HIGH ENDOTHELIAL VENULE BINDING AS A PREDICTOR OF THE DISSEMINATION OF PASSAGED MURINE LYMPHOMAS

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1 HIGH NDTHLIAL VNUL BINDING AS A PRDICTR F TH DISSMINATIN F PASSAGD MURIN LYMPHMAS BY RBRT F. BARGATZ,*"$ NRA W. WU,*,$ IRVING L. WISSMAN,* AND UGN C. BUTCHR*, * From the *Deprtment of Pthology, Stnford University Medicl Center, Stnford, Cliforni 9435; nd *The Veterns Administrtion Medicl Center, Plo Alto, Cliforni 9434 Lymphoid neoplsms disply wide spectrum of growth ptterns in vivo, from presenttion s solitry extrlymphoid tumor, to disseminted involvement of ll lymphoid orgns. Since norml lymphocytes re uniquely mobile cells throughout much of their life cycle, it is resonble to propose tht the dissemintion of mlignnt lymphocytes my reflect their expression of norml lymphocyte homing mechnisms. The migrtion nd trffic of norml lymphocytes to lymph nodes is controlled in lrge prt by the regulted expression of surfce receptors for high endothelil venules (HV), specilized venules tht medite the extrvstion of circulting lymphocytes from the blood into lymphoid orgns nd sites of chronic inflmmtion (reviewed in reference 1). By using quntittive in vitro ssy of lymphocyte binding to HV in frozen sections of lymphoid tissues (2-3), it hs been shown tht t lest two functionlly distinct nd independently regulted lymphocyte homing receptor/hv lignd recognition systems exist in the mouse, one controlling lymphocyte interctions with HV in the mucosl lymphoid orgns, Peyer's ptches, nd the other directing lymphocyte trffic to peripherl lymph nodes (4, reviewed in reference 1). Both functionl in vitro ssys nd immunologic studies with mabs to puttive homing receptors hve confirmed tht lymphoms cn lso express receptors for HV (1, 4). The present studies were undertken to exmine whether the expression of functionl homing receptors for HV might ply n importnt role in the in vivo behvior of neoplstic lymphocytes. We hve compred the ptterns of growth of severl HV-binding nd nonbinding murine lymphoms fter pssge into syngeneic recipients. The results indicte tht the bility to bind to HV either regultes the hemtogenous dissemintion of mlignnt lymphocytes to HV-bering orgns, or is coregulted with fctors determining this metsttic behvior. Mterils nd Methods Lymphoms. The lymphoms used in these studies re s follows: L1-2, n Abelson virus-induced C57L lymphom ; RAW112, n Abelson virus-induced BALB/c lym- This work ws supported by Ntionl Institutes of Helth grnt AI-19957, nd n wrd from the Veterns Administrtion to. C. Butcher, NIH fellowship CA-7879 to N. W. Wu, nd NIH grnt AI-972 to I. L. Weissmn.. C. Butche r is n stblished Investigtor of the Americn Hert Assocition. Journl of xperimentl Medicine - Volume 166 ctober Downloded from jem.rupress.org on Februry 23, 213

2 1126 HIGH NDTHLIAL VNUL BINDING AND LYMPHMA MTASTASIS phom ; AB 8.1/2 (Americn Type Culture Collection, Rockville, MD), n Abelsoninduced BALB/c lymphom ; L-4, chemicl crcinogen-induced C57BL lymphom ; BW5147 (Americn Type Culture Collection), n AKR T cell lymphom ; TK1, TK2, TK5, TK52, TK23, TK4, spontneous AKR/Cum T cell lymphoms ; TKJ43, n AKR/J lymphom; nd, n AKR/Cum B cell lymphom. In Vitro Assy of Lymphocyte Binding to HV. The bility of lymphom cells nd norml BALB/c mesenteric node lymphocytes (whose binding defines reltive dherence rtio [RAR] of unity) to dhere to HV ws determined in vitro s described previously (2, 4). Lymphoid tissues for sections nd control lymphocytes were from 6-8-wk-old BALB/c mice. Pssge of Lymphoms. Lymphoms were pssged by injection of 1' to 1' cells into the right thigh muscle of syngeneic mice. To improve the frequency of successful pssge, most recipients were given 4 rd whole body irrdition h before trnsfer of cells. Animls were killed 7-28 d lter, nd the indicted orgns were removed nd weighed. Blood, nticogulted with heprin, ws tken for white cell counts. White cell counts were performed by hemocytometer. Lrge blsts, infrequent in control blood, constituted minimum of 5% nd in most cses >8% of the circulting leukocytes in mice bering lymphoms. Histologic xmintion of Tissues. Peyer's ptches, spleens, femurs, nd selected lymph nodes from representtive recipients of ll lymphoms except TK2 nd TK52 were exmined histologiclly to ssess involvement by lymphom. Tissues were fixed in Tellyezhinski's fixtive nd processed routinely for sectioning nd stining with hemtoxylin nd eosin. Results nd Discussion Lymphoms were selected for the study becuse they displyed one or the other extreme in homing receptor expression (Tble I) : either filing to bind detectbly to HV (reltive dherence generlly <5% of tht of control lymph node cells), or binding extremely well to lymph node HV, to mucosl HV in Peyer's ptches, or to both HV clsses (dherence nerly equivlent to or exceeding tht of control mesenteric lymph node lymphocytes on one or both HV types). In xp. 1, when mice receiving the highest cell dose of syngeneic lymphom cells developed obvious lymphom (lrge plpble lymph nodes nd/or significnt locl mss in the thigh), ll nimls with detectble locl msses or lymph node enlrgement were killed nd tumor involvement t the site of injection nd in vrious lymphoid orgns ws ssessed grossly by weight. Animls without detectble disese t tht time were llowed to progress further before they were killed. In xp. 2, nimls receiving vrious tumor cell doses were killed only when gross involvement of lymph nodes or mssive involvement t the locl site developed. The results, presented in Tble I nd in Figs. 1 nd 2, demonstrte cler correltion between the expression of functionl HV-binding bility nd the in vivo metsttic ptterns of the trnsferred lymphoms. HV-binding lymphoms uniformly metstsized by the blood to give gross, symmetric enlrgement of ll lymph nodes. Peyer's ptches were lso observed to be enlrged by visul inspection. Histologiclly, lymph nodes were replced by lymphom cells, nd involvement of Peyer's ptches by ech of the HV-binding lymphoms except ws extensive, with totl effcement of the lymphoid rchitecture., B linege lymphom tht binds preferentilly to peripherl lymph node HV, involved Peyer's ptches foclly leving much of the rchitecture of norml lymphocyte domins intct. nly two of the HV-binding lymphoms, Downloded from jem.rupress.org on Februry 23, 213

3 I Published ctober 1, 1987 BARGATZ T AL l~u'1 CV ~D'+ ~ D ml~wdwo f 1~ 2.. GD GV.-+ 8Z v.. ~, CPI CJ d SD h 3 d y s V.Y t~ _ D.+ _ + ~ f ~ 7 M ".,.r.. n u'fl~ly u~ D T n ~ n ~.-+ M on ~f7 ~[f CJ i I ~~,C 1- n 7 W CJ.. ~ ~ n CV 7 on. i I I I I W~' ~~rcv u7 1~ H ~ o 47 Cp ~}'?'1 CV v c ~ " o r o :; -~ ^ ( n H1~1~1~ ~.n.n ~ ~n ~ n f I I I I c~' f ' 1 ~ w ~n o o o W ( Q7 ~ CV I4~I(~I I II ~ ~'.. '.....,..r CV CV GV CV., GV CV '+.. CV.. CV.r CJ...r CV V GV ~,C W ^Cb G C 2, 7J C ~+ +I +I -H +I W ~" wu Lm 3.Y. Y ~ Y ~C~ C W y ~ 5 Ir NT L w GV en C!' Y D " ~ U 9 YỴ+ c.4 I. 3 ~ o sl r on CJ o.fir ọ, Ṿ'. ~3 o I I Qn ell C4 N,.1 1,.1 L N.C v b mo'n~ o =' e I ~.C I I I I I ~~ 4 I d 3Y p ~ X Z Y u tp G T 3 Wo vwx~ U b N CJ m CI CV CV GV CV CI PI C p I I 77-1 I I I 1 W *4 n 4Ỵ Y "C rgv.~r. r7 z k C ~ L L _i+, t 3.C u..g u Downloded from jem.rupress.org on Februry 23, 213 o v o ~ H 41 -H -H -H ' - r o o. ~ ~ y on o QoG, u to-2 Cw ~.~ ~^. 4 C U A p Y v Y v ti u u u u ' Y o 'u 'Y u u u 'Y u n,wffff F FF F T zz. z n ~ c p ' o~^ C v y eti n Lo..1 ow FFF ~ Wx Z x FF F F =U Cj m -6oY''p.9~ ~ ~ o 3? v 3 c 3 Y c ww x " A o.. c ~ ~'Se o v.u C 4 ~.~ ''o' Yb 'G o ~oc c c Y c F3S. 3 ooef3-fu

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5 BARGATZ T AL Combined Peripherl Node Weights Binders (73 77[4 771 t o. Non Binders TK5 RAW112 HW5147 AB8.1/2 Controls BALB/c Control AKR Control WeightMg. b Binders w t o. Non Binders Controls c TKI43 TK23 TK4 TKI RAW112 BW5147 AB.In )N BALB/c Control AKR Control Binders TKJ43 TK23 TK4 w e. Non Binders T RAW112, BW AB8.1/2 Controls BALB/c Control. AKR Control. d Ileocecl Mesenteric Node Weights ` 4 5 Weight Mg. Injection Site Tumor Mss Weight Grms Circulting White Count Downloded from jem.rupress.org on Februry 23, 213 Binders TKJ43 TK23 TK4 TKl B t Non Binders TKS. RAW112., HW5147. ABB.1/2,. Controls BALB/cControi AKR Control 2 4 6' 8 1 Millions/M1. FIGUR 2. Men vlues from xp. 2, summrizing the mrked differences in the behvior of the nonbinding nd HV-binding lymphoms. The peripherl lymphnode dt () represent the dded weights of the slivry lymph nodes nd the brchil lymph nodes contrlterl to the injection site. (b) Combined ileocecl mesenteric lymph nodes. (c) Weight of tumor t the injection site in the right thigh, estimted bsed on physicl mesurement of the tumor. (d) Totl white cell count.

6 113 HIGH NDTHLIAL VNUL BINDING AND LYMPHMA MTASTASIS nd in two of six recipients TK4, produced significnt locl msses t the injection site. Nonbinding lymphoms, on the other hnd, produced little or no pprent chnge in distnt lymph node groups or in Peyer's ptches. The bsence of involvement of lymph nodes nd Peyer's ptches ws confirmed in histologicl exmintion of tissues from rndomly selected recipients of ech lymphom (except TK2 nd TK52, which were not studied histologiclly). A microscopic focus of lymphom ws seen in the mesenteric lymph node from single recipient of RAW 112. The predominnt mnifesttion of these nonbinding lymphoms ws mssive locl growth, with enlrgement of locl lymph nodes drining the site of tumor cell injection. The qulittive ptterns of growth observed were chrcteristic of the prticulr lymphom trnsferred, nd did not vry substntilly with cell dose or with time (up to 4 wk, the longest period studied). A slight increse in the frequency of contrlterl lymph node (combined xillry nd brchil) enlrgement in nimls receiving nonbinding lines ws pprent in xp. 1, prticulrly with the smllest doses nd longest times fter injection. In xp. 2, in which xillry nd brchil nodes were ssessed seprtely, it ws cler tht this effect ws limited to the xillry lymph node. It probbly represented lymphtic spred of lymphom cells cross the midline, since in no cse were the contrlterl slivry or brchil lymph nodes involved, nd xillry node enlrgement ws only observed in nimls exhibiting extensive nd grossly pprent lymphtic diltion with ssocited edem. In contrst to the generl symmetry exhibited by the HV-binding lymphoms, lymph node involvement by the nonbinding lymphoms ws lwys symmetricl. All lymphoms hd ccess to the bloodstrem. Both HV-binding nd nonbinding lines exhibited significntly elevted levels of white blood cells, the mjority of which were blsts (>8% in most cses). More importntly, histologic involvement of the bone mrrow ws universl, nd ll lymphoms cused splenic enlrgement, t lest in some recipients. Thus the expression of functionl homing receptors for HV my correlte with the bility of cells tht hve successfully entered the blood to extrvste into HV-bering orgns, rther thn primrily with the cpcity of lymphoms to enter the bloodstrem. Although some of the lymphoms exhibited significnt preference for binding to peripherl lymph node or to mucosl HV in vitro, ll HV-binding lymphoms in vivo involved the peripherl lymph nodes, the Peyer's ptches, nd the mesenteric lymph nodes (which express both peripherl nd mucosl binding specificities). This my reflect the fct tht binding is never completely specific for one or the other HV clss, or my rgue tht homing receptor expression is merely one importnt component of constelltion of co-regulted phenotypic fetures tht determines the locliztion nd growth of lymphoms. It should be pointed out, however, tht lymphoms TK1 nd TK4, which bind only poorly to peripherl lymph node HV in vitro, produced preferentil enlrgement of the mesenteric lymph nodes compred with the peripherl lymph node-preferentil lymphom (i.e., the rtio of weights of the mesenteric node, which contins HV cpble of binding mucos-specific cells, to peripherl nodes ws greter in the cse of the mucosl HV-specific lines). Furthermore, Downloded from jem.rupress.org on Februry 23, 213

7 BARGATZ T AL ll of the lymphoms tht bound well to mucosl HV cused totl replcement of the Peyer's ptch rchitecture histologiclly, wheres displyed only focl involvement. These observtions suggest tht receptor specificity my ply quntittive role in determining orgn involvement by doptively trnsferred lymphoms. More detiled studies of the kinetics of homing nd growth of pssged lymphoms, nd correltion of the initil presenttion of lymphoms with their HV-binding phenotype, must be undertken to understnd fully the role of homing receptors in the nturl progression nd spred of lymphoid mlignncies. Summry It hs long been postulted tht norml lymphocyte homing mechnisms help determine the metsttic spred of lymphoid neoplsms. The trffic of norml lymphocytes is controlled in prt by the regulted expression of surfce receptors for high endothelil venules (HV), specilized venules tht medite the extrvstion of circulting lymphocytes from the blood into lymphoid orgns nd sites of chronic inflmmtion. Here we hve compred the in vivo growth ptterns of HV-binding vs. nonbinding murine lymphoms pssged intrmusculrly into syngeneic recipients. We report tht lymphoms tht bind well to HV (s ssessed in quntittive in vitro ssy) disseminte widely vi the blood, involving ll lymph node groups symmetriclly. Although both HV-binding nd nonbinding lymphoms gin ccess to the blood, gross involvement of lymph nodes by nonbinding lymphoms is limited to nodes drining locl tumor t the site of injection, prominent feture of these lymphoms; distnt lymph nodes re not enlrged. The results suggest tht the expression of functionl receptors for HV either controls the hemtogenous dissemintion of mlignnt lymphocyte popultions to HV-bering orgns, or is coregulted with fctors determining this metsttic behvior. The findings support the concept tht norml lymphocyte homing mechnisms re importnt to the spred of leukemis nd lymphoms. Received for publiction 29 April 1987 nd in revisedform IJuly Downloded from jem.rupress.org on Februry 23, 213 References 1. Jlknen, S., R. Reichert,W. M. G. Glltin, I. L. Weissmn, nd. C. Butcher Homing receptors nd the control of lymphocyte migrtion. Immunol. Rev. 91 : Stmper, H. B., Jr., ndj. J. Woodruff Lymphocyte homing into lymph nodes: in vitro demonstrtion of the selective ffinity of recirculting lymphocytes for high endothelil venules. J. xp. Med. 144 : Butcher,. C., nd W. L. Ford Following cellulr trffic : methods of lbelling lymphocytes nd other cells to trce their migrtion in vivo. In Hndbook of xperimentl Immunology. 4th ed. D. Weir, L. A. Herzenberg, nd L. A. Herzenberg, editors. Blckwell Scientific Publictions, Plo Alto, CA Butcher,. C., R. G. Scolly, nd I. L. Weissmn rgn specificity of lymphocyte migrtion : medition by highly selective lymphocyte interction with orgn-specific determinnts on high endothelil venules. ur. J. Immunol. 1:556.