive immune cells
are thought to be important, such as psoriasis86, SLE87,
chronic obstructive pulmonary disease88 and chronic
severe asthma 89,90. In rheumatoid joints, numerous neutrophils accumulate, mainly in the synovial
fluid9296 but also in the pannuscartilage junctions92,
where they contribute to cartilage erosion by releasing proteolytic enzymes and toxic oxidative products.
Neutrophils are often the first cells to infiltrate the
synovial fluid and tissue in animal models, and depletion of neutrophils efficiently resolves the disease in
lymphocyte-independent arthritis models58,97. Many
treatments for arthritis, including glucocorticoids and
tumour-necrosis factor (TNF) blockers, also inhibit
neutrophil function, which probably contributes to
the therapeutic efficacy of these agents98,99.
The significant, albeit incomplete, reduction of leukocyte migration in response to a wide range of chemoattractants, such as chemokines, formylated bacterial
peptides and complement fragments, such as C5a,
both in vitro and in vivo, is a remarkable phenotype of
p110g-knockout mice3436. In these mice, compared with
wild-type mice, neutrophils and macrophages migrate
much less efficiently towards chemokines, such as
CC-chemokine ligand 3 (CCL3; also known as MIP1),
CCL5 (also known as RANTES), CXC-chemokine ligand 1
(CXCL1; also known as GRO and KC) and CXCL8 (also
known as IL-8), and chemoattractants, such as complement C5a and the bacterial peptide N-formyl-methionylleucyl-phenylalanine (fMLP), both in vitro and in vivo,
and they are not recruited to casein-induced peritonitis nor to Listeria monocytogenes or Escherichia coli
seeded in the peritoneal cavity in vivo3436. Other class I
PI3Ks do not compensate for the lack of PI3K, and
complete loss of class I PI3K activity does not exacerbate this phenotype, which indicates that PI3K is the
only PI3K isoform mediating migration towards such
chemoattractants3436,42.
The evidence for the involvement of PI3K in neutrophil function has been controversial. p110g-knockout

VOLUME 7 | MARCH 2007 | 195

2007 Nature Publishing Group

REVIEWS

Respiratory burst
A large increase in oxygen
consumption and reactive
oxygen species generation that
accompanies the exposure of
neutrophils to microorganisms
and/or inflammatory
mediators.

Pertussis toxin
Pertussis toxin blocks Gicoupled receptor signalling
(including chemokine-receptor
signalling) by catalysing ADP
ribosylation of the Gi subunit.

mice but not PI3K kinase-inactive knock-in mice show

impaired neutrophil infiltration in a lung sepsis model,
which indicates that bronchoalveolar leukocyte recruitment requires PI3K but not PI3K (REF. 44). However, a
selective PI3K inhibitor reduced neutrophil-mediated
lung inflammation in rats45. Differences between genetic
and pharmacological target inactivation, the complexity of animal models and species, a potential permissive
role of PI3K on PI3K-mediated migration in vivo and
residual non-selective inhibition of PI3K by the pharmacological treatment, might offer explanations for some
of the apparent discrepancies. The use of cell reconstitution has shown the importance of the endothelial-cell
component of PI3K activity, relative to its role in
neutrophils, in supporting neutrophil interactions with
the inflamed vessel wall100. The same authors show in
another study that endothelial-cell-expressed PI3K is
also important for neutrophil adhesion and subsequent
transendothelial migration in response to leukotriene B4
(LTB4)101. This indicates that the activity of both PI3K
and PI3K is required for efficient capture of neutrophils
by cytokine-stimulated endothelium, a prerequisite for
cell transmigration. So, PI3K and PI3K seem to coordinate neutrophil extravasation in vivo, although the
mechanism still needs to be fully elucidated.
Pre-exposure of neutrophils to TNF or LPS strongly
augments the generation of reactive oxygen species
(ROS) induced by subsequent stimuli such as chemoattractants102. A marked impairment of ROS production
by cytokine-primed neutrophils, in response to fMLP,
has been shown in the absence of PI3K (REFS 3436).
Similarly, pharmacological inhibition of PI3K with
selective inhibitors shows that this isoform has the pivotal role in the initiation of a biphasic pathway of ROS
production, induced by fMLP, in TNF-primed human
neutrophils. Indeed, even if the second phase of ROS
production is mediated by PI3K and, at least partially,
by PI3K and PI3K, both phases depend entirely on the
first phase of ROS production mediated solely by PI3K
activity102. It is therefore possible that both PI3K and
PI3K functions are required for the organization
and activation of the Phox (phagocyte NADPH oxidase)
complex, which is involved in ROS production, whereby
under certain conditions PI3K might function as a gatekeeper for maximum responses (FIG. 3). However, it is not
clear whether blockade with specific inhibitors of such a
gate-keeper role as that of PI3K in ROS generation will
be crucial for the treatment of inflammation in rheumatoid arthritis. Surprisingly, although TNF-primed mouse
bone-marrow-derived neutrophils exhibit similar patterns of biphasic PI3K activation and ROS production
in response to fMLP, as in human cells, these two phases
are substantially lower and mediated solely by PI3K
(REF. 102). Notably, PI3K does not have a crucial role
in respiratory burst in neutrophils responding to serumopsonized zymosan; this indicates that PI3K might not
be important for direct phagocytepathogen interactions34. In addition, PI3K also seems to be involved in
ROS production in endothelial cells downstream of TNF
and upstream of protein kinase C (PKC) and nuclear
factor-B (NF-B)103.

196 | MARCH 2007 | VOLUME 7

Recently, it was shown that the induction of apoptosis of inflammatory neutrophils by inhibiting cyclindependent kinases (CDKs) increases the resolution of
established joint inflammation, which further highlights
the crucial role of neutrophils in this process104. It seems
that eliminating neutrophils (for example, by CDK1/
CDK2 inhibitors) or preventing the sustained influx of
neutrophils to sites of joint inflammation (for example,
by PI3K and/or PI3K inhibitors) could be useful
therapeutic strategies against arthritic inflammation.
PI3K and PI3K function in mast cells. For many decades, mast cells have been known for their detrimental roles
in mediating allergic diseases105. However, the role and
importance of mast cells is not restricted to IgE-triggered
immune responses. Recently, more attention has been
paid to mast-cell function in autoimmune diseases,
including rheumatoid arthritis106. Accumulation of mast
cells and their secreted or degranulated products have
been found in rheumatoid synovial tissue and fluid107.
Not only are mast cells strategically localized along the
microvasculature in tissues, but also their multifunctional capacity to release, under some circumstances
independently of degranulation, vasoactive mediators,
such as TNF, vascular endothelial growth factor (VEGF),
tryptase, histamine and serotonin, together with various
pro-inflammatory cytokines and chemokines, means
they are important immunomodulatory cells in acute
and chronic inflammation108. The possibility that mast
cells have an early, coordinating role in the pathogenesis of rheumatoid arthritis has also been supported
by results obtained from studies of animal models55.
Many mediators, including stem-cell factor (SCF), and
immune complexes containing antigen and IgE or IgG,
affect mast-cell differentiation and activation. Activated
SCF receptors (KIT), the high-affinity Fc receptor for
IgE (FcRI) and Fc receptors for IgG (FcRs) all generate
PtdIns(3,4,5)P3 and PtdIns(3,4)P2 signals through PI3Ks.
A role for class I PI3K in mast-cell activation was also
shown by non-isoform-specific PI3K inhibitors, which
completely blocked antigen-triggered mast-cell degranulation109. However, which class I PI3K isoform is engaged
after ligand-specific mast-cell stimulation only became
clear recently (FIG. 4).
Both PI3K and PI3K are important in mast-cell
function. PI3K is required for SCF-induced and, in
part, IL-3-induced mast-cell proliferation, adhesion
and migration38. Mast-cell degranulation and cytokine
release in response to antigenIgE immune complexes
also depends on PI3K, at least partially38. Consequently,
PI3K kinase-inactive knock-in mice are largely
protected from passive cutaneous anaphylaxis38.
Interestingly, p110g-knockout mice are also resistant
to cutaneous anaphylaxis (oedema formation following
intradermal administration of adenosine) and passive
systemic anaphylactic shock39. Although PI3K is not
directly activated by FcRI and pertussis toxin only
partially blocks antigenIgE-triggered degranulation,
PI3K can amplify mast-cell function through secondary autocrine and paracrine loops, involving adenosinemediated, and probably also chemokine-mediated,

www.nature.com/reviews/immunol
2007 Nature Publishing Group

REVIEWS
TNFR1

Phox complex

Plasma membrane

PtdIns(4,5)P2
TRADD

PTK

PtdIns(3,4,5)P3

PI3K

P-REX1 RAC

Phox
p67

PtdIns(4,5)P2
Phox
p47

NADPH
Phox
NADP+ p40

PtdIns(3,4,5)P3
AKT PKC

p85 p110
PI3K

InsP3

DAG

PTK
PTEN

PKC

Ca2+

IKK

PKC

Class IB PI3K (primary

PtdIns(3,4,5)P3 response)

IKK IKK

H2O2

O2

PTP

SOD

p85 p110

Neutrophil

PI3K

Class IA PI3K (secondary

PtdIns(3,4,5)P3 response)

IB
p50 p65
NF-B

PtdIns(3,4,5)P3

PLC p101 p110

RIP
TRAFs

PtdIns(4,5)P2

AKT PDK1

Phox
gp91

Respiratory burst O2

Phox p22

fMLPR

Phagocytosis Degranulation

ROS

Cytokine production

Migration

Figure 3 | Model for cooperativity between PI3K and PI3K in respiratory burst in neutrophils. The multicomponent
phagocyte NADPH oxidase (Phox) complex catalyses the respiratory burst of neutrophils and other phagocytes.
Superoxide is generated after activation of gp91Phox (also known as NOX2)120. Activation of gp91Phox occurs after
assembly of the cytosolic regulatory proteins p40Phox, p47Phox, p67Phox and RAC at the plasma membrane. Stimulation
of tumour-necrosis factor (TNF)-primed neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP) results in rapid
and sequential accumulation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) (and/or phosphatidylinositol-3,4bisphosphate (PtdIns(3,4)P2) derived from it), which leads to the production of reactive oxygen species (ROS). The first
peak of PtdIns(3,4,5)P3 is mediated by the class I phosphoinositide 3-kinase (PI3K) P13K, whereas class IA PI3Ks (mostly
PI3K) are required for a second phase of PtdIns(3,4,5)P3 production. PI3K and the phospholipids can interact with the
Phox components in at least three ways: first, phosphatidylinositol-4,5-biphosphate PtdIns(4,5)P2 can directly bind to
p47Phox through its Phox domain, promoting the interaction with p40Phox (the Phox domain of p40 preferably binds to
PtdIns(3,4,5)P3; not depicted); second, PtdIns(3,4,5)P3 regulates protein kinases, such as AKT, PDK1 (3-phosphoinositidedependent protein kinase 1) and certain protein kinase C (PKC) isoforms, which then phosphorylate the autoinhibitory
domain of p47Phox, promoting the interaction with p22Phox; third, PtdIns(3,4,5)P3 and the G-protein subunits bind and
regulate the activity of P-REX1, a guanine-nucleotide-exchange factor that in turn activates the small GTPase RAC120123.
The mechanism by which fMLP and TNF activate class IA PI3Ks is not clear. Conceivably, TNF- and fMLP-stimulated
increase of tyrosine phosphorylation, either by activation of a protein tyrosine kinase (PTK) or by inactivation of protein
tyrosine phosphatase (PTP), can activate class IA PI3Ks. In addition, increased levels of intracellular hydrogen peroxide
(H2O2) generated by superoxide dismutase (SOD) inactivate PTPs, as well as phosphatase and tensin homologue (PTEN), by
oxidizing a cysteine residue in their catalytic site 124. DAG, diacylglycerol; IKK, inhibitor of NF-B (IB) kinase kinase; InsP3,
inositol-1,4,5-trisphosphate; NF-B, nuclear factor-B; O2; superoxide; PLC, phospholipase C; RIP, receptor-interacting
protein; TNFR1, TNF receptor 1; TRADD, TNFR1-associated via death domain; TRAF, TNFR-associated factor.

pertussis-toxin-sensitive GPCR activation39. In this

case, although it has not been directly compared, PI3K
activated by FcRI and PI3K subsequently activated
by pertussis-toxin-sensitive GPCRs might coordinate,
temporally and spatially, to achieve optimum mast-cell
function. Transient class-IA-dependent and subsequent
class IB (PI3K)-dependent activities, activated by a
receptor tyrosine kinase and by a GPCR, respectively,
have also been observed in FcR signalling in human
monocytes110.
Indeed, the data obtained so far indicate that the
initial stimulation of PI3K might directly or indirectly
increase intracellular levels of calcium, leading to an initial phase of degranulation, which subsequently activates

NATURE REVIEWS | IMMUNOLOGY

PI3K, allowing the generation of the full degranulation

response. The initial phase of degranulation that is triggered by antigenIgE immune complexes and mediated
by PI3K provides a limited release of GPCR agonists,
such as adenosine, that trigger autocrine and paracrine
positive-feedback loops, which lead to PI3K activation
and marked granule release39. Therefore, it seems conceivable that, at least in this scenario, PI3K functions
upstream of PI3K and that the two enzymes operate
episodically at two distinct but crucial levels of the same
signalling cascade in mast cells. In this way, PI3K and
PI3K may provide a signalling platform that integrates
distinct and multiple extracellular stimuli during
mast-cell activation. An alternative scenario has been

VOLUME 7 | MARCH 2007 | 197

2007 Nature Publishing Group

REVIEWS
FcRI

KIT

Ca2+

SC
F

F
SC

SOS

MAPKKK

PtdIns(3,4,5)P3
BTK

LYN

p85 p110

PtdIns(4,5)P2

PtdIns(3,4,5)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2

p110 p101 PLC

PI3K

SYK

P p85 p110
Y GAB2
Y GRB2
P
Y

PI3K

Y P
P P Y P
PLC

JAKs

P
P

PI3K

Ca2+

STATs

RAS

PtdIns(4,5)P2

A3AR

NTAL

P Y
P Y
Y

LAT

P Y
P Y
GRB2

Adenosine

SOCC

Plasma membrane

DAG

InsP3

PKC

Ca2+

InsP3

DAG

Ca2+

PKC

MAPKK

MAPK
Mast-cell
granule
Mast cell

Class IA (primary PtdIns(3,4,5)P3 response)

Class IB (secondary PtdIns(3,4,5)P3 response)

Proliferation Adhesion Degranulation Cytokine production Migration

Figure 4 | Model for cooperativity between PI3K and PI3K in degranulation in mast cells. After activation of the
high-affinity Fc receptor for IgE (FcRI), the adaptor molecules LAT (linker for activation of T cells) and NTAL (non-T-cell
activation linker) become tyrosine phosphorylated by the concerted action of LYN and SYK (spleen tyrosine kinase)125.
Phosphorylated tyrosine residues in LAT and NTAL provide docking sites for adaptor proteins, such as growth-factorreceptor-bound protein 2 (GRB2) and GRB2-associated binding protein 2 (GAB2), and key signalling enzymes, which
together form active signalosomes. Phospholipase C (PLC) binds to LAT, whereas phosphoinositide 3-kinase- (PI3K), by
the binding of its regulatory subunit p85 to the GRB2GAB2 complex, is recruited to NTAL (the regulation of PI3K by NTAL
in a LAT-independent manner has yet to be formally proven). PI3K generates phosphatidylinositol-3,4,5-trisphosphate
(PtdIns(3,4,5)P3), which activates Brutons tyrosine kinase (BTK), which in turn amplifies the activation of LAT-bound PLC.
PLC generates the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3) by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), and InsP3 initiates the release of calcium from internal stores. Emptied
stores trigger the activity of membrane-integral store-operated calcium channels (SOCCs)126. In addition, PtdIns(3,4,5)P3
binds directly to SOCCs. DAG activates conventional protein kinase C (PKC) isoforms that, together with free calcium,
triggers an initial wave of degranulation. The release of adenosine and, probably other G-protein-coupled receptor
(GPCR) ligands, subsequently activates the adenosine receptor 3A isoform (A3AR) and possibly other GPCRs, leading to
G-protein -subunit-mediated PI3K activation and a second wave of PtdIns(3,4,5)P3 production39. Sustained, high levels
of PtdIns(3,4,5)P3 and calcium cause maximum degranulation by mast cells39,127. In an alternative scenario, PLC mediates
the initial phase of calcium mobilization, independently of PI3K (REF. 128). Dimerization of KIT by stem-cell factor (SCF)
induces tyrosine autophosphorylation at several residues in the cytoplasmic domain, recruiting PI3K (and possibly also
PI3K and/or PI3K) and GRB2. This results in the production of PtdIns(3,4,5)P3 and activation of the RASMAPK (mitogenactivated protein kinase) pathway. PtdIns(3,4,5)P3 recruits BTK, which further activates PLC to produce InsP3 and release
calcium. KIT (receptor for SCF) also signals through the Janus kinase (JAK)signal transducer and activator of transcription
(STAT) pathway and, together with the MAPK pathway, contributes to mast-cell proliferation, differentiation, survival,
migration and cytokine production125. SOS, son of sevenless homologue.

proposed whereby PLC mediates the initial phase of

calcium mobilization, independently of PI3K (REF. 128).
After this first wave of calcium release, the coordinated
activation of PI3K and then PI3K would provide the
secondary response to sustain the intracellular calcium
levels that are required for full degranulation125.

198 | MARCH 2007 | VOLUME 7

So, in diseases such as rheumatoid arthritis, as well as

in asthma and other allergic and inflammatory diseases, in
which mast-cell activation can result from increased production of cytokines, chemokines and/or activators of
FcRs and FcRs, it will be of great interest to examine
the participation of both PI3K and PI3K.

www.nature.com/reviews/immunol
2007 Nature Publishing Group

REVIEWS
The proposed model (FIG. 4) of a temporarily coordinated contribution of PI3K and PI3K at different stages
of signalling, which ultimately causes complete mast-cell
activation and subsequent degranulation, resembles the
cooperativity observed between these PI3K isoforms for
optimum production of ROS following cytokine and
chemokine stimulation of neutrophils89 (FIG. 3).

Outlook
Our understanding of cytokine-controlled signaltransduction pathways that are implicated in rheumatoid arthritis has in the past led to drug development
programmes for targeting mitogen-activated protein
kinase and NF-B pathways, with the primary rationale
to intervene with the expression and/or action of proinflammatory cytokines and proteases111. Many such
signal-transduction-based small-molecule inhibitors
have been shown to work in experimental models of
rheumatoid arthritis. With the exception of the emerging
success of second-generation drugs that target p38, most
of these agents, however, have yet to show any beneficial
activity in patients.
Many chemokines are produced at high levels in
the rheumatoid synovium112. So, targeting chemokines
and/or chemokine receptors could be a potential
approach for treating chronic inflammatory disorders
like rheumatoid arthritis. Chemokine-receptor antagonists should, in principle, anergize leukocyte subpopulations that express the cognate receptor, which may
be advantageous in certain disease settings113. In other
circumstances, however, this could be insufficient
given the many cell types and ligands or receptors
that are involved in the inflammatory processes, and
the redundancy of the chemokines released at the sites
of inflammation. As PI3K is a shared downstream
component in chemokine signalling pathways, it might
represent an attractive alternative target for interfering
with excessive leukocyte activation and migration in

1.

2.
3.

4.

5.

6.

7.

8.

Fishman, M. C. & Porter, J. A. Pharmaceuticals: a new

grammar for drug discovery. Nature 437, 491493
(2005).
Cantley, L. C. The phosphoinositide 3-kinase pathway.
Science 296, 16551657 (2002).
Vanhaesebroeck, B. et al. Synthesis and function of
3-phosphorylated inositol lipids. Annu. Rev. Biochem.
70, 535602 (2001).
An excellent review of the molecular biology and
biochemistry of class I PI3Ks.
Foster, F. M., Traer, C. J., Abraham, S. M. & Fry, M. J.
The phosphoinositide (PI) 3-kinase family. J. Cell Sci.
116, 30373040 (2003).
Brachmann, S. M. et al. Role of phosphoinositide
3-kinase regulatory isoforms in development and actin
rearrangement. Mol. Cell. Biol. 25, 25932606 (2005).
Fruman, D. A. & Cantley, L. C. Phosphoinositide
3-kinase in immunological systems. Semin. Immunol.
14, 718 (2002).
A comprehensive review of the regulation,
function and receptor signalling of class I
PI3Ks (in leukocytes).
Jimenez, C., Hernandez, C., Pimentel, B. & Carrera,
A. C. The p85 regulatory subunit controls sequential
activation of phosphoinositide 3-kinase by Tyr kinases
and Ras. J. Biol. Chem. 277, 4155641562 (2002).
Vanhaesebroeck, B., Ali, K., Bilancio, A., Geering, B. &
Foukas, L. C. Signalling by PI3K isoforms: insights
from gene-targeted mice. Trends Biochem. Sci. 30,
194204 (2005).

9.

10.

11.

12.

13.

14.

15.

16.

chronic inflammation. Whether inhibition of PI3K is

a valid therapeutic approach for the treatment of human
rheumatoid arthritis will probably remain a working
hypothesis until the first compound shows efficacy in
Phase II human clinical trials. The function of PI3K
in autoimmune inflammatory diseases has not been
directly investigated.
The decisive role of PI3K and PI3K in the signalling and functions of T cells, B cells, neutrophils,
macrophages and mast cells strongly indicates that,
in addition to rheumatoid arthritis and SLE, these
enzymes are valid therapeutic targets for several other
inflammation-mediated diseases, such as multiple
sclerosis (in which T cells, B cells and mast cells are
implicated106,114,115), asthma (for which T cells and mast
cells are important116), chronic obstructive pulmonary
disease (which involves neutrophils, macrophages and
T cells88) and psoriasis (in which T cells, neutrophils
and macrophages are implicated86). Therefore, thorough
investigation of the functions of PI3K and PI3K in
relevant disease models, as well as in patient tissues, will
be crucial to validate them as targets for the treatment
of a range of inflammatory diseases.
Effective therapies for immune-mediated diseases
may need to provide therapeutic impact at various
phases of the multicellular disease process as in rheumatoid arthritis. Evidence so far suggests that PI3K and
PI3K operate as partners in distinct yet interdependent
signalling pathways in many immune cells; their finetuned integration gives rise to pro-inflammatory events
in the multistep pathogenic process of inflammation.
A key question that remains in the field is whether both
PI3K and PI3K are valid targets for the treatment of
chronic inflammatory diseases or whether one would be
more suitable than the other. In terms of drug efficacy
though, as the history of crime fighters attests, good
clinical benefits may require all partners in crime to be
successfully neutralized.

Voigt, P., Dorner, M. B. & Schaefer, M.

Characterization of p87PIKAP, a novel regulatory
subunit of phosphoinositide 3-kinase that is highly
expressed in heart and interacts with PDE3B. J. Biol.
Chem. 281, 99779986 (2006).
Stephens, L. R. et al. The G sensitivity of a PI3K is
dependent upon a tightly associated adaptor, p101.
Cell 89, 105114 (1997).
Suire, S. et al. p84, a new G-activated regulatory
subunit of the type IB phosphoinositide 3-kinase
p110. Curr. Biol. 15, 566570 (2005).
Brock, C. et al. Roles of G in membrane recruitment
and activation of p110/p101 phosphoinositide
3-kinase . J. Cell. Biol. 160, 8999 (2003).
Stephens, L. et al. A novel phosphoinositide 3
kinase activity in myeloid-derived cells is activated
by G protein subunits. Cell 77, 8393 (1994).
Leopoldt, D. et al. G stimulates phosphoinositide
3-kinase- by direct interaction with two domains of
the catalytic p110 subunit. J. Biol. Chem. 273,
70247029 (1998).
Bernstein, H. G., Keilhoff, G., Reiser, M., Freese, S. &
Wetzker, R. Tissue distribution and subcellular
localization of a G-protein activated phosphoinositide
3-kinase. An immunohistochemical study. Cell. Mol.
Biol. (Noisy-le-grand) 44, 973983 (1998).
Patrucco, E. et al. PI3K modulates the cardiac
response to chronic pressure overload by distinct kinasedependent and-independent effects. Cell 118, 375387
(2004).

NATURE REVIEWS | IMMUNOLOGY

17. Wetzker, R. & Rommel, C. Phosphoinositide 3-kinases

as targets for therapeutic intervention. Curr. Pharm.
Des. 10, 19151922 (2004).
18. Wymann, M. P., Zvelebil, M. & Laffargue, M.
Phosphoinositide 3-kinase signalling which way
to target? Trends Pharmacol. Sci. 24, 366376
(2003).
19. Wymann, M. P. et al. Wortmannin inactivates
phosphoinositide 3-kinase by covalent modification
of Lys-802, a residue involved in the phosphate
transfer reaction. Mol. Cell. Biol. 16, 17221733
(1996).
20. Davies, S. P., Reddy, H., Caivano, M. & Cohen, P.
Specificity and mechanism of action of some
commonly used protein kinase inhibitors. Biochem. J.
351, 95105 (2000).
21. Knight, Z. A. et al. Isoform-specific phosphoinositide
3-kinase inhibitors from an arylmorpholine scaffold.
Bioorg. Med. Chem. 12, 47494759 (2004).
22. Liu, Y. et al. Wortmannin, a widely used
phosphoinositide 3-kinase inhibitor, also potently
inhibits mammalian polo-like kinase. Chem. Biol. 12,
99107 (2005).
23. Clayton, E. et al. A crucial role for the p110 subunit
of phosphatidylinositol 3-kinase in B cell development
and activation. J. Exp. Med. 196, 753763 (2002).
24. Jou, S. T. et al. Essential, nonredundant role for the
phosphoinositide 3-kinase p110 in signaling by the
B-cell receptor complex. Mol. Cell. Biol. 22,
85808591 (2002).

VOLUME 7 | MARCH 2007 | 199

2007 Nature Publishing Group

REVIEWS
25. Okkenhaug, K. et al. Impaired B and T cell antigen
receptor signaling in p110 PI 3-kinase mutant mice.
Science 297, 10311034 (2002).
References 2325 report the important and
non-redundant role of PI3K in T-cell and B-cell
signalling.
26. Fruman, D. A. et al. Hypoglycaemia, liver necrosis
and perinatal death in mice lacking all isoforms of
phosphoinositide 3-kinase p85. Nature Genet.
26, 3793782 (2000).
27. Garcia, Z., Kumar, A., Marques, M., Cortes, I. &
Carrera, A. C. Phosphoinositide 3-kinase controls
early and late events in mammalian cell division.
EMBO J. 25, 655661 (2006).
28. Ueki, K. et al. Positive and negative roles of p85 and
p85 regulatory subunits of phosphoinositide 3-kinase
in insulin signaling. J. Biol. Chem. 278, 4845348466
(2003).
29. Ueki, K. et al. Increased insulin sensitivity in mice
lacking p85 subunit of phosphoinositide 3-kinase.
Proc. Natl Acad. Sci. USA 99, 419424 (2002).
30. Terauchi, Y. et al. Increased insulin sensitivity
and hypoglycaemia in mice lacking the p85
subunit of phosphoinositide 3-kinase. Nature Genet.
21, 230235 (1999).
31. Vanhaesebroeck, B., Rohn, J. L. & Waterfield, M. D.
Gene targeting: attention to detail. Cell 118,
274276 (2004).
32. Bi, L., Okabe, I., Bernard, D. J. & Nussbaum, R. L.
Early embryonic lethality in mice deficient in the
p110 catalytic subunit of PI 3-kinase. Mamm.
Genome 13, 169172 (2002).
33. Bi, L., Okabe, I., Bernard, D. J., Wynshaw-Boris, A. &
Nussbaum, R. L. Proliferative defect and embryonic
lethality in mice homozygous for a deletion in the
p110 subunit of phosphoinositide 3-kinase. J. Biol.
Chem. 274, 1096310968 (1999).
34. Hirsch, E. et al. Central role for G protein-coupled
phosphoinositide 3-kinase in inflammation. Science
287, 10491053 (2000).
35. Li, Z. et al. Roles of PLC-2 and-3 and PI3K in
chemoattractant-mediated signal transduction.
Science 287, 10461049 (2000).
36. Sasaki, T. et al. Function of PI3K in thymocyte
development, T cell activation, and neutrophil
migration. Science 287, 10401046 (2000).
References 3436 report the important and nonredundant role of PI3K in chemokine signalling,
leukocyte migration, oxidative burst and T-cell
activation and proliferation.
37. Del Prete, A. et al. Defective dendritic cell migration
and activation of adaptive immunity in PI3K-deficient
mice. EMBO J. 23, 35053515 (2004).
38. Ali, K. et al. Essential role for the p110
phosphoinositide 3-kinase in the allergic response.
Nature 431, 10071011 (2004).
Shows an essential and non-redundant role for PI3K
in mast-cell signalling and acute allergic responses.
39. Laffargue, M. et al. Phosphoinositide 3-kinase is an
essential amplifier of mast cell function. Immunity 16,
441451 (2002).
Shows an essential and non-redundant role for PI3K
in mast-cell signalling and acute allergic responses.
40. Ward, S. G. & Finan, P. Isoform-specific
phosphoinositide 3-kinase inhibitors as therapeutic
agents. Curr. Opin. Pharmacol. 3, 426434 (2003).
41. Barber, D. F. et al. PI3K inhibition blocks
glomerulonephritis and extends lifespan in a mouse
model of systemic lupus. Nature Med. 11, 933935
(2005).
42. Camps, M. et al. Blockade of PI3K suppresses joint
inflammation and damage in mouse models of
rheumatoid arthritis. Nature Med. 11, 936943
(2005).
References 41 and 42 use orally active smallmolecule inhibitors of PI3K to validate an
important role for PI3K in inflammatory diseases
such as SLE and rheumatoid arthritis.
43. Firestein, G. S. Inhibiting inflammation in rheumatoid
arthritis. N. Engl. J. Med. 354, 8082 (2006).
44. Thomas, M. J. et al. Airway inflammation:
chemokine-induced neutrophilia and the class I
phosphoinositide 3-kinases. Eur. J. Immunol. 35,
12831291 (2005).
45. Lee, K. S., Lee, H. K., Hayflick, J. S., Lee, Y. C. &
Puri, K. D. Inhibition of phosphoinositide 3-kinase
attenuates allergic airway inflammation and
hyperresponsiveness in murine asthma model.
FASEB J. 20, 455465 (2006).
References 44 and 45 describe the role of PI3K
versus PI3K in airway inflammation.

46. Firestein, G. S. Evolving concepts of rheumatoid

arthritis. Nature 423, 356361 (2003).
47. Firestein, G. S. The T cell cometh: interplay between
adaptive immunity and cytokine networks in
rheumatoid arthritis. J. Clin. Invest. 114, 471474
(2004).
48. Monach, P. A., Benoist, C. & Mathis, D. The role of
antibodies in mouse models of rheumatoid arthritis,
and relevance to human disease. Adv. Immunol. 82,
217248 (2004).
49. Benoist, C. & Mathis, D. A revival of the B cell
paradigm for rheumatoid arthritis pathogenesis?
Arthritis Res. 2, 9094 (2000).
50. Weyand, C. M., Seyler, T. M. & Goronzy, J. J. B cells in
rheumatoid synovitis. Arthritis Res. Ther. 7 (Suppl. 3),
912 (2005).
51. Feldmann, M. et al. Analysis of cytokine expression in
rheumatoid synovium has provided new insights into
the pathogenesis of rheumatoid arthritis and new
therapeutic opportunities. Transplant. Proc. 33,
20852086 (2001).
52. Feldmann, M. et al. Cytokine blockade in rheumatoid
arthritis. Adv. Exp. Med. Biol. 490, 119127 (2001).
53. Sarkar, S. & Fox, D. A. Dendritic cells in rheumatoid
arthritis. Front Biosci. 10, 656665 (2005).
54. Benoist, C. & Mathis, D. Mast cells in autoimmune
disease. Nature 420, 875878 (2002).
55. Lee, D. M. et al. Mast cells: a cellular link between
autoantibodies and inflammatory arthritis. Science
297, 16891692 (2002).
56. Nigrovic, P. A. & Lee, D. M. Mast cells in inflammatory
arthritis. Arthritis Res. Ther. 7, 111 (2005).
57. Edwards, S. W. & Hallett, M. B. Seeing the wood for the
trees: the forgotten role of neutrophils in rheumatoid
arthritis. Immunol. Today 18, 320324 (1997).
58. Wipke, B. T. & Allen, P. M. Essential role of neutrophils
in the initiation and progression of a murine model of
rheumatoid arthritis. J. Immunol. 167, 16011608
(2001).
59. Buckner, J. H. & Nepom, G. T. Genetics of rheumatoid
arthritis: is there a scientific explanation for the
human leukocyte antigen association? Curr. Opin.
Rheumatol. 14, 254259 (2002).
60. Jawaheer, D. et al. Dissecting the genetic complexity
of the association between human leukocyte antigens
and rheumatoid arthritis. Am. J. Hum. Genet. 71,
585594 (2002).
61. Weyand, C. M., Hicok, K. C., Hunder, G. G. & Goronzy,
J. J. The HLA-DRB1 locus as a genetic component in
giant cell arteritis. Mapping of a disease-linked
sequence motif to the antigen binding site of the
HLA-DR molecule. J. Clin. Invest. 90, 23552361
(1992).
62. Fitzgerald, J. E. et al. Analysis of clonal CD8+ T cell
expansions in normal individuals and patients with
rheumatoid arthritis. J. Immunol. 154, 35383547
(1995).
63. Hingorani, R. et al. Oligoclonality of V3 TCR chains in
the CD8+ T cell population of rheumatoid arthritis
patients. J. Immunol. 156, 852858 (1996).
64. Schmidt, D., Goronzy, J. J. & Weyand, C. M. CD4+
CD7 CD28 T cells are expanded in rheumatoid
arthritis and are characterized by autoreactivity.
J. Clin. Invest. 97, 20272037 (1996).
65. Chabaud, M., Fossiez, F., Taupin, J. L. & Miossec, P.
Enhancing effect of IL-17 on IL-1-induced IL-6 and
leukemia inhibitory factor production by rheumatoid
arthritis synoviocytes and its regulation by TH2
cytokines. J. Immunol. 161, 409414 (1998).
66. Kremer, J. M. et al. Treatment of rheumatoid arthritis
with the selective costimulation modulator abatacept:
twelve-month results of a phase iib, double-blind,
randomized, placebo-controlled trial. Arthritis Rheum.
52, 22632271 (2005).
67. Ruderman, E. M. & Pope, R. M. The evolving clinical
profile of abatacept (CTLA4-Ig): a novel
co-stimulatory modulator for the treatment of
rheumatoid arthritis. Arthritis Res. Ther. 7 (Suppl 2),
2125 (2005).
68. Mulherin, D., Fitzgerald, O. & Bresnihan, B. Synovial
tissue macrophage populations and articular damage
in rheumatoid arthritis. Arthritis Rheum. 39, 115124
(1996).
69. Yanni, G., Whelan, A., Feighery, C. & Bresnihan, B.
Synovial tissue macrophages and joint erosion in
rheumatoid arthritis. Ann. Rheum. Dis. 53, 3944
(1994).
70. Gonzalez-Garcia, A. et al. A new role for the p85phosphatidylinositol 3-kinase regulatory subunit
linking FRAP to p70 S6 kinase activation. J. Biol.
Chem. 277, 15001508 (2002).

200 | MARCH 2007 | VOLUME 7

71. Jimenez, C. et al. Identification and characterization of

a new oncogene derived from the regulatory subunit
of phosphoinositide 3-kinase. EMBO J. 17, 743753
(1998).
72. Rodriguez-Borlado, L. et al. Phosphatidylinositol
3-kinase regulates the CD4/CD8 T cell differentiation
ratio. J. Immunol. 170, 44754482 (2003).
73. Borlado, L. R. et al. Increased phosphoinositide
3-kinase activity induces a lymphoproliferative
disorder and contributes to tumor generation in vivo.
FASEB J. 14, 895903 (2000).
74. Xu, Y. & Wiernik, P. H. Systemic lupus erythematosus
and B-cell hematologic neoplasm. Lupus 10, 841850
(2001).
75. Di Cristofano, A. et al. Impaired Fas response and
autoimmunity in Pten+/ mice. Science 285,
21222125 (1999).
76. Barber, D. F. et al. Class IB-phosphatidylinositol
3-kinase (PI3K) deficiency ameliorates IA-PI3Kinduced systemic lupus but not T cell invasion.
J. Immunol. 176, 589593 (2006).
77. Reif, K. et al. Cutting edge: Differential roles for
phosphoinositide 3-kinases, p110 and p110, in
lymphocyte chemotaxis and homing. J. Immunol.
173, 22362240 (2004).
78. Curnock, A. P., Logan, M. K. & Ward, S. G. Chemokine
signalling: pivoting around multiple phosphoinositide
3-kinases. Immunology 105, 125136 (2002).
79. Okkenhaug, K. et al. The p110 isoform of
phosphoinositide 3-kinase controls clonal expansion
and differentiation of TH cells. J. Immunol. 177,
51225128 (2006).
80. Patton, D. T. et al. Cutting edge: The phosphoinositide
3-kinase p110 is critical for the function of
CD4+CD25+Foxp3+ regulatory T cells. J. Immunol.
177, 65986602 (2006).
81. Jasin, H. E. Autoantibody specificities of immune
complexes sequestered in articular cartilage of
patients with rheumatoid arthritis and osteoarthritis.
Arthritis Rheum. 28, 241248 (1985).
82. Edwards, J. C. et al. Efficacy of B-cell-targeted therapy
with rituximab in patients with rheumatoid arthritis.
N. Engl. J. Med. 350, 25722581 (2004).
83. Edwards, J. C. & Cambridge, G. B-cell targeting in
rheumatoid arthritis and other autoimmune diseases.
Nature Rev. Immunol. 6, 394403 (2006).
84. Bilancio, A. et al. Key role of the p110 isoform of
PI3K in B-cell antigen and IL-4 receptor signaling:
comparative analysis of genetic and pharmacologic
interference with p110 function in B cells. Blood 107,
642650 (2006).
85. Sujobert, P. et al. Essential role for the p110 isoform
in phosphoinositide 3-kinase activation and cell
proliferation in acute myeloid leukemia. Blood 106,
10631066 (2005).
86. Schon, M. P. & Boehncke, W. H. Psoriasis. N. Engl. J.
Med. 352, 18991912 (2005).
87. Cook, H. T. & Botto, M. Mechanisms of disease: the
complement system and the pathogenesis of systemic
lupus erythematosus. Nature Clin. Pract. Rheumatol.
2, 330337 (2006).
88. Shapiro, S. D. COPD unwound. N. Engl. J. Med. 352,
20162019 (2005).
89. Caramori, G., Pandit, A. & Papi, A. Is there a
difference between chronic airway inflammation in
chronic severe asthma and chronic obstructive
pulmonary disease? Curr. Opin. Allergy Clin. Immunol.
5, 7783 (2005).
90. Nathan, C. Neutrophils and immunity: challenges and
opportunities. Nature Rev. Immunol. 6, 17382 (2006).
91. Ley, K., Smith, E. & Stark, M. A. IL-17A-producing
neutrophil-regulatory Tn lymphocytes. Immunol. Res.
34, 229242 (2006).
92. Chatham, W. W. et al. Degradation of human articular
cartilage by neutrophils in synovial fluid. Arthritis
Rheum. 36, 5158 (1993).
93. Mohr, W., Kohler, G. & Wessinghage, D.
Polymorphonuclear granulocytes in rheumatic tissue
destruction. II. Demonstration of PMNs in rheumatoid
nodules by electron microscopy. Rheumatol. Int.
1, 2128 (1981).
94. Mohr, W., Pelster, B. & Wessinghage, D.
Polymorphonuclear granulocytes in rheumatic tissue
destruction. VI. The occurrence of PMNs in menisci
of patients with rheumatoid arthritis. Rheumatol. Int.
5, 3944 (1984).
95. Mohr, W., Westerhellweg, H. & Wessinghage, D.
Polymorphonuclear granulocytes in rheumatic tissue
destruction. III. An electron microscopic study of
PMNs at the pannus-cartilage junction in rheumatoid
arthritis. Ann. Rheum. Dis. 40, 396399 (1981).

www.nature.com/reviews/immunol
2007 Nature Publishing Group

REVIEWS
96. Mohr, W., Wild, A. & Wolf, H. P. Role of polymorphs in
inflammatory cartilage destruction in adjuvant arthritis
of rats. Ann. Rheum. Dis. 40, 171176 (1981).
97. Nandakumar, K. S., Svensson, L. & Holmdahl, R.
Collagen type II-specific monoclonal antibody-induced
arthritis in mice: description of the disease and the
influence of age, sex, and genes. Am. J. Pathol. 163,
18271837 (2003).
98. Capsoni, F. et al. Effect of adalimumab on neutrophil
function in patients with rheumatoid arthritis. Arthritis
Res. Ther. 7, R250R255 (2005).
99. Schramm, R. & Thorlacius, H. Neutrophil recruitment
in mast cell-dependent inflammation: inhibitory
mechanisms of glucocorticoids. Inflamm. Res. 53,
644652 (2004).
100. Puri, K. D. et al. The role of endothelial PI3K activity
in neutrophil trafficking. Blood 106, 150157 (2005).
101. Puri, K. D. et al. Mechanisms and implications of
phosphoinositide 3-kinase in promoting neutrophil
trafficking into inflamed tissue. Blood 103,
34483456 (2004).
References 100 and 101 indicate cooperativity
between PI3K and PI3K in neutrophil
extravasation.
102. Condliffe, A. M. et al. Sequential activation of class IB
and class IA PI3K is important for the primed
respiratory burst of human but not murine
neutrophils. Blood 106, 14321440 (2005).
Describes the cooperativity between PI3K and
PI3K in ROS production by neutrophils.
103. Frey, R. S. et al. Phosphatidylinositol 3-kinase
signaling through protein kinase C induces NADPH
oxidase-mediated oxidant generation and NF-B
activation in endothelial cells. J. Biol. Chem. 281,
1612816138 (2006).
104. Rossi, A. G. et al. Cyclin-dependent kinase inhibitors
enhance the resolution of inflammation by promoting
inflammatory cell apoptosis. Nature Med. 12,
10561064 (2006).
105. Okayama, Y. & Kawakami, T. Development, migration,
and survival of mast cells. Immunol. Res. 34, 97115
(2006).
106. Theoharides, T. C. & Cochrane, D. E. Critical role
of mast cells in inflammatory diseases and the effect
of acute stress. J. Neuroimmunol. 146, 112
(2004).
107. Olsson, N., Ulfgren, A. K. & Nilsson, G. Demonstration
of mast cell chemotactic activity in synovial fluid from
rheumatoid patients. Ann. Rheum. Dis. 60, 187193
(2001).
108. Galli, S. J., Nakae, S. & Tsai, M. Mast cells in the
development of adaptive immune responses. Nature
Immunol. 6, 135142 (2005).

109. Yano, H. et al. Inhibition of histamine secretion by

wortmannin through the blockade of
phosphatidylinositol 3-kinase in RBL-2H3 cells. J. Biol.
Chem. 268, 2584625856 (1993).
110. Melendez, A. J., Gillooly, D. J., Harnett, M. M. &
Allen, J. M. Aggregation of the human high
affinity immunoglobulin G receptor (FcRI) activates
both tyrosine kinase and G protein-coupled
phosphoinositide 3-kinase isoforms. Proc. Natl Acad.
Sci. USA 95, 21692174 (1998).
111. Hammaker, D., Sweeney, S. & Firestein, G. S.
Signal transduction networks in rheumatoid
arthritis. Ann. Rheum. Dis. 62 (Suppl. 2), 8689
(2003).
112. Szekanecz, Z., Szucs, G., Szanto, S. & Koch, A. E.
Chemokines in rheumatic diseases. Curr. Drug Targets
7, 91102 (2006).
113. Mackay, C. R. Chemokines: immunologys high impact
factors. Nature Immunol. 2, 95101 (2001).
114. Hemmer, B., Archelos, J. J. & Hartung, H. P. New
concepts in the immunopathogenesis of multiple
sclerosis. Nature Rev. Neurosci. 3, 291301
(2002).
115. Sospedra, M. & Martin, R. Immunology of multiple
sclerosis. Annu. Rev. Immunol. 23, 683747 (2005).
116. Busse, W. W. & Lemanske, R. F. Jr. Asthma. N. Engl. J.
Med. 344, 350362 (2001).
117. Wymann, M. P. & Marone, R. Phosphoinositide
3-kinase in disease: timing, location, and scaffolding.
Curr. Opin. Cell Biol. 17, 141149 (2005).
118. Stoyanov, B. et al. Cloning and characterization of a
G protein-activated human phosphoinositide-3 kinase.
Science 269, 690693 (1995).
119. Suire, S. et al. Gs and the Ras binding domain of
p110 are both important regulators of PI3K
signalling in neutrophils. Nature Cell Biol. 8,
13031309 (2006).
120. Lambeth, J. D. NOX enzymes and the biology of
reactive oxygen. Nature Rev. Immunol. 4, 181189
(2004).
121. Kanai, F. et al. The PX domains of p47phox and
p40phox bind to lipid products of PI(3)K. Nature Cell
Biol. 3, 675678 (2001).
Reports a direct link between PI3K and the Phox
complex.
122. Perisic, O. et al. The role of phosphoinositides and
phosphorylation in regulation of NADPH oxidase.
Adv. Enzyme Regul. 44, 279298 (2004).
123. Brown, G. E., Stewart, M. Q., Liu, H., Ha, V. L. &
Yaffe, M. B. A novel assay system implicates
PtdIns(3,4)P2, PtdIns(3)P, and PKC in intracellular
production of reactive oxygen species by the NADPH
oxidase. Mol. Cell 11, 3547 (2003).

NATURE REVIEWS | IMMUNOLOGY

124. Rhee, S. G. et al. Intracellular messenger function

of hydrogen peroxide and its regulation by
peroxiredoxins. Curr. Opin. Cell Biol. 17, 183189
(2005).
125. Gilfillan, A. M. & Tkaczyk, C. Integrated signalling
pathways for mast-cell activation. Nature Rev.
Immunol. 6, 218230 (2006).
126. Wymann, M. P. et al. Phosphoinositide 3-kinase : a
key modulator in inflammation and allergy. Biochem.
Soc. Trans. 31, 275280 (2003).
Describes the concept of cooperativity between
PI3K and PI3K in mast-cell signalling and function.
127. Hirsch, E. et al. Signaling through PI3K: a common
platform for leukocyte, platelet and cardiovascular
stress sensing. Thromb. Haemost. 95, 2935 (2006).
128. Tkaczyk, C., Beaven, M. A., Brachman, S. M.,
Metcalfe, D. D. & Gilfillan, A. M. The phospholipase
C1-dependent pathway of FcRI-mediated mast cell
activation is regulated independently of
phosphatidylinositol 3-kinase. J. Biol. Chem. 278,
4847448484 (2003).

Acknowledgements
We thank all our friends and colleagues, in particular,
B. Vanhaesebroeck, M. Wymann, A. Carrera, E. Hirsch,
R. Williams, P. Hawkins, L. Stephens, D. Lee and F. Rintelen
for many insightful discussions. We further thank
B. Vanhaesebroeck and R. Hooft for their kind and valuable
support during the preparation of this Review. We also
acknowledge the constructive and helpful suggestions provided by the anonymous referees. We are grateful to
C. Hebert for all the original graphic work and to all our
friends and colleagues for their outstanding support and
contributions to our research.

Competing interests statement

The authors declare competing financial interests: see web
version for details.

DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
CD28 | CTLA4 | FcRI | IL-2 | KIT | PI3K | PI3K | PI3K | PI3K |
PTEN | TNF

FURTHER INFORMATION
Poster:
PI3K signalling in immune cells: http://www.nature.com/nri/
posters/pi3k/index.html
Access to this links box is available online.

VOLUME 7 | MARCH 2007 | 201

2007 Nature Publishing Group