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1 In Vivo Administation of Antibody to Inteleukin-5 Inhibits Inceased Geneation of Eosinophils and Thei Pogenitos in Bone Maow of Paasitized Mice By Donna M. Rennick, LuAnn Thompson-Snipes, Robet L. Coffman, Bian. P. Seymou, John D. Jackson, and Susan Hudak Bone maow of mice paasitized with Nippostongylus basiliensis showed inceased numbes of eosinophils as ealy as 4 days afte infection. By day 7, thei bone maow also contained elevated numbes of pogenitos that fom small eosinophil colonies (2 to 5 cells) in soft aga cultues supplemented with inteleukin-5 (IL-5). Howeve, when mice wee infused with anti-il-5 antibodies at the time of infection, the numbe of ecognizable eosinophils pesent in bone maow emained low and eventually dopped below nomal levels. The antibody teatment also ARASITIC INFECTION is one of seveal disease P states that is often chaacteized by an acute blood and tissue eosinophilia. Although eosinophils ae the only cell type consistently found elevated in the blood of paasitized animals, tissues diectly involved in the infection show an inflammatoy esponse typified by lage cellula infiltates consisting of lymphocytes, mast cells, macophages, and neutophilic ganulocytes, as well as eosinophils. The composition of these infiltates togethe with the pofound blood eosinophilia suggest that factos such as inteleukin-5 (IL-5), ganulocyte-macophage colony-stimulating facto (GM- CSF), and inteleukin-3 (IL-3) may be inv1ved.l.~ Recently, we assessed the in vivo ole of IL-5 in a paasite-induced eosinophilia by giving a monoclonal anti-il- 5 antibody to mice infected with Nippostongylus basiliensis (Nb). Mice infected with this intestinal nematode develop a blood eosinophilia that peaks between 11 and 14 days. Because Nb lavae migate though and molt in the lung duing the fist few days of infection, this ogan becomes highly infiltated with inflammatoy cells, including lage numbes of eosinophils. hen Nb-infected mice wee teated with anti-il5 antibodies, they failed to develop the expected blood eosinophilia. Antibody teatment also inhibited the appeaance of lung eosinophils without affecting infiltating neutophilic ganulocytes and macophages. This study clealy showed that IL-5 is essential in suppoting inceased numbes of matue tissue and blood-bone eosinophils duing a paasite-induced eosinophilic esponse. Recently, simila esults wee obtained with mice infected with Schistosoma mansoni.6 In ode to futhe elucidate the in vivo elevance Fom the Depatment of Immunology. DNAX Reseach Institute of Molecula and Cellula Biology, Palo Alto, CA. Submitted Decembe ; accepted Mach Suppoted by Scheing-Plough Copoation. BloomJield, NJ. Addess epint equests to Donna M. Rennick. PhD, DNAX Reseach Institute of Molecula and Cellula Biology, 91 Califonia Ave. Palo Alto. CA The publication costs of this aticle wee defayed in pat by page chage payment. This aticle must theefoe be heeby maked advetisement in accodance with 18 U.S.C. section 1734 solely to indicate this fact. 199 by The Ameican Society of Hematology /9/762-6$3./ pevented inceased geneation of IL-5-esponsive pecusos capable of diffeentiating into matue eosinophils in liquid cultue and inhibited the geneation of pogenito cells capable of foming small eosinophil colonies o clustes in soft aga cultues. The esults of these in vivo expeiments diectly show that IL-5 is an essential egulatoy molecule equied fo the bone maow-dependent phase of a paasite-induced eosinophilia. 199 by The Ameican Society of Hematology. of IL-5, we have used the same system to study pecuso cell development in bone maow of infected mice. MATERIALS AND METHODS Gowth factos. Puified ecombinant mouse IL-3 (specific activity [sp act], 2 x lo8 U/mg) and IL-5 (sp act, 4 x lo6 U/mg) wee povided by Ds Jolanda Scheus and Janusz ideman, espectively (DNAX, Palo Alto, CA). One unit of IL-3 o IL-5 is defined as that amount of facto that stimulates half-maximal incopoation of H-thymidine by an appopiate facto-dependent cell line. Each facto was used at 1 U/mL. This amount was found to be at least two times highe than that equied to stimulate maximum colony fomation afte titation of each facto in semisolid bone maow cultues. Antibodies. Rat monoclonal antibodies (IgG2a) specific fo IL-5 (TRFK-5) wee pepaed as peviously descibed. Rat monoclonal antibodies (IgG2a) specific fo Escheichia coli beta galactosidase (GLll3) wee puified by Veax Cop (Lebanon, NH). The puity of the antibody pepaed by high-pefomance liquid chomatogaphy (HPLC) was geate than 95%. Infected mice. BALB/c mice, injected subcutaneously with thid-stage Nb lavae, eceived eithe no antibodies, an injection of anti-il-5 antibodies (TRFK-5), o an isotype-matched contol (GL113). Antibodies wee given intapeitoneally at 2 mg pe mouse. This amount of anti-il-5 antibody has been shown peviously to suppess completely Nb-induced blood eo~inophilia.~ At vaious time points, eosinophils wee quantified by pefoming total cell counts and diffeentials on stained cytocentifuged pepaations fom bone maow, peitoneal cavity, and blood. Eosinophil pogenito cell assays. On days 4, I, and 11 postinfection, fou mice fom each expeimental goup and fou nomal mice wee sacificed. Bone maow was havested fom thei femus, with an aveage cell ecovey of 1.8 x lo7 nucleated cells pe femu. Single cell suspensions wee pepaed individually fom one femu of each mouse and seeded at 2 x lo5 cells into tiplicate 1 ml cultues containing modified Iscove s medium (GIBCO, Gand Island, NY), 2% fetal calf seum, 5 bmol/l 2-mecaptoethanol,.3% (wt/vol) aga supplemented with puified ecombinant IL-5 (1 U/mL) o IL-3 (1 U/mL). Afte 6 days of incubation, the aga cultues wee fixed with 2.5% glutaaldehyde, washed with wate, tansfeed to gelatin-coated glass slides, and ai died. The slides wee then stained with.1% Luxol Blue and countestained with Maye s Hematoxylin. Peliminay expeiments showed that 5 to 6 days of incubation was optimal fo evaluating eosinophil colony fomation. Upon futhe incubation, colonies that contained geate than 5 cells at day 6 did not incease in numbe, although some continued to incease in size. The smalle colonies began to disintegate o the cells migated into the suounding aga leading to a diffuse backgound of Luxol Blue positive cells. 312 Blood, VOI 76, NO 2 (July 15). 199: pp

2 IN VIVO ADMINISTRATION OF ANTIBODY TO IL Liquid suspension cultues. Bone maow cells wee havested fom nomal mice and fom mice 7 days post-infection with Nb lavae. The cells wee suspended at 1 x lo5 cells/ml in tiplicate 1 ml liquid cultues of the basic medium descibed above. The cultues contained no gowth facto, o wee supplemented with IL-5 (1 U/mL) o IL-3 (1 U/mL). Afte 5 days of incubation, the total numbe of viable cells wee counted using dye exclusion and diffeential counts wee done on ight-giemsa-stained cytocentifuge pepaations. RESULTS Administation of anti-il-5 antibodies suppessed development of matue eosinophils and thei undiffeentiated pecusos in bone maow of Nb-infected mice. On aveage, the numbe of ecognizable eosinophils in nomal bone maow was 2.8% of all nucleated cells. The total numbe of eosinophils pe femu was found to be 5 x lo5 (Fig la, dashed line). Afte 4 days of infection with Nb lavae, matue and immatue eosinophils inceased almost sixfold in bone maow of paasitized mice (Fig 1). The bone maowassociated eosinophilia peaked by day 7, seveal days ealie than the maximum eosinophilic esponse occuing in the blood and peitoneal cavity (Fig 1B and C). hen Nbinfected mice eceived anti-il-5 antibodies, they not only failed to develop a peipheal eosinophilia, but they also failed to develop the expected numbes of matuing eosinophils in thei bone maow (Fig 1A). These esults showed that the absence of a blood and tissue eosinophilia was not simply due to the defective elease of matue eosinophils into ciculation. Instead, it appeaed that bone maow pecusos had failed to diffeentiate into matue foms in paasitized animals made IL-54eficient. If this wee the only effect of neutalizing IL-5 in paasitized mice, we easoned that thei eosinophil pecuso levels should still be simila to o even highe than that of the paasitized contols. To test this diectly, the numbe of pecusos in infected, anti-11-5-teated hosts was compaed with nomal mice and with infected mice left unteated o teated with contol antibodies. Pecuso levels wee oughly equated to the capacity of bone maow fom the vaious expeimental goups to give ise to matue eosinophils in cultues supplemented with IL-5. The numbe of eosinophils contained in the oiginal cell inoculum ( lo6 cells pe cultue) was detemined to be 2.8 x lo4 fo nomal mice, 14.8 x lo4 fo paasitized mice, x 1 O4 fo paasitized mice teated with anti-p-gal antibodies, and 2.6 x lo4 fo paasitized mice teated with anti-il-5 antibodies. It was found that all suspension cultues stimulated with IL-5 showed an absolute incease in the total numbe of eosinophils detected afte 7 days of cultue with IL-5 (Fig 2). Moeove, cultues established with bone maow of infected mice (+contol antibody teatment) poduced 3 to 4 times as many eosinophils as cultues established with nomal bone maow (Fig 2). In contast, cells fom paasitized mice injected with anti-il-5 antibodies poduced the same numbe of eosinophils as cells fom nomal mice, a value well below that obseved fo paasitized mice eceiving no antibodies o contol antibodies (Fig 2). Theefoe, it appeas that the elevated numbe of undiffeentiated pecusos associated with paasitic infection is diectly egulated by IL-5. L a 5 U. m\ x 2 \ - x U m - E \ P X sq A L Days afte infection Fig 1. Administation of anti-il-6 antibody damatically educes the numbe of eosinophils in bone maow (A) as well as peitoneal cavity (B) and blood (C) of paasitized mice. Female 12-week-old BALB/c mice wee injected subcutaneously with 75 thid-stage Nb lavae. On the same day (day ). mice wee injected intapeitoneally with eithe anti-il-5 (2 mg pe mouse) o an isotype-matched contol (1 mg pe mouse). Animals wee killed on days indicated, and the eosinophil numbes wee detemined in bone maow, peitoneal cavity, and blood. Data epesent mean values with standad deviations fo goups of fou mice. M, Nb-infected mice teated with anti-il-6 antibodies; -. Nbinfected mice teated with anti-&gal antibodies. The dashed line in each gaph epesents the aveage numbe of eosinophils obseved in nomal mice. Bone maow cells fom each expeimental goup wee also established in suspension cultues containing IL-3. Thee wee no significant diffeences in the poduction of eosinophils when compaing cultues established with bone

3 314 RENNICK ET AL Fig 2. Nb-infected mice teated with anti-il-5 antibodies have educed numbes of eosinophil pecusos that diffeentiate to matue cells in suspension cultues containing IL-5. Suspension cultues containing IL-5 wee established with a total of lo6 bone maow cells fom nomal mice and fom mice infected 7 days ealie with Nb lavae. Some of the infected mice wee teated with anti-il-5 o isotype-matched contol antibodies as peviously descibed. Data epesent mean values with standad deviations fo goups of fou mice. maow of nomal and Nb-infected mice (,anti-il-5 antibody teatment) (data not shown). EfSects of anti-il-5 antibody administation on geneation of eosinophil colony-foming cells in bone maow of Nb-infected mice. The numbes of eosinophil colonyfoming cells (Eo-CFCs) pesent in bone maow of nomal and Nb-infected mice ( L anti-il-5 antibody teatment) wee analyzed. These expeiments wee designed to detemine what effect neutalizing anti-il-5 antibodies would have on the geneation of eosinophil pogenitos duing the ealy phase of infection. Pogenitos wee enumeated by thei ability to fom eosinophil colonies in soft aga cultues containing an appopiate gowth facto. e found that bone maow fom nomal mice and fom mice 4 days postinfection contained equivalent numbes of Eo-CFCs when assayed in IL-5-stimulated cultues (data not shown). Howeve, bone maow havested fom mice 7 days post-infection showed a significant incease in Eo-CFCs capable of foming small colonies (2 to 5 cells) in esponse to IL-5 (Fig 3). Teatment of paasitized mice with anti-il-5 antibodies educed this numbe to that obtained with nomal mice. In contast, nomal and Nb-infected bone maow (k anti- IL-5 antibody teatment) showed no diffeence in the numbes of Eo-CFCs capable of foming lage IL-5-dependent colonies (geate than 5 cells). These esults show that the geneation of these paticula Eo-CFCs ae not affected by infection. Similaly, no diffeences between nomal and Nb-infected bone maow (* anti-il-5 antibody teatment) wee obseved with espect to numbes of IL-3-esponsive Eo-CFCs o Eo/mixed-CFCs (Table 1). Fig 3. Effects of anti-il-5 antibodies on in vivo geneation of eosinophil pogenitos in bone maow of Nb-infected mice. Seven days post-infection, bone maow cells fom each animal in each expeimental goup wee plated at 2 x 15/mL in tiplicate soft aga cultues containing IL-5. Eosinophil colonies wee identified by the pesence of Lux1 Blue staining cells and wee scoed accoding to size. Values epoted ae mean f SD of fou mice pe goup. Results ae typical of two independent expeiments. Cultues not supplemented with gowth facto failed to develop any eosinophil colonies.

4 ~~ IN VIVO ADMINISTRATION OF ANTIBODY TO IL Table 1. Effect of Anti-IL-5 Antibodies on In Vivo Geneation of Eosinophil Pogenitos in Nb-Infected Mice IL-3-Stimulated Colonies Containing: Dono Teatment EofMixed >5 Cells 2-5 Cells Nomal 3. f f f 2.9 Nb 3. * f f 7.3 Nb + anti-il f f f 1. Nb + anti--gal 3.3 f f f 2.4 Results show numbe of eosinophil colonies x lo*/femu and ae expessed as mean f 1 SD fo goups of fou mice. Data ae epesentative of two sepaate expeiments. In a sepaate expeiment, we found that bone maow havested 14 days afte mice wee infected (k anti-il-5 antibodies) gave the same esults as bone maow sampled 7 days afte infection (k anti-il-5 antibodies) (data not shown). DISCUSSION e have peviously shown that IL-5 plays an essential ole in sustaining the blood and tissue eosinophilic esponse of mice infected with Nippostongylus basilien~is.~ In this study, the impotance of IL-5 to eosinophil development in bone maow of paasitized mice was evaluated. e found inceased numbes of matuing eosinophils in bone maow 4 days afte Nb infection (Fig 1). The bone maow eosinophilia peaked by day 7, seveal days ealie than that occuing in the peiphey. By day 7, it was detemined that bone maow of Nb-infected mice also contained highe than nomal numbes of IL-5-esponsive eosinophil pecusos as identified by thei ability to apidly diffeentiate into matue foms in cultue (Fig 2). Simila esults have been epoted using mice infected with othe helminth^.^.'*^ hen Nbinfected mice wee teated with anti-il-5 antibodies, thee was a damatic dop in pecuso levels and in the numbe of ecognizable eosinophils. In fact, the absolute numbes of matuing eosinophils pesent in the bone maow of these teated mice dopped below the levels detected in bone maow of nomal mice (Fig 1). This esult suggested that IL-5 may be necessay fo teminal cell diffeentiation, an activity of IL-5 documented by numeous in vito 1.12 Howeve, ou esults futhe suggested that IL-5 is necessay fo the inceased geneation of pecuso cells usually found in bone maow of paasitized mice (Fig 2). Thus, IL-5 not only suppots cell matuation but also egulates the size of the undiffeentiated pecuso cell pool. To continue this line of investigation, we next detemined what effect anti-il-5 antibodies would have on the ability of paasitized mice to geneate inceased numbes of Eo-CSFs. Results of clonal assays showed that giving neutalizing antibodies deceased Eo-CFC levels to that obseved in uninfected mice. Specifically, anti-il-5 antibody-teated mice showed educed numbes of IL-5-esponsive pogenitos capable of foming small colonies o clustes (2 to 5 cells) (Fig 3). Pesumably, these ae elatively matue cells compaed with those that fom lage colonies (geate than 5 cells) in esponse to IL-5. Inteestingly, the numbe of lage IL-5-esponsive colony foming cells was not significantly elevated duing infection, no was it affected by teating paasitized mice with anti-il-5 antibodies (Fig 3). Although the elatively immatue lage colony foming cells have acquied IL-5-esponsiveness in vito, appaently thei in vivo geneation fom an even ealie pogenito was not dependent on IL-5. It was also found (Table 1) that giving anti-il-5 antibodies to paastized mice did not affect the in vivo geneation of IL-3-esponsive eosinophil pogenitos epesenting an even ealie stage in the developmental Both of these esults ae in ageement with those of in vito studies, showing that IL-5 egulates only late stages of eosinophil development. In summay, the pesent studies clealy show that IL-5 is sufficient and necessay to sustain the bone maow eosinophilic esponse associated with helminth infection. Duing a paasite-induced esponse, IL-5 functions as moe than just a matuation and suvival facto; it also expands the size of the eosinophil pecuso compatment by stimulating the inceased geneation of matue pogenito cells with a limited polifeative potential (2 to 5 cells pe colony). Thus, IL-5 augments the geneation of its own final tagets, those exhibiting dependence on IL-5 fo teminal diffeentiation (summaized in Fig 4). These esults ae consistent with the pedicted physiologic actions of IL-5 based on many infomative in vito st~dies.~,'~,'~-~~ Although the pesent expeiments show that IL-5 is equied fo the eosinophilia manifested by paasitized mice, they do not ule out a ole fo IL-3 o GM-CSF. The ability of these two factos to induce a pofound blood and tissue eosinophilia has been documented in othe expeimental system^.'^-^' The possibility that IL-3 and/o GM-CSF ae involved in ealie stages of a paasite-induced eosinophilia may be esolved by using specific neutalizing antibodies in studies simila to those descibed hee. ACKNOLEDGMENT The authos thank Ds Jolanda Scheus and Janusz ideman fo poviding puified gowth factos and D J.A. aitz fo helpful pimitive Eo-CFU - Inhibited by teatment with anti-ll-5 antibodies /\ Fig 4. Scheme showing effects of anti-il-5 antibodies on eosinophil development in paasitized mice. Bold aows denote steps in eosinophil develealy Eo-CFU matue opment whee IL-5-dependent 4 late EO-CFU II) 4 eosinophils amplification of cell poduction IL-5 esponsive IL-5 esponsive occus in the bone maow of (>5 cells/colony) (2-5 cells/ colony) Paasitized mice.

5 316 RENNICK ET AL comments. e also thank Dan Finn fo help in the pepaation of the manuscipt. REFERENCES 1. Sandeson CJ, aen DJ, Stath M: Identification of a lymphokine that stimulates eosinophil diffeentiation in vito. Its elationship to inteleukin 3, and functional popeties of eosinophils poduced in cultues. J Exp Med 162:6, aen DJ, Mooe MAS: Synegism among inteleukin I, inteleukin 3, and inteleukin 5 in the poduction of eosinophils fom pimitive hemopoietic stem cells. J Immunol 14:94, Metcalf D, Begley CG, Johnson GR, Nicola NA, Vadas MA, Lopez AF, illiamson DJ, ong GG, Clak SC, ang EA: Biologic popeties in vito of ecombinant human ganulocytemacophage colony stimulating facto. Blood 67:37, Rennick DM, Lee FD, Yokota T, Aai KI, Canto H, Nabel GJ: A cloned cdna encodes a multilineage gowth facto: Multiple activities of inteleukin 3. J Immunol 134:91, Coffman RL, Seymou BP, Hudak S, Jackson J, Rennick D: Antibody to inteleukin-5 inhibits helminth-induced eosinophilia in mice. Science 245:38, She A, Coffman RL, Hieny S, Scott P, Cheeve A: Inteleukin-5 (IL-5) is equied fo the blood and tissue eosinophilia but not ganuloma fomation induced by infection with Schistosoma mansoni. PNAS 87:61, Schumache JH, O Gaa A, Shade B, van Kimmenade A, Bond M, Mosmann TR, Coffman RL: The chaacteization of fou monoclonal antibodies specific fo mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunoabsobent. J Immunol 141:1576, Stath M, Sandeson CJ: Detection of eosinophil diffeentiation facto and its elationship to eosinophilia in Mesocestoides Coti-infected mice. Exp Hematol 14:16, Ruscetti F, Cypess RH, Chevenick PA: Specific elease of neutophilic- and eosinophilic-stimulating factos fom sensitized lymphocytes. Blood 47:757, Yamaguchi Y, Suda T, Suda J, Eguchi M, Miua Y, Haada N, Tominaga A, Takatsu K: Puified inteleukin 5 suppots the teminal diffeentiation and polifeation of muine eosinophilic pecusos. J Exp Med 167:43, Sonoda Y, Aai N, Ogawa M: Humoal egulation of eosinophilopoiesis in vito: Analysis of the tagets of inteleukin-3, ganulocyte-macophage colony-stimulating facto (GM-CSF), and inteleukin-5. Leukemia 3:14, Sandeson CJ, Campbell HD, Young IC: Molecula and cellula biology of eosinophil diffeentiation facto (inteleukin-5) and its effects on human and mouse B cells. Immunol Rev 12:3, Lopez AF, Begley DG, illiamson DJ, aen DJ, Vadas MA, Sandeson CJ: Muine eosinophil diffeentiation facto. An eosinophil-specific colony-stimulating facto with activity fo human cells. J Exp Med 163:185, Cluttebuck EJ, Sandeson CJ: Human eosinophil hematopoiesis studied in vito by means of muine eosinophil diffeentiation facto (IL-5): Poduction of functionally active eosinophils fom nomal human bone maow. Blood 71:646, Donahue RE, ang EA, Stone DK, Kamen R, ong GG, Sehgal PK, Nathan DG, Clak SC: Stimulation of haematopoiesis in pimates by continuous infusion of ecombinant human GM-CSF. Natue 321:872, Metcalf D, Begley CG, Johnson GR, Nicola NA, Lopez AF, illiamson DJ: Effects of puified bacteially synthesized muine multi-csf (IL-3) on hematopoieis in nomal adult mice. Blood 68:46, Goopman JE, Mitsuyasu RT, DeLeo MJ, Oette DH, Golde D: Effect of ecombinant human ganulocyte-macophage colonystimulating facto on myelopoiesis in the acquied immunodeficiency syndome. N Engl J Med 317:593,1987