Fever-range thermal stress promotes lymphocyte trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism

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1 Fever-rnge therml stress promotes lymphocyte trfficking cross high endothelil venules vi n interleukin 6 trns-signling mechnism Qing Chen 1, Dniel T Fisher 1, Kristen A Clncy 1, Jen-Mrc M Guguet 2, Wn-Cho Wng 1, Emily Unger 1, Stefn Rose-John 3, Ulrich H von Andrin 2, Heinz Bumnn 4 & Shron S Evns 1 Fever is n evolutionrily conserved response during cute inflmmtion, lthough its physiologicl enefit is poorly understood. Here we show therml stress in the rnge of fever tempertures incresed the intrvsculr disply of two gtekeeper homing molecules, intercellulr dhesion molecule 1 () nd CCL21 chemokine, exclusively in high endothelil venules (HEVs) tht re chief portls for the entry of lood-orne lymphocytes into lymphoid orgns. Enhnced endothelil expression of nd CCL21 ws linked to incresed lymphocyte trfficking cross HEVs. A ifurction in the mechnisms controlling HEV dhesion ws demonstrted y evidence tht the therml induction of ut not of CCL21 involved n interleukin 6 trnssignling pthwy. Our findings identify the HEV xis s thermlly sensitive lert system tht heightens immune surveillnce during inflmmtion y mplifying lymphocyte trfficking to lymphoid orgns. The continuous recircultion of lymphocytes cross specilized high endothelil venules (HEVs) in lymphoid orgns is criticl for the mintennce of immune homeostsis nd immune surveillnce. HEVs lined y cuoidl endothelil cells re distinguished from fltwlled vsculr eds throughout the ody y their ility to support efficient extrvstion of lymphocytes into underlying tissues 1. Lymphocyte entry cross HEVs involves highly ordered sequence of dhesion events tht includes tethering nd rolling long vessel wlls; chemokine-dependent ctivtion; firm rrest; nd trnsendothelil migrtion 2 4. Primry tethering nd rolling of nive nd centrl memory lymphocytes in HEVs of peripherl lymph nodes (s) nd mesenteric lymph nodes (MLNs) is initited vi the enggement of silomucin-like endothelil molecules, collectively clled peripherl lymph node ddressin (PNAd), y L-selectin on lymphocytes 2,3,5. Mucosl ddressin cell dhesion molecule 1 (MAdCAM-1) on HEVs of MLNs nd Peyer s ptches lso supports primry dhesion through interctions with L-selectin or 4 7 integrin on circulting lymphocytes 2. Secondry firm rrest is triggered minly y interctions etween CCL21 chemokine displyed on the lumenl surfces of HEVs with G 1 protein coupled CCR7 chemokine receptors on lymphocytes 2 4. Chemokine ctivtion increses the ffinity of leukocyte function ssocited ntigen 1 (LFA-1) for its endothelil lignds, intercellulr dhesion molecule 1 () nd ICAM-2, which hve redundnt functions during stedy-stte trfficking cross HEVs 2,3. The finl process of trnsendothelil migrtion in HEVs is not fully understood, ut is suggested y in vitro models to involve molecules locted in interendothelil junctions, including, ICAM-2, CD31 nd junctionl dhesion molecules 1 nd 2 (refs. 3,6). Locl nd systemic increses in temperture re crdinl fetures of inflmmtion. The evolutionrily conserved ferile response hs een linked to improved survivl during infection in endothermic nd ectothermic species 7 1. A key unresolved issue reltes to the physiologicl enefit of fever. Studies hve suggested tht ferile tempertures provide dnger signl tht moilizes the entry of lood-orne lymphocytes into secondry lymphoid orgns, where the proility of encountering cognte ntigens or pthogens is enhnced. Tempertures tht mimic ferile episodes (38 4 1C) ct directly on T lymphocytes nd B lymphocytes to enhnce L-selectin-dependent nd 4 7 integrin dependent homing cross lymph node nd Peyer s ptch HEVs Therml modultion of L-selectin dhesion involves tightly orchestrted trns-signling mechnism initited y enggement of the glycoprotein gp13 signl-trnsducing suunit y interleukin 6 (IL-6) nd solule form of the IL-6 receptor- inding suunit (sil-6r) 13. Those findings support the present view tht IL-6 trns signling provides moleculr switch tht governs lymphocyte trfficking during cute inflmmtion or chronic inflmmtory disorders 14,15. Although ferile tempertures cn improve the vsculr delivery of inflmmtory cells to tissues y regulting hemodynmic prmeters 1 Deprtments of Immunology, Roswell Prk Cncer Institute, Bufflo, New York 14263, USA. 2 The CBR Institute for Biomedicl Reserch nd Deprtment of Pthology, Hrvrd Medicl School, Boston, Msschusetts, 2115, USA. 3 Deprtment of Biochemistry, Christin Alrechts University Kiel, D-2498 Kiel, Germny. 4 Moleculr nd Cellulr Biology, Roswell Prk Cncer Institute, Bufflo, New York 14263, USA. Correspondence should e ddressed to S.S.E. (shron.evns@roswellprk.org). Received 19 June; ccepted 4 Octoer; pulished online 5 Novemer 26; doi:1.138/ni146 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1

2 PP SPL Liver Pnc such s vsodiltion nd lood flow, the contriution of therml stress to endothelil dhesion remins mostly unexplored. The inding function of gtekeeper cuoidl HEVs is ugmented y therml stress in the rnge of fever tempertures in vivo 16,17,wheresferile tempertures do not influence the ility of squmous endothelium of nonlymphoid orgns to support leukocyte dhesion in vivo or in vitro 16,18,19. Here we report tht the therml element of fever controls the moleculr mechnisms tht underlie firm dhesion nd TRITC PNAd (6 ) Cells ound/hev Cells/HEV Cells in strom/unit re B6 C3H MLN SCID Time (min) BALB/c 6 PBS TNF BALB/c Figure 1 Fever-rnge therml stress enhnces HEV dhesion nd homing of lymphocytes to lymphoid orgns with HEV structures. () In vitro frozensection dherence ssy of the inding of splenocytes to HEVs in pooled s from individul normothermic control mice (), -treted mice or TNF-treted mice. Arrowheds in representtive photomicrogrphs (left) indicte toluidine-stined splenocytes ound to individul HEVs in s from BALB/c mice. Right, quntifiction (men ± s.e.m.) of cells ound to HEVs (n ¼ HEVs per mouse). B6, C57BL/6; SCID, severe comined immunodeficiency. () In vivo short-term (1-hour) homing of fluorescence-leled splenocytes to secondry lymphoid orgns (pooled s, MLNs, Peyer s ptches (PP) nd spleen (SPL)) nd nonlymphoid orgns (liver nd pncres (Pnc)) in individul normothermic control BALB/c mice or -treted BALB/c mice. Left, representtive photomicrogrphs of s. Right, quntifiction (men ± s.e.m.) of clcein-leled cells in orgn cryosections, y fluorescence microscopy (n ¼ 1 fields per mouse)., P o.1, versus. Scle rs (,), 5 mm. Dt re representtive of three or more () or ten() independent experiments. trnsendothelil migrtion of lood-orne lymphocytes cross HEVs. We identified n integrted function for IL-6 trns signling s regultor of trfficking y demonstrting tht IL-6 sil-6r medited therml induction of -dependent extrvstion of lymphocytes selectively in HEVs of lymphoid orgns. Our results support the ide tht HEVs ct s sentinels during ferile inflmmtory responses y heightening trfficking of nive nd centrl memory lymphocytes to secondry lymphoid orgns. RESULTS Therml stress stimultes lymphocyte-hev interctions We used severl experimentl pproches to ssess the effects of therml stress on HEV dhesion independently of other fctors tht my lso influence trfficking, such s hemodynmic prmeters. In the first series of studies, we treted mice for 6 h with whole-ody hyperthermi () to rise the core temperture to the rnge of fever tempertures ( fever-rnge ; 39.5 ±.5 1C). We quntified the inding ctivity of individul HEVs y n in vitro ssy in which we llowed splenocytes (from untreted mice) to dhere to HEVs in II III V I II SEV V IV II SEA IV III II Sticking frction (%) Rolling frction (%) I II III IV Venulr order V Figure 2 Fever-rnge therml stress enhnces lymphocyte-hev interctions. () Kinetic nlysis of lymphocyte homing. s were isolted 5 6 min fter doptive trnsfer of TRITC-leled splenocytes (red) into normothermic control BALB/c mice or -treted BALB/c mice. Tissue cryosections (left) re stined with ma specific for PNAd to visulize HEVs (green) nd re representtive photomicrogrphs of TRITC-leled cells ssocited with HEVs or infiltrted into the tissue prenchym t 6 min (6 ); right, quntifiction of those cells y fluorescence microscopy. Dt (men ± s.e.m.) re of ten fields nlyzed of pooled s from individul mice nd re representtive of three or more independent experiments. () Intrvitl microscopy (left) of the interctions of clcein-leled splenocytes with the lymph node venulr tree of -treted mouse, showing the vsculr structure, including the superficil epigstric rtery (SEA), superficil epigstric vein (SEV) nd venulr rnches (I V) in n inguinl lymph node (Supplementry Video 1 online). Right, rolling frctions nd sticking frctions in normothermic control nd -treted C57BL/6 mice. Dt (men ± s.e.m.) re from three mice per tretment group in three independent experiments. Scle rs, 5 mm () or 2 mm ()., P o.1, nd, P o.1, normothermic versus. 2 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

3 CCL21 ICAM-2 PNAd Merged Liver Figure 3 Fever-rnge therml stress selectively increses the expression of nd CCL21 on HEVs. Expression of trfficking molecules in normothermic control or -treted BALB/c mice. () Scnning confocl microscopy of cryosections dully stined for (red) nd PNAd (green). () Intrvsculr stining of, CCL21 or ICAM-2 in s or liver y intrvenous injection of primry ntiody into mice fter het tretment; orgns otined 2 min lter were counterstined with fluorochrome-conjugted secondry ntiody. There ws no immunofluorescence stining with isotype-mtched control ntiody (dt not shown). Scle rs (,), 5 mm. Arrowheds indicte HEVs with wek stining of or CCL21. (c) Quntittive imge nlysis of the immunofluorescence intensity of PNAd (from tissue-section stining) nd, CCL21 nd ICAM-2 (from intrvsculr stining) in PNAd + cuoidl HEVs. Horizontl xes, fluorescence intensity; verticl xes, pixels with ech intensity; numers in plots, men fluorescence intensity. All dt re representtive of three or more independent experiments. orgn cryosections from control mice t norml temperture ( normothermic mice) or het-treted mice. Fever-rnge incresed the ility of HEVs to support lymphocyte dhesion in severl immunocompetent mouse strins (C57BL/6, C3H nd BALB/c) s well s in mice with severe comined immunodeficiency tht lcked B lymphocytes nd T lymphocytes (Fig. 1). We detected similr increses in HEV dhesion in response to therml stress or to the dministrtion of recominnt tumor necrosis fctor (TNF; Fig. 1), potent stimultor of vsculr dhesion in vitro nd in vivo 2. We next exmined the therml regultion of lymphocyte trfficking in short-term (1-hour) in vivo homing ssys in which we leled splenocytes ex vivo with fluorescent trcking dyes (TRITC or clcein) nd then injected the cells intrvenously into normothermic control or -treted BALB/c mice. We llowed recipient mice to equilirte to norml ody temperture efore doptively trnsferring leled cells to reverse the therml effects on lood flow nd to void het ctivtion of L-selectin or 4 7 integrin dhesion in leled indictor cells Therml stress cused n increse of pproximtely twofold in the trfficking of leled cells selectively in secondry lymphoid orgns with HEVs (s, MLNs nd Peyer s ptches) ut not in spleen or extrlymphoid orgns (such s liver or pncres), which lck HEV structures (Fig. 1 nd Supplementry Fig. 1 online). tretment did not lter the composition of cells tht trfficked to s, suggesting tht homeosttic trfficking mechnisms were mintined. Thus, therml stress did not result in enrichment for specific leukocyte popultions tht gined entry to s, including T cells (CD4 + nd CD8 + ) tht trffic more efficiently thn B cells (B22 + ) 21,22, or monocytes (CD11 hi Gr1 lo ) nd neutrophils (Gr1 hi ), which were essentilly excluded 2,3 (Supplementry Fig. 1). Nive cells (L-selectinhigh CD44 lo ) or centrl memory cells (L-selectin-high CD44 hi ) constituted the min T cell susets tht homed to s in control nd hyperthermic conditions, wheres effector-memory T cells (L-selectinlow CD44 hi ; Supplementry Fig. 1) nd ctivted T cell popultions (L-selectin-low CXCR3 hi CCR7 lo ; dt not shown) were excluded from this site. We ssessed the influence of therml stress on the kinetics of lymphocyte interctions with gtekeeper HEVs fter intrvenous injection Fluorescence of fluorescence-leled splenocytes into normothermic or treted mice. We counterstined tissue sections with monoclonl ntiodies (mas) specific for molecules on HEVs (PNAd) or Peyer s ptch HEVs (MAdCAM-1) to determine whether leled cells were locted in HEVs or were extrvsted into the tissue prenchym. We found tht tretment incresed the numer of lymphocytes ssocited with HEVs (Fig. 2) nd Peyer s ptch HEVs (Supplementry Fig. 2 online) within 5 min of trnsfer nd tht the increse in lymphocyte-hev interctions ws sustined over 1 h. Notly, more lymphocytes completed the multistep process culminting in extrvstion into the strom of s or Peyer s ptches y 3 min in -treted mice thn in normothermic control mice t 1h(P o.2; Fig. 2 nd Supplementry Fig. 2) PTX: CCL21: CCL21 A: c Pixels PNAd : 9 : 88 CCL : 64 : 11 + : 48 : 98 ICAM-2 : 75 : 78 Figure 4 CCL21 is required for the therml stimultion of lymphocyte trfficking cross HEVs. Short-term (1-hour) homing of TRITC-leled splenocytes. Cells were pretreted with (+) or without ( ) pertussis toxin (PTX) or were pretreted with (+) or without ( ) desensitizing concentrtion of CCL21 efore doptive trnsfer into normothermic control or -treted BALB/c mice. Alterntively, mice were treted for 2 min with (+) or without ( ) CCL21-neutrlizing ntiody (CCL21 A) efore cell trnsfer. Isotypemtched control ntiody did not ffect lymphocyte homing (dt not shown). Cryosections of pooled s from individul mice were counterstined for PNAd, nd TRITC-leled cells infiltrting the prenchym were quntified y fluorescence microscopy (n ¼ 1 fields per mouse)., P o.1, function-locking regent versus untreted control. Dt (men ± s.e.m.) re representtive of three or more independent experiments. NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 3

4 c ma: ICAM-2 ma: MLN PP SPL MLN PP SPL ma: MLN PP SPL ICAM-2 ma: Control α-tnf α-il-1β α-ifn-γ α-il-6 Figure 5 is required for the therml enhncement of trfficking cross HEVs. Short-term (1-hour) homing of TRITC-leled splenocytes in normothermic control or -treted BALB/c mice. At 2 min efore doptive trnsfer, recipient mice were treted with function-locking mas (+) specific for nd ICAM-2 () or for only () or ICAM-2 only (c) or with isotype-mtched control ntiody ( ). TRITC-leled cells in tissue cryosections from individul mice were quntified y fluorescence microscopy (n ¼ 1 fields per mouse)., P o.1, function-locking ma versus control ntiody. Dt (men ± s.e.m.) re representtive of three or more independent experiments. We used intrvitl microscopy of inguinl lymph nodes to pinpoint the nture of the dhesive interctions regulted y therml stress (Fig. 2). High-order (III V) postcpillry HEVs detected in this orgn preprtion reside in the prcorticl region, wheres low-order (I nd II) flt-wlled venules drin into the superficil epigstric vein in the medull 23. pretretment did not ffect the frequency of rolling interctions throughout the venulr tree (Fig. 2). However, het tretment sustntilly incresed the frction of cells tht trnsitioned from primry rolling to secondry firm sticking exclusively in order III V venules (HEVs). The velocity of nonintercting lymphocytes in order III venules ws equivlent in normothermic nd treted mice (1,195 ± 34 mm/s nd 1,13 ± 549 mm/s, respectively (men ± s.e.m.); n 4 6 cells nlyzed in three experiments), indicting tht the increse in sticking interctions could not e ttriuted to sustined effects of het pretretment on lood flow. Thus, therml stimultion of lymphocyte homing in lymphoid orgns is correlted with enhnced secondry firm dhesive interctions selectively in HEVs. Therml stress enhnces nd CCL21 disply in HEVs To investigte the moleculr sis of the therml stimultion of lymphocyte-hev interctions, we exmined the expression of trfficking molecules known to prticipte in the multistep dhesion sequence in HEVs. Two-color confocl immunofluorescence microscopy of tissue cryosections showed tht fever-rnge therml stress did not lter the reltive expression of PNAd or MAdCAM-1, which medite primry tethering nd rolling in or Peyer s ptch HEVs, respectively (Fig. 3 nd Supplementry Fig. 2). In contrst, the expression of, which prticiptes in secondry dhesion nd trnsendothelil migrtion, ws enhnced considerly y het in PNAd + nd MAdCAM-1 + HEVs (Fig. 3 nd Supplementry Fig. 2). We detected similr stining in HEVs fter therml stress or systemic dministrtion of recominnt TNF (Supplementry Tle 1 online). Although TNF tretment upregulted indiscrimintely on the vessels of ll orgns exmined, the response to therml stress ws site specific, such tht only HEVs of lymphoid 6 Cells oud/hev A: Control TNF IL-1β IFN-γ IL-6 Figure 6 IL-6 medites the therml stimultion of expression nd homing of lymphocytes cross HEVs. Cytokine-neutrlizing ntiodies to TNF, IL-1, interferon-g (IFN-g) or IL-6 or isotypemtched control ntiody (Control) were dministered intrvenously 3 min efore tretment of BALB/c mice. () Immunofluorescence stining of (red) in cryosections. Arrowheds indicte HEVs with wek stining., ntiody to. Scle r, 5 mm. () In vitro dherence of splenocytes to HEVs. Dt re men ± s.e.m. of HEVs nlyzed in pooled cryosections from individul mice. (c) Short-term (1-hour) homing of clcein-leled splenocytes in tissue cryosections from individul mice, quntified y fluorescence microscopy. Dt re men ± s.e.m. of ten fields nlyzed from individul mice. A, ntiody., P o.1, neutrlizing ntiody versus control ntiody. Dt re one representtive stining () or experiment (,c) of three or more independent experiments. c MLN PP SPL IL-6 A: ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

5 WT CCL21 Il6 / WT Il6 / WT Il6 / 5 Pixels : 44 : 96 Fluorescence : 46 : 47 : 45 : 74 Fluorescence orgns hd incresed expression (Supplementry Tle 1). tretment did not lter the vsculr expression of other dhesion molecules tht medite trnsendothelil migrtion, including CD31 (Supplementry Tle 1) nd junctionl dhesion molecules 1 nd 2 (dt not shown). Therml stress lso did not modify the HEV expression of molecules ssocited with inflmmtory responses, such s vsculr cell dhesion molecule 1 (Supplementry Tle 1), E-selectin nd Duffy ntigen relted receptor for chemokines (dt not shown), which hs een linked to regultion of the ctivity nd/or vilility of inflmmtory chemokines (CXCL1, CXCL5, CCL2, CCL5 nd CCL7) ut not of homeosttic chemokines (CCL21, CCL19, CXCL12 nd CXCL13) 3,4,24. We did intrvsculr stining in situ y injecting -specific ma into the vsculr comprtments of recipient mice, then counterstining tissue sections with fluorescence-leled detection ntiody. Hyperthermi ugmented expression on the lumenl nd junctionl surfces of high endothelil cells (HECs; Fig. 3), which re the contct sites for lood-orne lymphocytes undergoing firm dhesion or trnsendothelil migrtion. Therml stress lso sustntilly incresed intrvsculr presenttion of the homeosttic chemokine CCL21 (Fig. 3), the principl chemokine required for the trnsition of lymphocytes from primry rolling interctions to secondry rrest in HEVs, without ffecting the wek-to-nondetectle stining for other homeosttic chemokines (CCL19, CXCL12 nd CXCL13) on HEVs (dt not shown). Quntittive imge nlysis showed tht therml stress incresed the men fluorescence stining intensity for nd CCL21 on PNAd + cuoidl HEVs y out 2-fold nd 1.5-fold, respectively, wheres PNAd nd ICAM-2 stining ws unchnged (Fig. 3c). The finding tht the intrvsculr density of ICAM-2 ws not ltered estlished tht pretretment did not generlly increse the ccess of mas to epitopes in the HEV microcomprtment (Fig. 3,c). There ws no chnge in the lumenl presenttion of (Fig. 3) or CCL21 (dt not shown) on the vessels of extrlymphoid orgns such s liver. Consistent with the oservtion tht therml stress ltered nd CCL21 expression only in HECs tht constitute Pixels : 46 : 76 SPL SPL Figure 7 Therml induction of nd lymphocyte homing does not occur in IL-6-deficient mice. () Intrvsculr stining of (red) nd CCL21 (green) in s from normothermic control or -treted Il6 / nd wild-type (WT) C57BL/6 mice. Arrowheds indicte HEVs with wek stining of or CCL21. Scle rs, 5 mm. Bottom, quntittive imge nlysis of nd CCL21 stining: horizontl xes, fluorescence intensity; verticl xes, pixels with ech intensity; numers in plots, men fluorescence intensity. () Short-term (1-hour) homing of TRITC-leled splenocytes in tissue cryosections from individul Il6 / nd wild-type mice with () or without () tretment, quntified y fluorescence microscopy. Dt re men ± s.e.m. of ten fields nlyzed from individul mice., P o.1, versus. All dt re representtive of three or more independent experiments. minor popultion in nodl tissues (less thn 4% of stroml cell rich frctions 25 ), we did not detect chnge in the totl mounts of those proteins in whole- lystes (Supplementry Fig. 3 online). These dt suggested tht incresed intrvsculr disply of CCL21 nd contriutes to enhnced lymphocyte trfficking cross HEVs in response to therml stress. Enhnced homing depends on CCL21 nd We used multipronged pproch to determine the reltive contriutions of CCL21 nd its lignd, CCR7, to improved trfficking y therml stress. We initilly used pertussis toxin, n irreversile inhiitor of G 1 protein signling, to glolly inhiit chemokinedependent entry of lymphocytes into lymphoid orgns. Pretretment of fluorescence-leled splenocytes with pertussis toxin fully locked their ility to extrvste in s in oth normothermic nd hyperthermic conditions (Fig. 4). To directly trget the CCR7 chemokine receptor for CCL21, we pre-exposed fluorescence-leled splenocytes to high concentrtion of recominnt CCL21 (1 mg/ ml) ex vivo efore trnsfer into recipient mice (Fig. 4). Tht tretment did not lter the surfce expression of L-selectin or LFA-1 on lymphocytes (dt not shown). As reported efore 26, CCR7 desensitiztion y tht strtegy reduced the homing of lymphocytes to s (Fig. 4) nd Peyer s ptches (dt not shown) in normothermic conditions, leit only prtilly, which proly reflected incomplete desensitiztion or resensitiztion in vivo. The extent of reduction in lymphocyte trfficking ws similr in normothermic nd -pretreted mice, indicting common CCR7-dependent mechnism during trfficking cross HEVs in oth temperture conditions. Lymphocyte extrvstion in control nd het-treted mice ws lso reduced sustntilly y complementry strtegy trgeting CCL21 in vivo with functionlocking ntiody (Fig. 4). We conclude tht CCR7-CCL21 is the min chemokine receptor chemokine pir responsile for improved lymphocyte entry cross HEVs in response to therml stress. Those oservtions rised the issue of whether CCL21 induction y therml stress ws the sole determinnt of physiologicl importnce for homing in HEVs or if het-inducile contriuted to this NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5

6 CCL21 PBS sgp13 PBS sgp13 c Cells ound/hev MLN PP SPL sgp13: + + sgp13: Figure 8 IL-6 trns signling medites the therml enhncement of expression nd lymphocyte homing in HEVs. ( c) Immunofluorescence (), dherence () nd homing (c) of cells from normothermic control nd -treted BALB/c mice injected intrvenously with solule gp13 (sgp13; +) or vehicle control (PBS; ) 3 min efore initition of tretment. () Immunofluorescence stining of (red) nd CCL21 (green) in cryosections. () In vitro dherence of splenocytes to HEVs. Dt re men ± s.e.m. of HEVs nlyzed in pooled cryosections from individul mice. (c) In vivo short-term (1- hour) homing of clcein-leled splenocytes to lymphoid orgns from individul mice., P o.1, solule gp13 versus PBS control. Dt re men ± s.e.m. of ten fields nlyzed from individul mice. (d) Intrvsculr stining of in s nd process. We ddressed tht issue y ssessing the reltive requirements for LFA-1 nd its cognte receptors, nd ICAM-2, in shortterm homing studies. Comined ma lockde of nd ICAM-2 reduced lymphocyte trfficking cross HEVs considerly in oth normothermic nd hyperthermic conditions (Fig. 5), prlleling results otined with n LFA-1-locking ma (dt not shown). Independent ma lockde of or ICAM-2 hd only modest effects on trfficking to lymphoid orgns in normothermic conditions (Fig. 5,c), supporting reports tht those molecules cn sustitute for ech other s HEV lignds for LFA-1 during stedy-stte homing in HEVs 27,28. Interference with ctivity, either y neutrlizing ma (Fig. 5) or y genetic trgeting in -deficient mice (Supplementry Fig. 4 online), completely prevented the therml stimultion of trfficking to s, MLNs nd Peyer s ptches. In contrst, functionl inhiition of ICAM-2 filed to reduce trfficking cross HEVs in -treted mice (Fig. 5c). We confirmed the requirement for in het-induced HEV dhesion y frozen-section in vitro dherence ssy (Supplementry Fig. 4). Although this ssy minly mesures PNAd-dependent dhesion in HEVs in norml conditions 5, LFA-1 ICAM dependent inding interctions hve een chrcterized in HEVs of inflmed lymph nodes with this method 29. Evidence tht HEVs of control nd hyperthermi-treted mice supported equivlent PNAd-dependent dhesion fter concomitnt lockde of nd ICAM-2 in frozen-section ssys (Supplementry Fig. 4) ws consistent with intrvitl studies (Fig. 2) demonstrting tht therml stress did not ugment primry dhesive interctions in HEVs. These results collectively demonstrted tht is the preferentil inding prtner d Control Pncres e Pixels for LFA-1 in HEVs in therml conditions nd tht high-density presenttion of CCL21 is not sufficient, in the sence of, to support improved trfficking to lymphoid orgns. IL-6 trns signling medites enhnced trfficking Inflmmtory cytokines, including TNF, IL-1, interferon-g nd IL-6, re potent stimultors of endothelil expression of nd inflmmtory chemokines in vitro or in vivo 3 (Supplementry Tle 1). To determine if those cndidte cytokines regulte HEV dhesion during therml stress, we treted mice with cytokineneutrlizing mas efore inititing nd then exmined expression of or CCL21 y HEVs. There ws therml induction of in HEVs (Fig. 6) nd Peyer s ptch HEVs (dt not shown), despite the presence of mas specific for TNF, IL-1 or interferon-g, wheres there ws no induction in mice treted with n IL-6 function locking ma. Similrly, only IL-6-neutrlizing ma locked the therml enhncement of HEV dhesion detected y frozen-section in vitro dherence ssys (Fig. 6). The IL-6-neutrlizing ma lso prevented the therml stimultion of lymphocyte homing cross HEVs in s, MLNs nd Peyer s ptches (Fig. 6c). Improved trfficking ws not ccompnied y mesurle increse in totl nodl content of IL-6 during het tretment (IL-6 in s of normothermic control mice nd in mice fter tretment t 2, 4 nd 6 h: 1.1 ±.2 pg/mg totl protein,.9 ±.2 pg/mg totl protein,.6 ±.1 pg/mg totl protein nd 1. ±.2 pg/mg totl protein, respectively (men ± s.e.m.); n ¼ 3 mice). We confirmed the requirement for IL-6 in IL-6-deficient mice, s therml stress filed to induce expression or lymphocyte trfficking in HEVs in Sline ActD Fluorescence : 45 : 1 H-IL-6: 11 : 44 : 46 H-IL-6: 43 Sline ActD CCL21 : 62 : 95 H-IL-6: 6 : 61 : 59 H-IL-6: 6 pncret from BALB/c mice treted with hyper-il-6 (H-IL-6) nd from control BALB/c mice. (e) Imge nlysis quntifiction of the intrvsculr stining intensity of nd CCL21 in HEVs of pooled s from individul BALB/c mice given ctinomycin D (ActD) or vehicle control (Sline) intrperitonelly 3 min efore tretment for 6 h with or hyper-il-6. Horizontl xes, fluorescence intensity; verticl xes, pixels with ech intensity; numers in plots, men fluorescence intensity. Arrowheds (,d) indicte HEVs with wek stining of or CCL21; scle rs (,d), 5 mm. Dt re one representtive stining (,d) or experiment (,c,e) of three or more independent experiments. 6 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

7 these mice (Fig. 7,). In contrst, IL-6-deficient mice (Fig. 7) nd wild-type mice treted with IL-6-neutrlizing ma (dt not shown) hd norml CCL21 induction in response to therml stress. IL-6 regultion of occurs in primry venulr endothelil cells in vitro tht lck the memrne form of the IL-6R inding suunit y mens of trns-signling mechnism tht depends on the vilility of oth IL-6 nd solule form of IL-6R 14,31,32.Totest whether IL-6 trns signling contriutes to -medited homing during therml stress in vivo, we dministered recominnt solule gp13 efore tretment. Solule gp13 competitively inhiits trns signling y IL-6 sil-6r without interfering with the clssicl signling pthwy medited y memrne-nchored IL-6R 14,15. Solule gp13 fully locked the therml induction of expression on HEVs ut did not prevent CCL21 upregultion (Fig. 8). Moreover, disruption of IL-6 trns signling y solule gp13 prevented the therml enhncement of HEV dhesion detected in frozen-section in vitro dherence ssys s well s trfficking cross HEVs in vivo without ffecting the seline ctivity of HEVs in normothermic conditions (Fig. 8,c). To determine whether therml regultion of trfficking molecules in HEVs involves trnscriptionl control, we treted mice with ctinomycin D, phrmcologicl inhiitor of trnscription in vivo 33.We mpped the requirement for trnscriptionl events in the IL-6 trnssignling pthwy leding to induction in prllel studies tht exmined the effect of ctinomycin D on IL-6 trns-signling responses triggered directly y hyper-il-6, recominnt fusion protein in which IL-6 is covlently linked to sil-6r 34. We detected similr induction on HEVs in response to hyper-il-6 or therml stress (Fig. 8d,e). However, in contrst to the site-specific response of therml stress, ws lso widely induced y hyper-il-6 on vessels of extrlymphoid orgns (such s pncres, liver, kidney nd hert; Fig. 8d nd dt not shown). Actinomycin D rogted upregultion t ll vsculr sites in response to therml stress or hyper-il-6 (Fig. 8e nd dt not shown) without disrupting cellulr processes tht do not depend on new trnscription, such s ctivtion of downstrem signling molecules (such s STAT3) y hyper-il-6 (dt not shown). The therml enhncement of intrvsculr CCL21 in HEVs ws lso sensitive to ctinomycin D tretment (Fig. 8e). Consistent with the oservtion tht the therml upregultion of CCL21 did not involve endogenous IL-6 trns-signling mechnisms, dministrtion of hyper-il-6 filed to increse the presenttion of CCL21 on HEVs (Fig. 8e). Overll, these dt support model wherey the vsculr-specific effects of fever-rnge therml stress on ICAM- 1-medited lymphocyte trfficking cross HEVs re orchestrted y IL-6 trns signling, wheres IL-6 is dispensle for enhnced intrvsculr disply of CCL21 (Supplementry Fig. 5 online). DISCUSSION Secondry lymphoid orgns re strtegiclly positioned to provide the first line of defense ginst invding pthogens. Microes entering the skin re source of ntigens tht re trnsported y Lngerhns cells to drining lymph nodes through the fferent lymphtics. These ntigen-presenting cells loclize proximl to HEVs 3, the min gtewys for recirculting B lymphocytes nd T lymphocytes. Enteric ntigens hve immedite ccess to gut-ssocited Peyer s ptches nd MLNs. The continul flux of pthogen-derived ntigens nd immune cells through lymphoid orgns increses the proility tht cognte ntigens will e encountered y rre ntigen-specific lymphocytes present t frequency of only 1 in to cells. Our study hs provided insight into the eneficil mechnism of ction of the therml component of fever tht hs generlly een relegted to ystnder function during infection nd inflmmtion. A centrl finding ws tht fever-rnge therml stress ugmented the cpture efficiency of gtekeeper HEVs for nive nd centrl memory lymphocytes y incresing the intrvsculr density of CCL21 nd. Notly, the pproximtely twofold increse in recruitment induced y therml stress represented profound enhncement in n lredy efficient process in which out one of every four lymphocytes tht enter HEVs completes the multistep cscde leding to extrvstion 1,3. Thus, the cute effects of fever on lymphocyte trfficking would e predicted to enhnce sustntilly the repertoire of ntigenspecific B lymphocytes nd T lymphocytes tht screen lymphoid orgns for the presence of foreign ntigens. Tht mechnism complements the stimultory effects of therml stress on the migrtion of skin-derived Lngerhns cells to lymph nodes 1,35. Our findings expnd on the long-recognized function of lymph nodes in moilizing the immune system during inflmmtion 3,36. Locl relese of TNF or CCL4 y mst cells t sites of cteril chllenge or ntigen stimultion enhnces T cell entry into drining lymph nodes 37,38. Virl infection hs lso een reported to mplify CD4 + nd CD8 + T cell recircultion y incresing the dimeter of the feeding rterioles tht supply postcpillry HEVs in drining lymph nodes (from out 1 mm to15mm) 39. A fundmentl difference etween our findings nd pulished results otined in inflmed lymph nodes reltes to the scope of the vsculr response. Locl inflmmtion t sites of infection or ntigen chllenge influences trfficking only in drining lymph nodes 3,36. In contrst, fever-rnge, which mirrors the therml component of systemic fever, ugments the recruitment properties of the HEV xis throughout the ody. Heightened immune surveillnce of distl secondry lymphoid orgns during fever would provide sustntil dvntge y protecting the host ginst widespred dissemintion of rpidly multiplying infectious gents. A notle distinction is tht systemic therml stress mintins homeosttic trfficking mechnisms, wheres locl tissue inflmmtion opens the gtewy for CCR7 cell types normlly excluded from trfficking cross HEVs in drining lymph nodes 3,36. Thus, therml stress minly improves the recruitment of nive nd centrl memory CD4 + nd CD8 + T cells nd B cells cross lymph node HEVs without disproportiontely enhncing the homing of monocytes, neutrophils, effector-memory T cell susets or ctivted T cells. The mintennce of homeosttic lymphocyte recircultion y therml stress is explined y the nture of the trfficking molecules upregulted on HEVs. Fever-rnge preferentilly enhnced the intrvsculr density of two molecules involved in homeosttic trfficking (CCL21 nd ). Those dt re consistent with in vitro studies showing tht CCL21 nd ct coopertively to optimize LFA-1 inding ctivity CCL21 functions in dosedependent wy in vitro to ctivte inside-out signling, leding to lignd-independent conformtionl chnges tht convert LFA-1 from low-ffinity stte to n intermedite-ffinity stte 4,42. Trnsition of LFA-1 to high-ffinity stte depends on outside-in signling initited y enggement of LFA-1 y through mechnism tht is highly dependent on density 4,41. A centrl finding of our study ws tht CCL21 ws necessry ut not sufficient to support improved trfficking cross HEVs in response to therml stress. Interference with -dependent dhesion rogted the therml effects on trfficking despite high intrvsculr density of CCL21 nd the vilility of ICAM-2. The criticl requirement for during lymphocyte egress cross HEVs demonstrted y therml stress ws unexpected, given tht nd ICAM-2 cn sustitute for ech other during stedy-stte trfficking in normothermic conditions 27,28. Fundmentl differences NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 7

8 etween nd ICAM-2 my explin why is the preferentil inding prtner for LFA-1 during therml responses. The high intrvsculr density of resulting from therml stimultion in vivo could theoreticlly fvor the formtion of stle dimers nd higher-order multimers, s demonstrted iochemiclly in endothelil cells in vitro fter upregultion y TNF or y genetic overexpression 43,44. Notly, ICAM-2 cnnot form dimers, which my relte to intrinsic differences in the hydrophoicity of the N-terminl regions of ICAM-2 nd (refs. 43,45). dimer formtion reinforces moleculr interctions with high-ffinity LFA-1 conformers; thus, the ond lifetimes of monomeric nd dimeric for LFA-1 re out 25 s nd 33 s, respectively 46. The regultion of density y therml stress my lso ffect its ility to redistriute to cup-like structures tht re proposed to provide trction for cells undergoing trnsmigrtion 47,48. In contrst to, ICAM-2 is present in only moderte mounts in endothelil microdomins juxtposed to LFA-1 on migrting lymphocytes 47. Our studies here hve demonstrted nonredundnt function for IL-6 trns signling in controlling lymphocyte ccess to lymphoid orgns during therml stress. Disruption of IL-6 trns-signling mechnisms prevented the therml upregultion of on HEVs nd enhncement of lymphocyte homing. It remins to e determined if incresed expression results from the direct enggement of gp13 molecules on HECs y IL-6 sil-6r nd susequent STAT3-medited ctivtion of the promoter, s reported for primry endothelil cells (humn umilicl vein endothelil cells) in vitro 3 32, or if other IL-6-responsive intermediry cells prticipte in tht response. The finding tht ctinomycin D inhiited upregultion on HEVs y oth therml stress nd hyper-il-6 indicted trnscriptionl component of the pthwy downstrem of gp13 ligtion y IL-6 nd sil-6r tht controls new synthesis. Our results excluding the possiility of involvement of IL-6 sil-6r during therml induction of the homeosttic chemokine CCL21 re not entirely unexpected, s IL-6 trns signling hs een linked minly to regultion of the production of inflmmtory chemokines (CCL2, CCL8, CXCL5 nd CXCL6) in vitro or in vivo 14,15. The mechnisms controlling CCL21 expression on the lumenl surfce of HEVs re unknown 3,4.Dtindictingthtthe therml control of intrvsculr presenttion of CCL21 depended on newly initited trnscriptionl events without ltering the totl nodl content of CCL21 re consistent with scenrio in which chemokine synthesis is induced only in limited cell popultions (such s HECs or perivsculr cells) or in which het controls the trnscription of s-yetunidentified molecules involved in the trnsport, presenttion or turnover of tht homeosttic chemokine 3,4. Mny lines of evidence indicte tht the IL-6 trns-signling response initited y therml stress involves locl microenvironmentl control of dhesion in discrete vsculr eds. Notly, the therml effects on firm sticking of lymphocytes were restricted to high-order (III V) venules (HEVs) in s. Tht result ws in contrst to studies demonstrting downstrem low-order (I II) segments in the sme venulr tree tht were refrctory to therml stress. Immunolocliztion studies estlished tht upregultion of occurred solely in the numericlly minor popultion of HECs without ffecting the totl content in nodl tissues. Moreover, ws not induced indiscrimintely y hyperthermi in vessels of extrlymphoid orgns. A possile key to the tight control of vsculr-specific responses lies in the dul requirement for IL-6 nd sil-6r in the initition of therml responses. Notly, the squmous endothelium of extrlymphoid orgns is le to upregulte in response to IL-6 trns signling initited y hyper-il-6. One possile explntion for the finding tht therml upregultion of occurs exclusively in HEVs without ffecting the overll IL-6 concentrtion in lymphoid orgns is tht site-specific vsculr responses to hyperthermi re dictted y the locl iovilility of IL-6 nd/or sil-6r in microntomiclly restricted sites. Mny cell types re potentil source of IL-6 in lymphoid orgns, including monocytes, lymphocytes, dendritic cells, endothelil cells, firolsts or pericytes locted in the firoreticulr network surrounding HEVs, wheres sil-6r derives minly from leukocytes 13,49. In conclusion, we hve provided evidence tht tempertures in the rnge of physiologicl fever ct s n lert system to enhnce the frequency of the homeosttic recircultion of lymphocytes cross HEVs. The ferile response involves n integrted function for IL-6 trns signling in controlling not only HEV dhesion, s reported here, ut lso the inding function of the L-selectin homing receptor in lymphocytes 9,13,17. Our findings enlrge on the function of IL-6 trns signling in controlling lymphocyte trfficking during cute nd chronic inflmmtion 14,15.IL-6 sil-6r ctivity hs een linked to T cell recruitment t sites of cute inflmmtion s well s to the pthogenesis of chronic inflmmtory disorders, including inflmmtory owel disese, rheumtoid rthritis, peritonitis nd dietes 14,15. Thus, our finding tht IL-6 regultes -dependent lymphocyte trfficking in the context of cute ferile responses my hve rod relevnce to the mechnisms underlying chronic inflmmtion. METHODS Mice. The following ge-mtched (8 12 weeks of ge) femle mice were purchsed from Jckson Lortory or Tconic: BALB/c, C3H, C57BL/6, severe comined immunodeficient (BALB/c ckground), IL-6-deficient (B6.129S2- Il6 tm1kopf /J on C57BL/6 ckground) nd -deficient (Icm1 tmjegr /J on C57BL/6 ckground). Mice were mintined in pthogen-free rrier conditions. All niml protocols were pproved y the Roswell Prk Cncer Institutionl Animl Cre nd Use Committee. Tretment with fever-rnge, cytokine-neutrlizing regents, recominnt cytokines or trnscription inhiitor. Mice were treted with fever-rnge (core temperture of 39.5 ±.5 1C for 6 h) y eing plced in n environmentl chmer t C (model BE5; Memmert) s descried 16,35. Normotherml control mice (core temperture, 36.8 ±.2 1C) were mintined t 22 1C in drkened cinet for the experimentl period. Mice were injected intrperitonelly with 1 ml sterile sline to void dehydrtion, nd the core tempertures of sentinel mice in ll experimentl groups were monitored with sucutneously implnted microchip thermotrnsponder (14 mm 2.2 mm; implnted 1 week or more efore tretment) nd progrmmle dt-cquisition system (Bio Medic Dt Systems). Neutrlizing mas specific for IL-6, TNF, IL-1, interferon-g (1 mg/mouse in 2 ml PBS; R&D Systems) or recominnt solule gp13 (2.5 mg/mouse in 25 ml PBS; R&D Systems) were injected intrvenously into mice 3 min efore tretment. Mice were injected intrperitonelly with 1 ml of recominnt TNF (1 mg/kg ody weight in sterile sline; R&D Systems) or hyper-il-6 (8 mg/kg ody weight) 6 h efore orgns were collected. Detils for the production of hyper-il-6 re in the Supplementry Methods online. Actinomycin D (Sigm) ws injected intrperitonelly (2 mg/kg ody weight in 1 ml sline, dose reported to rrest trnscription in vivo 33 ) 3 min efore the initition of or hyper-il-6 tretment. Frozen-section in vitro dhesion ssy. Lymphocyte dhesion to HEVs ws evluted y frozen-section in vitro dhesion ssy s descried 11,13,16. A totl of mouse splenocytes were overlid onto 12-mm-thick cryosections of s (pooled inguinl, rchil, xillry, scitic, superficil nd deep cervicl nodes) from normothermic control mice or -treted mice. Selected lymphoid tissue specimens were locked with ma specific for PNAd (MECA79; 5 mg/ml), (3E2; 5 mg/ml), ICAM-2 (3C4; 5 mg/ml) or isotype control ntiody from BD Biosciences. The ssy ws done t 4 1C for 3 min with 8 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

9 mechnicl rottion (112 r.p.m.; DS-5 Oritl Shker; VWR). After removl of nondherent cells, sections were fixed in 3% glutrldehyde nd were stined with.5% toluidine. Lymphocyte dhesion ws quntified y light microscopy (Olympus, Spectr Services) for nlysis of totl of HEVs per lymphoid tissue smple. For consistency in doule-lind evlutions, HEVs were quntified only if they contined one dherent cell or more. In vivo homing ssy. Homing of lymphocytes to lymphoid nd nonlymphoid orgns ws ssessed y short-term homing ssy 16,17. Mouse splenocytes t density of cells per ml were leled for 2 min t 37 1C with 18 mg/ml of TRITC (tetrmethylrhodmine-6-isothiocynte; Moleculr Proes) or 1 mg/ml of clcein (Moleculr Proes) in RPMI 164 medium (Invitrogen), nd leling ws stopped y centrifugtion through cushion of FCS (Invitrogen). Equivlent numers of leled cells (1 1 7 to cells in 3 ml PBS) were injected intrvenously into -treted mice nd normothermic control mice, nd orgns were collected 1 h fter cell trnsfer unless otherwise indicted. s consisted of pooled inguinl, rchil, xillry, scitic, superficil nd deep cervicl nodes. The numer of fluorescence-leled cells ws quntified (y reserchers linded to smple identity) with BH2/ RFL fluorescence microscope (Olympus Opticl) in ten fields or more (unit re of ech field,.34 mm 2 ) of nonsequentil cryosections 9 mm inthickness. The percentge of fluorescence-leled cells in single-cell suspensions of recipient lymphoid orgns ws lso nlyzed y flow cytometry with FACScn (BD Biosciences). Equivlent results were otined y microscopic quntifiction nd flow cytometry; ll results were confirmed y oth methods. The constnt rtio (out 1:1) of trnsferred cells in spleens of control mice nd -treted mice provided n internl reference stndrd for the numer of input cells in homing studies. Detils of tretment with dhesion-locking ntiodies, chemokines nd pertussis toxin re provided in the Supplementry Methods. For studies involving the homing of n enriched popultion of CD4 + or CD8 + T cells tht ws L-selectin-low, CXCR3 hi, mouse splenocytes were ctivted for 2 d in vitro with plte-ound ma to mouse CD3 (145-2C11; BD Biosciences), followed y tretment for 3 d with recominnt IL-2 (12.5 ng/ml; R&D Systems) 5. Flow cytometry. Multiprmeter flow cytometry ws used for phenotyping of TRITC-leled cells efore or fter trnsfer in single-cell suspensions of s nd spleen from recipient mice. Detils re provided in the Supplementry Methods. Intrvitl microscopy. Intrvitl microscopy of inguinl s ws done s descried 17,23. C57BL/6 mice were nesthetized y intrperitonel injection of 1 mg/ml of xylzine nd 1 mg/ml of ketmine (1 ml per kg ody weight). The left inguinl lymph node ws exposed in n dominl skin flp nd the surrounding ftty tissue ws removed to expose the lymph node microvsculture. Approximtely clcein-leled splenocytes were injected through ctheter inserted into the right femorl rtery nd were visulized with customized intrvitl microscopy system (Spectr Services). Brightfield microscopy ws used to identify the vsculr structure nd lood flow sttus in venulr rnches; fluorescent microscopy ws used to visulize clcein-leled cells. At the end of the oservtion period, 15-kilodlton fluorescein isothiocynte conjugted dextrn (1 mg/ml; Moleculr Proes) ws injected to define the venulr structure. All imges were cptured with n EB chrgecoupled device cmer (Hmmtsu Photonics) nd were recorded with digitl videocssette recorder (DSR-11, Sony) for nlysis of cell ctivity. The rolling frction ws defined s the percentge of totl cells pssing through the vessel tht trnsiently intercted with HEVs during the oservtion period 17,23. The sticking frction ws defined s the percentge of rolling cells tht dhered to HEVs for 3 s or more. For rolling nd sticking frctions, dt were generted from three normothermic control mice (venules nlyzed: order I, 3; order II, 8; order III, 15; order IV, 16; order V, 12) nd three -treted mice (venules nlyzed: order I, 4; order II, 7; order III, 11; order IV, 13; order V, 11). The velocity of more thn 2 nonintercting (fst-moving) cells in order III venules ws mesured in ech mouse. Immunofluorescence nlysis. Orgns were emedded in optimum cutting temperture compound (Skur Finetek). Tissue cryosections 9 mm in thickness were fixed for 1 min t 2 1C in methnol/cetone (3:1), nd the expression of homing molecules ws detected y immunofluorescence stining. Detils of the procedures for stining of orgn cryosections or intrvsculr stining re in the Supplementry Methods. Digitl imges were cptured with n Olympus BX5 upright fluorescence microscope equipped with SPOT RT cmer (Spectr Services). Confocl imges were otined with the Leic TCS SP2 spectrophotometer confocl microscope. All imges were cptured with identicl exposure times nd imge settings in ech experiment. Imges were nlyzed with ImgeJ softwre 51 ( for determintion of the reltive fluorescence stining intensity for trfficking molecules on ll HEVs in single 9-mm-thick cross-section of 14 pooled s (pired inguinl, rchil, xillry, scitic, superficil nd deep cervicl nodes). For normliztion of the vrying sizes of individul HEVs in cryosections, fluorescence intensity is expressed in terms of pixels (reflecting fixed unit re). For this, PNAd + cuoidl structures were circled (with Intuos3 USB Pen Tlet; Wcom) nd ech pixel in those defined regions ws ssigned fluorescence intensity vlue (sed on scle from to 255). Histogrms represent the dt from ll pixels nlyzed (rnge, to )for the totl HEVs per cryosection (rnge, to ); similr numers of HEVs nd pixels were nlyzed for ech tretment condition in individul experiments. Immunolots nd enzyme-linked immunosorent ssy. s were sonicted to otin totl tissue lystes. For immunolot nlysis of CCL21, equl mounts of protein (2 mg) were frctionted y reducing SDS-PAGE (12% crylmide), nd memrne lots were proed with ntiodies specific for CCL21 (R&D Systems) or -ctin (Sigm). Recominnt CCL21 (R&D Systems) ws used s positive control. protein in tissue lystes ws mesured y enzyme-linked immunosorent ssy (R&D Systems). IL-6 concentrtions in tissue lystes were mesured y multiplex immunossy (Luminex 1) using mouse Fluorokine MAP ssys (R&D Systems). Phosphorylted STAT3 ws detected y immunolot nlysis 13 in liver nd spleen extrcts of mice pretreted with ctinomycin D for vrious times (,.5, 1, 2 nd 4 h), followed y 3 min of tretment with hyper-il-6. Sttisticl nlysis. Sttisticl nlysis used the unpired, two-tiled Student s t-test. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMES We thnk J.D. Blck nd E.A. Repsky for discussions nd comments on the mnuscript; M. Miysk nd T. Tnk (Osk University) for ntiserum to mouse Duffy ntigen relted receptor for chemokines; P.K. Wllce nd E.A. Timm for ssistnce with flow cytometry of leukocyte susets; nd E.L. Hurley for technicl support for confocl microscopy. Supported y the US Ntionl Institutes of Helth (CA79765 nd CA9445 to S.S.E.; AI61663 nd AI69259 to U.V.A.; DK33886 nd CA8558 to H.B.; nd CA1656 to Roswell Prk Cncer Institute), the Deprtment of Defense (W81XWH to Q.C.), the Roswell Prk Allince Foundtion (to S.S.E.), the Leukocyte Migrtion Core of the Hrvrd Skin Disese Reserch Center (P3 AR to U.H.v.A.) nd the Deutsche Forschungsgemeinschft (Bonn, Germny; SFB414, TPB5 to S.R.J.). AUTHOR CORIBUTIONS Q.C. nd S.S.E. conceptulized nd designed the reserch; S.S.E. supervised the reserch; Q.C. did ll experiments unless stted otherwise; D.T.F. contriuted to the experimentl design for quntittive imge nlysis nd did the phenotypic nlysis in short-term homing ssys nd enzyme-linked immunosorent ssy for ; K.A.C ssisted in immunofluorescence stining nd kinetic nlysis in short-term homing ssys; E.U. contriuted to the nlysis of stining; W.-C.W. did frozen-section dherence ssys nd provided technicl ssistnce for orgn retrievl; U.H.v.A. nd J.-M.G. helped with intrvitl microscopy studies; S.R.J. provided the hyper-il-6 expression construct; H.B. provided recominnt hyper-il-6 nd contriuted to discussions regrding IL-6 regultion of lymphocyte trfficking; nd ll uthors contriuted to discussions nd to the preprtion of the mnuscript. COMPETING IERESTS STATEME The uthors declre tht they hve no competing finncil interests. NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 9