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1992 Immunology: Correction Proc. Natl. Acad. Sci. USA 86 (1989) Correction. In the article "Thymotaxin: A thymic epithelial peptide chemotactic forT-cell precursors" by Beat A. Imhof, Marie-Ange Deugnier, Jeanne-Marie Girault, Serge Cham- pion, Chantal Damais, Tsunetoshi Itoh, and Jean Paul Thi- ery, which appeared in number 20, October 1988, of Proc. Natl. Acad. Sci. USA (85, 7699-7703), the authors request that the following change be noted. The sentence beginning on line 15 of the abstract should read "The phenotype of the responding cells is Thy-l+, CD4- and CD8-." Downloaded by guest on September 28, 2021 Proc. Nati. Acad. Sci. USA Vol. 85, pp. 7699-7703, October 1988 Immunology Thymotaxin: A thymic epithelial peptide chemotactic for T-cell precursors (thymus/T-celi differentiation in vitro/bone marrow in vitro/stem cells) BEAT A. IMHOF*, MARIE-ANGE DEUGNIER*, JEANNE-MARIE GIRAULT*, SERGE CHAMPION*t CHANTAL DAMAIS*, TSUNETOSHI ITOH§, AND JEAN PAUL THIERY* *Centre National de la Recherche Scientifique, Ecole Normale Supdrieure, Physiopathologie du DMveloppement, 46 rue d'Ulm, 75230 Paris Cedex 05, France; tCentre National de la Recherche Scientifique, Roussel Uclaf, 93230 Romainville, France; and §Tohoku University School of Medicine Seiryo-cho, Sendai 980, Japan Communicated by Alfred Jost, June 17, 1988

ABSTRACT The embryonic thymus is seeded by invading dual function in controlling the oriented migration and by hemopoietic precursor cells that differentiate intrathymically inducing a transient invasive behavior (15). into T lymphocytes. We have recently reported that avian Recently, a rat thymic cell line (IT-45 R1) was established thymic epithelial cells secrete chemotactic peptides, which and shown to possess all the properties ofepithelial cells (17). provoke oriented migration of hemopoietic precursor cells in These cells show morphological and functional aspects ofthe vitro. The established rat thymic epithelial cell line IT-45 R1 superficial cortex epithelium. By immunofluorescence, thy- produced a polypeptide that resolves as a single band in the mulin as well as thymopoietin can be detected in the cyto- region of 11 kDa on NaDodSO4/polyacrylamide gels. This plasm ofIT-45 R1 cells (18). These cells secrete thymulin, and molecule, which we have named thymotaxin, induced a chemo- their conditioned medium contains this thymic hormone as a tactic response in a subpopulation of hemopoietic cells from biologically active molecule. In the present study, we show juvenile rat bone marrow. Responding cells were generated by that this rat thymic cell line produces chemotactic molecules short-term coculture ofrat bone marrow hemopoietic cells with comparable to the avian system. Hemopoietic precursor cells mouse bone marrow stroma in a steroid-free medium. Cells responding to the chemotactic peptides are found in the bone selected in a chemotactic chamber have a lymphoid or blast cell marrow of young rats. An in vitro culture system of bone morphology. The phenotype of the responding cells is Thy-i +, marrow cells grown on mouse stroma using steroid-free sera CD4+ and CD8 -. In contrast, CD8 T-lymphocyte differenti- allowed the enrichment of responding cells. The responding ation antigen was expressed after coculture with embryonic cells predominantly had a lymphoid phenotype and were thymic monolayers, suggesting that the responding cells cor- devoid of T- or B-cell differentiation markers. A significant respond to the precursors colonizing the thymus. percentage ofthese hemopoietic cells selected by the chemotactic peptides were able to express T-cell markers when The thymic microenvironment plays a crucial role in the grown in contact with primary cultures of embryonic rat differentiation of immigrated hemopoietic precursors into T thymus. cells (1-5). Direct cell-cell interaction as well as soluble factors are involved in the establishment of the different MATERIALS AND METHODS T-cell lineages (6-10). Thymic epithelial cells of the super- Animals. Male WAG Wistar rats and male Swiss mice, 3- ficial cortex and of the medulla have attracted particular 4 weeks old, were routinely used for bone marrow prepara- interest since they produce functional thymic hormones such tion. as thymopoietin, thymosin, or thymulin (11, 12). Besides the Hemopoietic Precursor Cells. Bone marrow cells were secretion of hormones that play important roles in the prepared from the femur cavity and loaded onto bovine differentiation of hemopoietic precursors, thymic epithelial serum albumin (Path-O-cyte-5, Miles) diluted to 28% with cells are involved in the recruitment of T-cell precursors to phosphate-buffered saline (PBS) and centrifuged at 1000 x g for 30 min. Floating cells were washed with Dulbecco's the thymus. Avian thymic epithelial cells, prepared from modified Eagle's medium (DMEM; GIBCO) and used for quail embryos, secrete peptides that are chemotactic for chemotactic migration assays. hemopoietic precursor cells (13, 14). Quail precursors can be Chemotactic Migration Assays. For migration assays, Boy- selected by chemotactic migration in vitro toward these den chambers (100 p.1) were used as described (14). The cells peptides in Boyden chambers. These quail cells subsequently were counted with a Coulter Counter ZM equipped with a still have the capacity to colonize a xenogeneic chicken channelizer 256; the counting window was 5-9 pum and thymus in vitro. When this colonized thymus is grafted back aperture size was 140 pm. Approximately 5 x 105 bone to a chicken embryo, the quail hemopoietic precursors marrow cells or 2-10 x 104 cultured cells were loaded in the mature into T cells. In avian embryos, cells responding to upper compartment of Boyden chambers. Testing media thymic chemotactic peptides are found in the bone marrow were placed into the lower blind well chamber, which was (15); early on, however, they may also originate from discrete separated from the cell-containing compartment by Nucle- hemopoietic foci that are dispersed in the embryo (16). To pore filters (pore size, 5 pum; Nuclepore, Pleasanton, CA). reach the thymus, these hemopoietic cells extravasate from After 3 hr incubation at 370C, the cells of the upper compart- blood vessels, traverse the perithymic mesenchyme, and ment were removed by washing five times with PBS. The finally invade the thymic basement membrane before insert- chamber was then centrifuged at 100 x g to detach weakly ing themselves between epithelial cells. The chemotactic adherent cells from the lower surface of the filter, the peptides are directly involved in these events by exerting a Nuclepore filter was discarded, and the migrated cells were

The publication costs of this article were defrayed in part by page charge Abbreviation: IL-1, interleukin 1. payment. This article must therefore be hereby marked "advertisement" tPresent address: Universite de Reims, Sciences exactes et natu- in accordance with 18 U.S.C. §1734 solely to indicate this fact. relles. Moulin de la Housse, 51062 Reims, France.

7699 7700 Immunology: Imhof et al. Proc. Natl. Acad. Sci. USA 85 (1988) collected from the lower compartment and suspended in 10 Thymocyte Activation and Cytotoxicity Used as Detection ml of Isoton II (Coultronics, Margency, France). Aliquots Assays for Interleukin 1 (IL-1) Activity. Activation of thy- (500 gI) were assayed on the Coulter Counter. mocytes was performed as described (21). Briefly, 1.5 x 106 For cytological analysis, hemopoietic cells were cytocen- thymocytes were cultured in 96-well plates for 48 hr in the trifuged in a Cytospin 2 (Shandon, Cheshire, England). presence of phytohemagglutinin P (1 Ag/ml) (Wellcome). Centrifuged cells were incubated in concentrated May- After 48 hr, 1 p1Ci of [methyl-3H]thymidine (specific activity, Grunwald solution (Merck), followed by staining in 7% 1 Ci/mmol; 1 Ci = 37 GBq; Commissariat a l'Energie Giemsa solution. For determination of the cytological type, Atomique, Saclay, France) was added to each culture well at least 200 cells per slide were analyzed. and the cells were incubated for an additional 20 hr. Cyto- Cultured Hemopoietic Precursor Cells. Short-term cultures toxicity mediated by IL-1 was performed on L929-a target were prepared according to Hayashi et al. (19) with the cells that were maintained at 104 cells per well. Twenty-four following modifications: adherent feeder cells were obtained hours later, serial 1:3 dilutions of monocyte conditioned by culture of total mouse bone marrow cells in 5 ml of medium or testing medium were added to the cells with Iscove's modified Dulbecco's medium (IMDM; GIBCO), actinomycin D (1 jug/ml) (Sigma). The plates were reincu- 20% horse serum (GIBCO) at 370C, 7.5% CO2 in a humidified bated for 20-24 hr at 370C. Thereafter, medium was removed at a per per atmosphere density of 107 cells 5 ml 25-cm2 and the cells were stained with 0.5% crystal violet dye for 15 culture flask (Nunc). After 3 days, an additional 5 ml of fresh min. A unit of cytotoxic activity is defined by the reciprocal medium was added and, at day 6, the medium was renewed. of the dilution that causes a 50o reduction in cell number At day 10, the flasks were washed twice with 5 ml of IMDM under conditions of the assay. and the medium was replaced by IMDM containing 20%o fetal calf serum (Flow Laboratories) preabsorbed on charcoal RESULTS (Norit A, Serva, Heidelberg) and Dextran K70 (Pharmacia) to Chemotactic Migration of Hemopoietic Precursor Cells remove steroid hormones, as described (19), and 1% steroid- Toward Thymic Conditioned Medium. The most free synthetic serum Ultroser SF (IBF, Villeneuve-la-Ga- Epithelial abundant source of hemopoietic precursors was the bone renne, France). Rat bone marrow cells enriched for hemo- marrow of 3-week-old rats. Total marrow cells con- poietic precursors on 28% bovine serum albumin centrifuga- bone tained three major cell size tion gradients were then cocultured with the adherent mouse populations defined by their using a Coulter Counter The first cell feeder layer (3 x 106 cells per 7 ml per flask). Culturing for channelizer 256 (Fig. la). 3 days produced weakly adherent and floating cells with a population, with an average diameter of 4-4.5 ,um, contained mostly erythrocytes and erythroid precursors; the second diameter of 6.2 gm that were used for migration assays. Differentiation of Hemopoietic Cells on Thymic Tissue from corresponded to cells having a diameter of 5.5 ,um and Rat Embryos. Thymuses of 14- to 15-day-old rat embryos contained mostly cells of the lymphoid type (22); the third taken just at the onset of colonization were explanted, cut size class, constituted by cells with a mean diameter of 7 Am, into two parts, and cultured in microwell dishes (96 wells; was mainly of the myeloid type. After centrifugation on a Nunclon, Delta, Nunc) for 3 days in DMEM containing cushion of 28% bovine serum albumin, lymphoid precursor D-valine (GIBCO), 15% fetal calf serum (Flow Laboratories), cells and other immature cells were found in the floating layer ascorbic acid (50 ,ug/ml), reduced glutathione (100 ,ug/ml), corresponding to 10% of the total cells (Fig. lb). All mature insulin (10,ug/ml), and NADH (10,ug/ml) (20). After 3 days, erythrocytes, polymorphonuclear leukocytes, granulocytes, the adherent tissue was washed and the medium was ex- and mastocytes were found in the pellet. When 5 x 10' bone changed by IMDM containing 20% steroid-free fetal calf serum and 1% synthetic serum Ultroser SF. Hemopoietic cells (105) that had migrated were added to the culture. At day _ 80- a 3, cells were harvested and analyzed for their T-cell surface c ...a.....c antigens. 0 60- was on Immunolabeling. Immunofluorescence performed cm0) cell suspensions by using the following mouse monoclonal 40- :::::: :-:...C:---:::--:E.:: ..:: .:..:::::X.::::::-:::---::...... antibodies at dilutions indicated by the supplier: Ox-6 (major 20- : ...... :::::::::::::::::::...... 0 CK : . :. : : : : : : : .: ,-.....:...... :.:..::-1 . . , A, 1 histocompatibility complex class II), Ox-7 (Thy-1), Ox-8 ....l ...... A§,

(CD8), W3-25 (CD4), and Marm 4 (IgM), all from Serotec l- .l . (Oxon, England). Fluorescein-coupled goat anti-mouse im- d e munoglobulin (Nordic, Tilburg, the Netherlands) was used as E 40 c second antibody (1:100). 1 : 2 1 1 Purification of Thymic Chemotactic Peptides. IT-45 R1 cells A' 20 were cultured in DMEM/20% fetal calf serum (Flow Labo- -;.,0 --1 - * - 7 ...kl|--l-f ratories) in plastic roller bottles (850 cm2; Corning). When the 5 6 7 8 9 5 6 7 8 9 cells reached subconfluency at a density of 1.1 x 10' cells per cell diameter (,um) cm2, medium was exchanged for 200 ml ofserum-free DMEM per roller bottle. After conditioning for 3 days, the superna- FIG. 1. Histogram of bone marrow cells before and after in vitro tant was centrifuged, and filtered, and 1 liter was loaded at 80 migration. The diameters of rat bone marrow cells were analyzed on ml/hr onto five Sep-Pak C18 cartridges (Millipore, Waters a Coulter Counter channelizer 256. (a) Fresh bone marrow cells of Associates). The material was eluted from Sep-Pak by 1.5 ml 3-week-old rats. (b) Bone marrow cells enriched for hemopoietic of 80% acetonitrile and loaded onto a Sephadex G-50 (Phar- precursor cells on a bovine serum albumin gradient. (c) Hemopoietic macia) column (1.5 x 100 cm) equilibrated with PBS and run precursor cells from the bovine serum albumin gradient after cocul- at 10 ml/hr. Fractions (2.5 ml) were lyophilized to allow ture on mouse bone marrow stroma for 3 days. Cells with a mean better storage, reconstituted with H20, diluted in DMEM to diameter of 6.2,um were greatly enriched. (d) Hemopoietic precur- the original volume of conditioned medium, and tested for sors after bovine serum albumin gradient that migrated toward IT45 R1 thymic epithelia conditioned medium. The two cell populations chemotactic activity on hemopoietic precursors in Boyden detected by window settings 1 and 2 migrated specifically. The chambers. Aliquots were analyzed on silver-stained NaDod- smaller erythroid lineages also migrated toward control medium and S04/polyacrylamide gels. Peptide concentrations were de- were therefore not counted. (e) Cultured cells that migrated toward termined by the Bio-Rad protein assay. IT45 R1 conditioned medium. Immunology: Imhof et A Proc. Natl. Acad. Sci. USA 85 (1988) 7701 marrow cells, 10 times enriched for precursors and lymphoids Table 1. Discrimination between chemotactic and chemokinetic on bovine serum albumin, were exposed to IT-45 Ri thymic activity of IT-45 R1 conditioned medium on lymphoid type cells epithelial cell-conditioned medium in Boyden chambers, 1.7 Migrated + 0.18% of these cells migrated toward chemotactic factors lymphoid present in this medium. A histogram of cells after chemo- Upper well Lower well cells tactic migration performed on the channelizer 256 showed two migrating cell populations defined by their size (Fig. ld). Control medium Control medium 129 ± 51 These cells were further analyzed after cytocentrifugation Control medium Conditioned medium 358 + 8 and Giemsa-May-Grunwald staining. Large cells (8 ,um on Conditioned medium Control medium 193 ± 58 Coulter Counter) were mostly of the myeloid type but Conditioned medium Conditioned medium 109 ± 42 contained 23% immature blasts, and the medium sized cells Thymic epithelial cell conditioned medium was prepurified on a (6 ,m) contained cells of the lymphoid type. Using an Sep-Pak cartridge and the eluted activity was reconstituted to the original volume by DMEM. Control medium was DMEM. Tests were appropriate window setting on the channelizer 256, it was performed in Boyden chambers with 2.5 x 104 cultured bone marrow possible to distinguish migration of the two major cell cells. Migrated cells were counted and analyzed for their phenotype subpopulations. The 6-,um cell population migrated specifi- by May-Griinwald-Giemsa staining. Lymphoid type cells migrated cally toward thymic epithelial cell conditioned medium, chemotactically toward IT-45 R1 products. Standard deviation was whereas large cells also migrated toward components in the given for three experiments. Phenotype of responding cells was fetal calf serum (data not shown). Therefore, the migration determined by counting at least 250 cells that migrated in preparative effect ofsmall 6-,m cells that contain lymphoids was used for Boyden chambers. subsequent studies. Short-Term in Vitro Culture ofHemopoietic Precursors from chemotactically toward these thymic epithelial cell products Bone Marrow. In an attempt to further enrich hemopoietic (Table 1). For further purification, cartridge eluted material cells that migrate specifically toward thymic factors, rat bone was loaded onto a Sephadex G-50 column. All fractions were marrow cells after bovine serum albumin gradient centrifu- diluted with DMEM to the original volume ofthe conditioned gation were cocultured with mouse bone marrow monolayer medium and tested for chemotactic migration of cultured in the presence of 20% steroid-free fetal calf serum supple- bone marrow cells in Boyden chambers. Chemotactic activ- mented with 1% steroid-free synthetic serum. A cell popu- ity was found in two zones of 11 and 4 kDa (Fig. 3A). Cells lation of intermediate cell size (diameter, 6.2 ,um) appeared that migrated toward the different column fractions were after 24 hr of culture and persisted for several days (Fig. ic). analyzed for their morphological phenotype by May- Most of these cells weakly adhered to feeder cells (Fig. 2). Grunwald-Giemsa staining. The migration profile for lym- Upon exposure to the thymic epithelial cell-derived medium, phoid type cells corroborated with the active fractions that 7% of these cells migrated in Boyden chambers at day 3 of have been found by counting migrated cells by the Coulter culture (Fig. le). Migration competent precursors are there- Counter (Fig. 3A). The purified 11-kDa material was much fore 41-fold enriched under these culture conditions com- more selective than the crude conditioned medium (75 vs. pared to those found in freshly prepared total bone marrow 51% of migrated cells were of lymphoid type, respectively; cells. Noticeably, the contaminating erythroid precursor Table 2). The purified chemotactic activity was further cells disappeared from cultured cells, and cells of the lym- analyzed by NaDodSO4/gel chromatography. Silver-staining phoid type with a diameter of 6 Am were enriched in such identified the 11-kDa material as a single distinct band, which populations (Fig. lc; Table 1). As much as 60%o of migrated we have named thymotaxin. Full chemotactic activity was cells express the Thy-1 molecule, whereas no T-cell (CD4, achieved by using thymotaxin at 14 ng/ml, whereas Sep-Pak CD8) or B-cell differentiation markers (IgM, major histocom- B patibility complex class II) were detectable on these cultured A bone marrow cells, suggesting an immature phenotype. 6000 Isolation ofthe Chemotactic Activity Secreted by the Thymic Epithelial Cell Line IT45 R1. For rapid concentration, serum- 4000 24 kDainI, free IT-45 Ri thymic epithelial cell-conditioned medium was 2000 passed through Sep-Pak C18 cartridges. The cartridges were eluted with (A8a 0 12.4 kDa- 80% acetonitrile and aliquots were lyophilized 0)4) -_o-thymota xin and reconstituted to the original volume by fresh DMEM. 400 00 7.4 kDaj- Checkerboard analysis in Boyden chambers confirmed that ._ cultured bone marrow cells of the lymphoid type migrated 2000 2 '~~~~~~~~' a b c 40 45 50 55 60 Fraction number FIG. 3. Purification ofchemotactic peptides produced by thymic epithelial cells. Conditioned medium concentrated and desalted on Sep-Pak cartridges and fractionated on Sephadex G-50. (A) All fractions were tested for chemotactic migration of cultured bone marrow cells in Boyden chambers (total cells). Phenotype of mi- £, .41 grated cells was analyzed after cytocentrifugation and May- Grunwald-Giemsa staining (lymphoid cells). Eluted material from FIG. 2. Rat hemopoietic precursor cells cocultured on mouse two column regions corresponding to 11 and 4 kDa showed chemo- bone marrow stroma. Bone marrow cells of 3-week-old rats were tactic activity for lymphoid type cells. Standard deviation was enriched for hemopoietic precursor cells on a bovine serum albumin calculated from three experiments. (B) NaDodSO4/PAGE of three gradient and cocultured on mouse bone marrow stroma in steroid- representative column fractions followed by silver-staining tech- free serum. Cells with an average diameter of 6.2 Itm were predom- niques. Lanes a-c correspond to an active and to control fractions inant after 3 days of culture. These precursors were round shaped shown in A. Thymotaxin that eluted in column fraction c could be and adhered around stromalAcells.,~~~~~~~~.4 identified as a distinct band in the region of 11 kDa. 7702 Immunology: Imhof et al. Proc. Natl. Acad. Sci. USA 85 (1988) Table 2. Cytological phenotype of migrated cells Cell source Immat. Erythr. Myel. Monoc. Lymph. blasts prog. prog. PMN macro. Before migration After BSA gradient 29 1 12 2 46 + 3 9 ± 3 2 ± 1 0 After 3-day culture 41 5 2 2 0 28 ± 2 17 ± 4 12 ± 3 After migration toward Total IT-45 R1 medium 51 6 0 0 12 ± 5 35 ± 8 2 ± 1.7 Thymotaxin 75 7 0 0 17 ± 8 9 ± 6 2 ± 1 DMEM control 22 0 0 67 6 5 Bone marrow-derived cells were taken before and after chemotactic migration toward IT-45 R1 conditioned medium, purified thymotaxin, and control DMEM. These cells were then cytocentrifuged and stained in May-Grunwald and Giemsa solution. Numbers represent the percentage of at least 250 counted cells per experiment and standard deviation was given for three independent experiments. Lymphoid type cells were enriched after migration toward the 11-kDa peptide thymotaxin. Lymph., lymphoid cells, Immat. blasts, immature blast cells that did not contain any granules in the cytoplasm and were probably not committed cells; Erythr. prog., erythroid progenitor cells; Myel. prog., myeloid progenitor cells; PMN, polymorphonuclear leukocytes; Monoc. macro., monocytes and macrophages; BSA, bovine serum albumin. eluted material at 25 ,ug/ml or undiluted conditioned medium lymphocyte activation on fresh thymocytes nor cytotoxic (protein concentration, 1.03 mg/ml) was necessary. activity on L929-a target cells (same test used for detection Human IL-1 gives rise to biologically active degradation of tumor necrosis factor activity) was found (Fig. 4). peptides in the same size range that were detected for In Vitro Differentiation of Hemopoietic Precursors. The thymotaxin and the 4-kDa products (23). Furthermore, IL-1 rudiments of 14-day-old rat embryonic thymus were explant- is chemotactic for mature T and B lymphocytes (24). There- ed and cultured in microwell dishes. At this stage ofembryo- fore, recombinant human IL-1 was tested for chemotactic genesis, the rat thymus contains only very few lymphocytes. migration of day 3 cultured bone marrow cells in Boyden In a medium specially designed for this purpose (14, 20), chambers. None of the tested IL-1 concentrations (from 10 epithelial cells form a halo around the explants after 3 days ,ug to 0.1 ng) attracted cultured bone marrow cells (data not of culture (Fig. 5a). Cultured hemopoietic precursors that shown). responded to thymotaxin were cocultured on these thymic Purified thymotaxin and the 4-kDa material were then monolayers after a change to steroid-free medium for an tested for two known biological IL-1 activities. Neither additional 3-day period (Fig. Sb). Cocultured cells expressed de novo the CD8 antigen detected by Ox-8 antibodies (Fig. 5 c and d; 13.5% Ox-8 positive cells). This T-cell antigen was 25 1 totally absent before coculturing on embryonic thymus. Neither migrated hemopoietic cells cultured alone on tissue culture plastic in the same medium, migrated cells toward 20 0 DMEM control medium, nor the thymic tissue alone pro- x duced cells bearing T-cell markers (unpublished data).

_ 1 =w o- CD -0 O- = CD00 10

>-S_

S 5

1 2 4 16 1 2 4 16 inverted dilution inverted dilution (x 1000) (x 1000) FIG. 4. Detection of IL-1 activities in purified IT-45 Ri-derived FIG. 5. Differentiation of hemopoietic precursors by coculture peptide fractions. (A) Mouse thymocyte activation served as detec- with embryonic thymic monolayers. Embryonic thymuses of 14- to tion assay for IL-1 activity. (B) Cytotoxic activity was tested by using 15-day-old rats were explanted and cultured for 3 days before L929 target cells. o, Positive controls: IL-1 activity produced by coculture with hemopoietic cells was initiated. (a) A monolayer of lipopolysaccharide-stimulated monocytes; *, thymotaxin; A, thymic epithelial cells grew out of the organ. (b) Coculture of migrated chemotactic peptides of 4 kDa; o, negative controls: fraction that hemopoietic cells with embryonic thymus. (c and d) Phase contrast eluted at 80 ml from the G-50 column and that showed no chemotactic and corresponding immunofluorescence for the T-cell antigen CD8 of activity. In both assays, the thymic chemotactic molecules were migrated hemopoietic precursors after differentiation by coculture negative at dilutions of optimal chemotactic activity. on thymus. Immunology: Imhof et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7703 DISCUSSION Thy-1 molecules are expressed by precursors that give rise to Our results suggest that in mammals a chemotactic mecha- a variety of hemopoietic lineages. Recent experiments in our nism underlies the colonization ofthe thymus by hemopoietic laboratory showed that a minor population of fresh bone precursors. The polypeptide thymotaxin, produced by a rat marrow cells enriched by chemotactic migration toward thymic epithelial cell line, attracted hemopoietic precursors thymotaxin can also differentiate into T cells in coculture present in the juvenile bone marrow in an in vitro migration with thymic stroma. The responding cells did not contain the assay. Responding cells ofthe bone marrow were enriched in granulocyte, erythroid, macrophage colony-forming cells a short-term coculture system by using steroid-free serum on (GEM-CFC) as assayed in semisolid cultures in the presence a mouse bone marrow feeder layer. These migratory precur- of IL-1, IL-3, and erythropoietin (unpublished data). There- sors had a lymphoid cytology and acquired a T-cell marker fore, thymotaxin might specifically attract committed pre-T when cocultured on monolayers of embryonic thymus. cells; alternatively, it is intriguing to consider that it acts on In our assays, molecules with different size secreted by the stem cells, which could not reach the GEM-CFC stage in our thymic epithelium induced a chemotactic migration on he- semisolid culture system. mopoietic precursors. IL-1 produces a similar molecular We are especially grateful to Monique Denoyelle for excellent mass pattern of peptides as we described for the thymic technical assistance, to Dr. Paule Echinard-Garin for the animal epithelial cell-derived substances (23). In addition, IL-1 has facilities, and to Catherine Dargemont and Thiery Rabilloud for a chemotactic effect on NaDodSO4/PAGE and silver staining. We thank Dr. Jelena Gavri- differentiated lymphoid cells (24), and lovic for critical reading of the manuscript and Charles Dinarello for it can be produced by different epithelial cells such as A431 providing us with human recombinant IL-1. This work was supported carcinoma or human keratinocytes (25). The rat thymic by grants from Centre National de la Recherche Scientifique peptides were tested for IL-1 activity. Both tests lead to the (950914), Institut National de la Santd et de la Recherche Mddicale conclusion that IL-1 activity did not corroborate with the (CRE 86-4015), the Ligue Francaise contre le Cancer, the Associa- biologically active thymotaxin. Production of thymotaxin tion pour la Recherche sur le Cancer, the Fondation pour la seems to be restricted to thymic epithelial cells because other Recherche Medicale, and the March of Dimes Birth Defects (1-993). epithelial cell lines, several carcinomas, a thymic cell line 1. Ritter, M. A. (1978) Immunology 34, 69-76. with a fibroblastic phenotype, and spleen cells do not 2. Ezine, S., Weissman, I. L. & Rouse, R. V. (1984) Nature (London) produce it (unpublished data). 309, 629-631. The 3. Hirokawa, K., Sado, T., Kudo, S., Kamisaku, H., Hitomi, K. & 6.2-,Im hemopoietic precursors responded to the Utsuyama, M. (1985) J. Immunol. 134, 3615-3624. thymic peptides precisely at 3 days of culture. In prolonged 4. Le Douarin, N. M. (1984) Cell 38, 353-360. cultures, these precursors disappeared and a larger cell type, 5. Jotereau, F., Henze, F., Salomon-Vie, V. & Gascan, H. (1987) J. expressing high peroxidase activity was generated; these Immunol. 138, 1026-1030. larger cells responded to a variety of molecules other than 6. Scheid, M. P., Goldstein, G. & Boyse, E. A. (1978) J. Exp. Med. those 147, 1727-1743. specifically secreted by the thymic epithelium (data not 7. Bach, J.-F. (1983) Clin. Immunol. Allergy 3, 133-156. shown). Media selective for the emergence of different 8. Trainin, N., Handzel, Z. T. & Pecht, M. (1985) Thymus 7, 137-150. hemopoietic lineages have been developed. We have modi- 9. Schuurman, H. J., Van de Wijngaert, F. P., Delvoye, L., Broek- fied the xenogeneic culture system described by Hayashi et huizen, R., McClure, J. E., Goldstein, A. L. & Kater, L. (1985) al. (19) since it was suggested that pre-T cells could be Thymus 7, 13-23. preferentially enriched under these conditions. In contrast to 10. Farr, A. G., Anderson, S. K., Marrack, P. & Kappler, J. (1985) Cell 43, 543-550. the steroid-rich Dexter cultures, which give rise to myeloid 11. Savino, W. & Dardenne, M. (1984) Eur. J. Immunol. 14, 987-991. cells (26), steroid low culture conditions favor the mainte- 12. Gay-Bellite, V., Boumsell, L., Caillon, B. & Bernard, A. (1986) nance or the development of lymphoid cells (19, 27). So far, Thymus 8, 201-223. culture conditions allowing the propagation of a pure popu- 13. Ben Slimane, S., Houllier, F., Tucker, G. C. & Thiery, J. P. (1983) lation ofcommitted pre-T cells are not yet available, although Cell DiJffer. 13, 1-24. cells with different of maturation in the or 14. Champion, S., Imhof, B. A., Savagner, P. & Thiery, J. P. (1986) degrees myeloid Cell 44, 781-790. lymphoid lineage can be maintained (28). It is remarkable that 15. Savagner, P., Imhof, B. A., Yamada, K. M. & Thiery, J. P. (1986) Dexter cultures, when shifted to the Whitlock-Witte condi- J. Cell Biol. 103, 2715-2727. tion, provide pre-B cells after 2 weeks (28, 29). These results 16. Le Douarin, N. M., Dieterlen-Lievre, F. & Oliver, P. D. (1984) Am. may reflect the survival of multipotential stem cells for J. Anat. 170, 261-299. lymphoid and myeloid lineages. In the present work, the rat 17. Itoh, T., Aizu, S., Kasahara, S. & Mori, T. (1982) Biomed. Res. 2, bone marrow on mouse stroma 11-19. cells cultured in steroid-free 18. Savino, W., Itoh, T., Imhof, B. A. & Dardenne, M. (1986) Thymus medium may lead after short-term culture to a high percent- 8, 245-256. age of immature lymphoid cells capable of responding to the 19. Hayashi, J., Medlock, E. S. & Goldschneider, I. (1984) J. Exp. thymic chemotactic peptides. However, this response was Med. 160, 1622-1639. limited to day 3 ofculture. This could be due to (i) an eventual 20. Small, M., Barr-Nea, L. & Aronson, M. (1984) Eur. J. Immunol. 14, limitation of responsiveness that corroborates with a defined 936-942. 21. Damais, C., Riveau, G., Parant, M., Gerota, J. & Chedid, L. (1982) early state of differentiation of pre-T cells and that cultured Int. J. Immunopharmacol. 4, 451-458. cells in many systems undergo such first events very rapidly 22. Greiner, D. L., Goldschneider, I. & Barton, R. W. (1982) J. Exp. (30), (ii) cells that only in a certain state of the cell cycle Med. 156, 1448-1460. express receptors for thymic chemotactic peptides, (iii) the 23. Auron, P. E., Warner, S. J. C., Webb, A. C., Cannon, J. G., fact that the lymphoid cells were diluted very rapidly by Bernheim, H. A., McAdam, K. J. P. W., Rosenwasser, L. J., Lo cells. short-term Preste, G., Mucci, S. F. & Dinarello, C. A. (1987) J. Immunol. 138, fast-growing myeloid Nevertheless, cul- 1447-1456. tured bone marrow cells of lymphoid type facilitated our 24. Miossec, P., Yu, C. L. & Ziff, M. (1984) J. Immunol. 133, 2007-2011. migration assay. It made the assay more reproducible be- 25. Sauder, D. N., Mounessa, N. L., Katz, St. I., Dinarello, C. A. & cause of the increased percentage of migrating cells, and it Gallin, I. G. (1984) J. Immunol. 132, 828-832. made it more independent from the condition of animal 26. Dexter, T. & Testa, N. G. (1976) Methods Cell Biol. 14, 387-395. keeping; often bone marrow cells from rats stressed by 27. Whitlock, C. A. & Witte, 0. N. (1982) Proc. Natl. Acad. Sci. USA no 79, 3608-3612. transportation longer responded; however, these animals 28. Johnson, A. & Dorshkind, K. 0. (1986) Blood 68, 1348-1354. were able to recover after 2 or 3 days. 29. Dorshkind, K. (1986) J. Immunol. 136, 422-429. Thymotaxin selects for small Thy-1 + lymphoid cells, 30. Ziller, C., Dupin, E., Brazeau, P., Paulin, D. & Le Douarin, N. M. which could acquire a T-cell marker in vitro. In rats, the (1983) Cell 32, 627-638.