Immunology: Correction Proc
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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 chemo- tactic 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).