been devised. In a recent issue of Science,Christopherson et al.3 now present evidencethat a different strategy may help to over-come the limitations imposed by insufficientnumbers of HSCs. These authors have founda way of treating HSCs from mice so that thecells can engraft the bone marrow of intra-venously injected recipients more efficiently(Fig. 1).

The strategy hinged on the membrane-bound protease CD26, present on the surfaceof some hematopoietic cells. For their trans-plantation experiments, the authors incu-

N E W S A N D V I E W S

VEGF in the mouse lung; as might beexpected, this treatment promoted markedneovascularization and edema in the lung.But VEGF expression had far more profoundconsequences, dramatically affecting multiplemarkers of lung inflammation, increasing thenumber of infiltrating leukocytes, promotinglung remodeling (mainly increasing collagendeposition, myocyte hyperplasia and forma-tion of mucus-secreting goblet cells), andincreasing the responsiveness of the lung tostimuli (airway hyper-responsiveness).

The investigators went on to show that theVEGF transgenic mice overproduced IL-13,known to direct production of mucus, adhe-sion molecules and chemokines in airwaycells. Moreover, the ability of VEGF to inducemucus was abrogated in IL-13-deficientmice. These results dovetail with prior obser-

vations that IL-13 induces VEGF overexpres-sion10, suggesting that VEGF and IL-13 par-ticipate in a feedback loop.

Investigating the broader effect of VEGF, theinvestigators showed that transgenic mice haveelevated numbers of activated dendritic cellsand that they develop an exaggerated allergicimmune response when exposed to a respira-tory antigen. Finally, the authors implicatedVEGF in the development of antigen-inducedexperimental asthma. They first localizedVEGF production to airway epithelium andTH2 cells. They then showed that abrogation ofVEGF function partly protected the mice fromcertain features of experimental asthma,including inflammatory cell numbers, airwayhyper-responsiveness and IL-13 production.

These results draw attention to the role ofVEGF in promoting tissue remodeling andantigen sensitization in the lung (Fig. 1 and 2).Perhaps induction of VEGF in early life maypromote subsequent development of antigensensitization and TH2-associated diseases.Recently, Whitsett and colleagues also overex-pressed VEGF in the lung with the same trans-genic system as Lee’s group11. Theinvestigators did not focus on immunity, and,unlike Lee et al., who induced VEGF after amonth, they induced the gene right after birth.Alveolar leakage and alterations in pulmonaryvasculature development were observed. Butthe mice also experienced pulmonary hemor-rhage, hemosiderosis, emphysema (air spaceenlargement) and death11. Thus, VEGF over-expression in the early postnatal lung can pro-mote lung disease well beyond TH2-associated

disease11. Indeed, VEGF has been shown toincrease in a variety of TH1-associated states;even Lee et al. show that TH1 cells expressVEGF, albeit at lower levels than TH2 cells.

These results are notable on severalgrounds, and have immediate clinical impli-cations. Anti-VEGF therapies have recentlybeen approved for the treatment of patientswith renal cell carcinoma, and it will beimportant to examine respiratory parametersin patients currently being treated for cancer.The results of the study by Lee et al.4 shouldalso propel the design of studies specificallyaimed at assessing the role of VEGF thera-peutics in allergic inflammation. In the past,there was reluctance to use cancer therapeu-tics for the treatment of allergic disease,mainly because of toxicity. But there is a highdegree of in situ cell proliferation in the asth-matic lung. Indeed, a number of rapidlyemerging, relatively safe cancer therapeuticsmay soon benefit allergy sufferers.

1. Bach, J.F. N. Engl. J. Med. 347, 911–20 (2002).2. Nathan, C. Nature 420, 846–852 (2002).3. Umetsu, D.T., McIntire, J.J., Akbari, O., Macaubas, C.

& DeKruyff, R.H. Nat. Immunol. 3, 715–720 (2002).4. Lee, C.G. et al. Nat. Med. 10, 1095–1103 (2004).5. Busse, W.W. & Lemanske, R.F. Jr. N. Engl. J. Med.

344, 350–362 (2001).6. Lange, P., Parner, J., Vestbo, J., Schnohr, P. & Jensen,

G. N. Engl. J. Med. 339, 1194–1200 (1998).7. Hoshino, M., Nakamura, Y. & Hamid, Q.A. J. Allergy

Clin. Immunol. 107, 1034–1038 (2001).8. Kanazawa, H., Nomura, S. & Yoshikawa, J. Am. J.

Respir. Crit. Care Med. 169, 1125–1130 (2004).9. Hoshino, M., Takahashi, M. & Aoike, N. J. Allergy Clin.

Immunol. 107, 295–301 (2001).10. Corne, J. et al. J. Clin. Invest. 106, 783–791 (2000).11. Le Cras, T.D. et al. Am. J. Physiol. Lung Cell. Mol.

Physiol. 287, L134–L142 (2004).

1042 VOLUME 10 | NUMBER 10 | OCTOBER 2004 NATURE MEDICINE

Hematopoietic stem cells get closer to the boneConnie Eaves

Clinical transplants of hematopoietic stem cells have a large spectrum of therapeutic applications, but these can belimited by insufficient stem cell numbers and the current inability to expand them in vitro. Inactivation of anenzyme that impedes stem-cell homing to the bone marrow may offer a simple alternative.

Connie Eaves is at the Terry Fox Laboratory, British

Columbia Cancer Agency, Vancouver, British

Columbia, V5Z 1L3 Canada.

e-mail: ceaves@bccrc.ca

Hematopoietic stem cells (HSCs) are rarecells that persist throughout adult life in thebone marrow. Some of these cells continu-ously detach from their ‘niche’ at the surfaceof the bone to enter the blood, from whichthey then colonize other sites1,2. The enor-mous regenerative potential of HSCs, cou-

pled with their ability to rehome to the bonemarrow from the circulation, is the clinicalbasis of HSC transplants. These transplantshave helped thousands of patients withhematologic malignancies and hold promisefor the treatment of many other disorders.

Unfortunately, even for established clinicalapplications, the number of HSCs available isoften limiting. In the last decade, impressiveprogress was made in the detection, purifica-tion and molecular characterization of HSCs;nevertheless, medically useful procedures forexpanding human HSCs in vitro have not yet

Figure 2 An airway from an IL-13 transgenic mousemodel of asthma. Features include prominentmucus-producing epithelial cells, peribronchialinflammation, and airway wall thickening.

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bated HSC-enriched populations briefly withinhibitors of CD26, or used HSCs fromCD26-deficient mice. They then askedwhether this treatment would alter the num-bers of cells required to protect injectedrecipients from an otherwise lethal dose ofradiation. Inactivation of CD26 significantlyincreased the protective function of the cellstransplanted. Their ability to produce newblood cells in the transplanted hosts was alsoincreased.

The agents used to inhibit CD26 were sim-ple, readily synthesized peptide derivativesand their effects were obtained after only 15minutes of exposure, suggesting readyextrapolation to clinical protocols. Theeffects seen were also sustained and could beobtained with unpurified cells, thus addingfurther to the attractiveness of the approach.

The authors also attempt to provide amechanistic explanation for these observa-tions. CD26 is a widely expressed membrane-bound ectopeptidase that removesN-terminal residues from certain proteins.One of its substrates is stromal-derived fac-tor-1 (SDF-1, also called CXCL12)4, achemokine produced at high levels in thebone marrow and believed to be a keychemoattractant for circulating HSCs pass-ing through the marrow cavity5,6. CD26 isexpressed on a primitive subset ofhematopoietic cells that also express high lev-els of CXCR4, the receptor for SDF-1 (ref. 7).When SDF-1 is cleaved by CD26, SDF-1 losesits chemotactic action on primitivehematopoietic cells in vitro3,7. CD26 produc-tion by intravenously transplanted HSCsmight therefore prevent some of them frombeing induced to migrate into the bone mar-row space. This hypothesis ledChristopherson et al. to test whether the HSCactivity of mouse bone marrow cells could beenhanced by CD26 inactivation.

This hypothesis implies that standardtransplant assays underestimate the totalnumber of cells in the bone marrow withHSC potential, as has been widely believedfor many years, based on the assumption thatonly a fraction of HSCs would be detected insuch assays. But recent experiments withtransplants of highly purified populations ofHSCs now call this assumption into ques-tion. These studies have demonstrated that asmany as 40–90% of irradiated mice injectedwith single cells from these highly purifiedpopulations can regenerate new blood cellsfor many months8,9. Because previous workhas indicated that these populations alsocontain the majority of all detectable HSCs, itwould appear that no more than 10–60% ofHSCs are not detected in standard assays.

Therefore, two possible explanations forthe findings reported by Christopherson etal.3 need to be considered. One explanation isthat CD26 inhibition does not affect thenumber of HSCs that home to the bone mar-row, but instead enhances their individualregenerative activity (Fig. 1).

A second possibility is that there aremany more cells with HSC potential thanpreviously thought, but these normallyelude detection because their expression ofCD26 prevents re-entry into the bone mar-row from the circulation. This latter expla-nation is more consistent with the findingof Christopherson et al. that inhibition ofCD26 increases the proportion of cells in anHSC-enriched population that are found inthe bone marrow 24 hours after injection(Fig. 1). If the second explanation is cor-rect, however, then we must imagine a newclass of ‘mystery’ HSCs, severalfold morefrequent than those described to date andcontained within the larger population ofcells with features typical of cells withshort-lived repopulating activity. Future

NATURE MEDICINE VOLUME 10 | NUMBER 10 | OCTOBER 2004 1043

analysis of the exact cell types affected byCD26 inhibition should allow these ques-tions to be resolved.

More important from a clinical perspec-tive will be analogous in vivo experimentswith primitive hematopoietic cells fromhumans and nonhuman primates. Suchstudies will determine whether CD26 block-ade is likely to translate into similar results inpatients. Experiments have already shownthat blockade of CD26 on primitive humanumbilical cord blood cells can enhance theirchemotactic response to SDF-1 in vitro7.Thus, there is some basis for anticipatingthat the in vivo reconstituting activity ofthese cells will be similarly affected. If real-ized, this strategy could have importantimplications. At the very least, it may offer away to enable the use of cord blood trans-plants to be extended from pediatric to adultpatients. It could also help to improve out-comes in cancer patients who might benefitfrom high doses of chemotherapy but fromwhom adequate harvests of HSCs cannot beobtained. The future transplantation of

SDF-1

Bl od

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Control: No CD26 inhibitor

Treatment with CD26 inhibitor

Scenario A

Scenario B

Irradiation

HSCs

Bone Marrow cells

+ 24 hours + 6 months

Bon Ma row

Figure 1 Home run for HSCs. Christopherson et al.3 incubated mouse bone marrow cells with inhibitors ofCD26 before injecting the cells into irradiated hosts. This treatment enhanced the numbers of mature bloodcells produced by the transplanted cells several-fold. This result was correlated with an increased ability ofthe treated cells to home to the marrow space within 24 hours and to be attracted in vitro toward a source ofthe chemokine SDF-1. Local levels of SDF-1 in the bone marrow are thought to promote HSC migration intothis tissue. CD26 expression on HSCs may thus regulate their engraftment potential by inactivating the SDF-1 produced in the bone marrow. Not yet resolved is whether CD26 inhibition allows an increased number ofinitially transplanted cells to be detectable as HSCs (scenario A) or whether CD26 inhibition increases theregenerative ability of the known HSC population (scenario B).

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genetically corrected autologous HSCs intopatients with somatic cell diseases provides athird setting in which the approachdescribed by Christopherson et al. couldhave an impact. In such applications, achiev-ing adequate chimerism will be essential toobtaining permanent cures10,11. Thus a sim-ple method of bumping up the number of

HSCs that engraft could make the differencebetween cure and failure.

1. Arai, F. et al. Cell 118, 149–161 (2004).2. Wright, D. E. et al. Science 294, 1933–1936

(2001).3. Christopherson, K. W. et al. Science 305,

1000–1003 (2004).4. Proost, P. et al. FEBS Lett. 432, 73–76 (1998).5. Ara, T. et al. Immunity 19, 257–267 (2003).

6. Kollet, O. et al. Blood 100, 2778–2786 (2002).7. Christopherson, K. W. et al. J. Immunol. 169,

7000–7008 (2002).8. Uchida, N. et al. Exp. Hematol. 31, 1338–1347

(2003).9. Matsuzaki, Y. et al. Immunity 20, 87–93

(2004).10. Pawliuk, R. et al. Science 294, 2368–2371

(2001).11. Imren, S. et al. Proc. Natl. Acad. Sci. USA 99,

14380–14385 (2002).

1044 VOLUME 10 | NUMBER 10 | OCTOBER 2004 NATURE MEDICINE

Bone marrow spawns brain killersCostantino Iadecola

The reduction in blood flow to the brain that causes a stroke triggers a deadly cascade of events that can lead tobrain death. Studies in mice show that activation of an adenosine receptor on neutrophils invading the brain fromthe blood contribute to the damage (pages 1081–1087).

Ischemic stroke is a devastating brain diseasethat afflicts millions worldwide1. It is causedby a sudden occlusion of a cerebral artery thatcuts off the blood supply to a restricted regionof the brain. The loss of cerebral blood flow, orcerebral ischemia, leads to permanent braindamage and neurological deficits, includingparalysis, impaired vision, inability to speak,loss of balance and cognitive deficits.

Although some of the molecular events thatlead to brain death occur within the ischemicbrain, factors outside the brain also partici-pate. In this issue, Yu et al.2 take a close look atextrinsic events whose contribution to injuryhas been controversial—the invasion of circu-lating white blood cells into the injured brain.The authors show that much of the damageinduced by cerebral ischemia stems from acti-vation of the adenosine A2A receptor (A2AR)on bone marrow–derived cells, mainly neu-trophils, that enter the brain and enhance theinflammatory response to ischemia.

Prior to these studies, clear-cut evidence fora pathogenic role of infiltrating blood-bornecells had been missing, and the specific factorsregulating their deleterious effect wereunknown. Although there was evidence thatblocking the entry of neutrophils reducedstroke damage3, it had also been proposedthat neutrophils enter the brain ‘after the fact,’mainly to remove dead cells and set the stagefor tissue remodeling4. The findings of Yu etal. directly implicate infiltrating neutrophilsin the mechanisms of the injury and identify

A2ARs as a critical factor in the pathogenicprocess.

The authors created chimeric mice inwhich A2AR-deficient bone marrow cells weretransplanted in wild-type mice and, con-versely, wild-type bone marrow was trans-planted in A2AR-deficient mice. Thisexperimental design allowed the investigatorsto specifically examine the role of A2ARs on

bone marrow–derived cells, including neu-trophils. Mice were then subjected to cerebralischemia by transient occlusion of the middlecerebral artery. Yu et al.2 found that the braininfarct resulting from middle cerebral arteryocclusion was markedly reduced in A2AR-nullmice transplanted with A2AR-null bone mar-row, a finding anticipated based on a previousstudy5. However, in A2AR-null mice trans-

Costantino Iadecola is in the Division of

Neurobiology, Department of Neurology and

Neuroscience, Weill Medical Center of Cornell

University, New York, New York 10021, USA.

e-mail: coi2001@med.cornell.edu

A2A receptor

BM-derived cell

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ATP

ADP

IL1IL6IL12ROS?COX2? Damage

Adenosine

Adenosine Vasodilation (A2A)

aggregation (A2A)

Glu release (A1) Glu release (A2A)

Cerebral blood vessel

Figure 1 Adenosine aggravates neutrophils. Cerebral ischemia induces migration of circulating neutrophilsacross cerebral blood vessels and into the ischemic brain. The ischemia-induced energy deficit also leads torapid breakdown of ATP and extracellular accumulation of large amounts of adenosine. Adenosine receptors(only A1 and A2A receptors are shown for simplicity) are present in brain cells (neurons, glia, vascular cells)and on circulating bone marrow–derived cells, including neutrophils. In this issue, Yu et al. demonstratethat adenosine injures the ischemic brain by activating A2A receptors (A2AR) on infiltrating bonemarrow–derived cells, mainly neutrophils. These receptors contribute to ischemic injury by enhancing theformation of inflammatory mediators, including cytokines (IL-1, IL-6 and IL-12) and, possibly,cyclooxygenase-2 (COX-2) and reactive oxygen species (ROS). Neuronal A2ARs also play a role, seeminglymore minor, by enhancing glutamate (glu) release. The deleterious effects of A2AR activation in neutrophilsovershadow beneficial effects deriving from the cerebrovascular actions of these receptors, and from theattenuation in glutamate release mediated by neuronal A1R.

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