THE ANATOMICAL RECORD 221:714-719 (1988) In Vivo Homing of Thymus-Enriched Bone Marrow Cells KURT G. KLUSSMANN AND JACK L. HAAR Department of Anatomy, Division of Immunology, Medical College of Virginia, Virginia Commnwealth University, Richmond, Virginia 23298 ABSTRACT A population of adult CBNJ mouse bone marrow (BM)cells enriched by in vitro migration to supernatant prepared from neonatal thymus was labeled with a DNA-binding fluorochrome, Hoechst dye No. 33342 (H33342). Labeled cells were injected into irradiated recipients in order to compare the in vivo localization of the migration-enrichedBM (MEBM)cells to the localization of injected nonenriched BM (NEBM) cell controls. A characteristic difference in the distribution of localized cells was observed in the spleen but not in other lymphoid organs. At 2 hr after injection the MEBM cells were located in the marginal zones surrounding the periarterial lymphoid sheaths (PALS)of the splenic white pulp. At 6 hr after hjection the MEBM cells were seen distributed between marginal zones and the PALS and by 16 hr they had localized almost exclusively in the white pulp. In contrast, the NEBM cells were located in the marginal zones or red pulp for the duration of the experiment. These observations show that the MEBM cells home selectively to T-cell areas of the spleen. Direct immunofluorescentmonoclonal antibody staining of H33342labeled cells obtained from the recipient spleens at 16hr demonstrated that the MEBM cells were negative for Thy-1 antigen, indicating that acquisition of Thy-1 was not prerequisite to the observed homing. The results are compared to known localization patterns of mature lymphocytes. The lymphoid system of mammals is an orderly arrangement of cells and tissues distributed throughout the body. The lymphoid organs, interconnected with cells circulating through blood, lymph, and connective tissue, are composed mainly of bone marrow (BM), thymus, spleen, lymph nodes, and subepithelial tissues of the gastrointestinal and respiratory tracts. Considerable information exists concerning the origin and distribution of cells in lymphoid organs, including the areas occupied by lymphocyte subsets (reviewed in Osmond, 1985). For example, mature T and B cells can be located in splenic periarterial lymphoid sheaths (PALS) and marginal zones, respectively, by a variety of methods (Jonjic et al., 1987; Rolstad et al., 1986; Tabibzadeh and Gerber, 1986; Brenan et al., 1985; Timens and Poppema, 1985; Murray et al., 1984; Rouse et al., 1984; Witmer and Steinman, 1984; Kumararante and MacLennon, 1981; Veerman and van Ewijk, 1975). Questions that naturally arise in the study of lymphocyte localization address specific homing mechanisms and the developmental stages a t which a homing capacity is acquired. In the case of T cells it is known that prethymic cells, derived during embryogenesis from fetal yolk sac and liver (Hemmingson and Alm, 1973; Moore and Metcalf, 1970) and then BM in the developing mammal (Rosenthal et al., 1983; Ford et al., 1966; Moore and Owen, 1967b),enter the thymus, where they undergo maturation into potentially immunocompetent cells (Moore and Owen, 1967a), which enter the circulation and localize, at least transiently, throughout the 0 1988 ALAN R. LISS, INC. lymphoid system (Le Gross et al., 1983; Rocha et al., 1983). It is uncertain whether cells of the T-cell lineage must first enter the thymus before acquiring the ability to home to lymphoid tissues, although experiments that might favor such a conclusion are reported (Rouse et al., 1984; Carrol and de Sousa, 1983; Le Gross et al., 1983; Van Ewijk et al., 1982). Ideally, prethymic stem cells would be isolated, labeled, and injected into recipients where their localization could be observed. However, the complete isolation of prethymic BM cells, or prothymocytes, defined by their ability to repopulate the T-cell compartments of irradiated recipients, has not yet been accomplished (reviewed in Silverstone and Cayre, 1985). In an effort to determine what signals prethymic cells to enter the thymus, several studies utilizing in vitro migration assays in mice (Haar and Loor, 1981; Haar et al., 1987; Taubenberger and Haar, 1987a)b) and in birds (Champion et al., 1986) have demonstrated the ability of factors derived from the thymus to act as chemoattractive agents to subpopulations of BM cells. Received September 21, 1987; accepted December 9, 1987. Address reprint requests to Dr. Jack L. Haar, Department of Anatomy, P.O. Box 709, MCV Station, Richmond, VA 23298. HOMING OF THYMUS ENRICHED BONE MARROW CELLS 715 Such cells appear to be the progenitors of mature T cells; they home to the thymus in vivo and they lack surface antigens characteristic of mature T cells (but have been shown to develop mature T-cell markers after arriving in the thymus in the avian system). From those studies we conclude that thymic epithelium produces chemoattractive peptides that may specifically recruit BM precursors destined to develop into mature T cells, which in turn localize according to their role in lymphoid tissues. Moreover, analogous or homologous mechanisms are working in birds and mice. The present study demonstrates utilization of the in vitro migration assay of Haar and Loor (1981) to collect migration-enriched BM (MEBM)cells. The in vivo homing of injected MEBM cells labeled with H33342 (according to Brenan et al., 1985) in lymphoid organs of irradiated recipients is compared with the homing of nonenriched BM (NEBM) cells. A characteristic difference in homing by MEBM cells is seen in the spleen: MEBM cells home to T-dependent areas. The results are discussed in terms of the homing and distribution of mature lymphocytes. lowing modifications: Chemotactic chambers (Nuclepore 440900) with upper and lower well volumes of 0.2 ml separated by a 13-mm diameter, 5-pm pore size filter (Nucleopore 110413)were used. The bottom (blind)wells were filled with 0.2 ml TS (experimental) or 0.2 ml IMDM alone (control). To the upper wells, 0.2 ml of the BM suspension (containing 1 x lo6 cells) was added. Chambers were incubated for 90 min at 37°C in a humidified atmosphere of 5% CO, in air. After incubation the cells that migrated to the bottom wells were collected, washed in PBSBSA, and counted in a hemacytometer, and their viability was ascertained by trypan blue exclusion. A migration index for each group of chambers was calculated as the percentage of the cells applied to the top well that were collected in the bottom well. The pooled MEBM cells from wells containing TS were pelleted in preparation for labeling. In order to determine what direct influenceTS might have on NEBM cells, which were not subjected to the migration assay, some NEBM cells were incubated in TS (1 x lo6 cells per 1 ml) for 90 min at 37°C prior to labeling. MATERIALS AND METHODS Animals Two populations of MB cells were labeled with H33342 by the method outlined by Brenan and Parish (1984): 1) MEBM cells and 2) NEBM cells-BM cells that were not subjected to the enrichment migration. Prior to labeling, all cells had been washed twice in PBSBSA and pelleted. In all experiments (1-4) x 106 pelleted MEBM cells and an equal number of NEBM cells were resuspended in a 6-pglml solution of H33342 in phosphatebuffered saline (PBS) to a concentration of 1.5 x lo6 cells per 1 ml. The suspensions were incubated for 15 min at 37°C. Labeling was stopped by adding cold PBS. The cells were washed twice in PBS and resuspended at a concentration of 1 x 108 cells per 1ml for injection. Age- and sex-matched syngeneic 6 to 8-week-old adult CBA/J mice of both sexes were used as BM cell donors and recipients. Recipients were irradiated (900 rads) with a Picker teletherapy radiation unit equipped with a cobalt 60 gamma ray source (Neutron Products, Inc.) 24 hr prior to injection. Neonatal (4- to 6-day-old)CBA/ J mice were used to prepare thymus supernatant (TS). Thymus Supernatant Preparation The procedures followed to make TS as an attractant for BM cells were those previously reported by Haar and Loor (1981),with the following modifications: The thymus was removed from decapitated neonatal mice and cut into fragments (8-10 equal pieces per thymic lobe) under a dissecting microscope. Fragments from 3-4 neonates were placed in 35-mm petri dishes containing 3 ml Iscove's modified Dulbecco's Medium (IMDM) (Gibco 430-2200) and incubated at 37" for 48 hr. After incubation, the culture medium was centrifuged to remove cells and debris, sterilized by filtration through a 0.22-pm disposable filter assembly (Gelman, 41921, and stored a t - 70°C until used. Bone Marrow Cell Preparation Femora and tibiae were dissected from adult mice sacrificed by cervical dislocation. Cells were obtained by flushing the marrow cavity with 5 ml phosphatebuffered saline with 0.5% added bovine serum albumin (PBSBSA). Following sedimentation for 5 rnin at lg, erythrocytes were lysed with 0.05 M Tris-NH,C1 buffer, washed in PBSBSA, and counted in a hemacytometer; viability was determined by trypan blue exclusion. Cells were resuspended in IMDM at a concentration of 5 x 106 viable cells per 1 ml in preparation for migration to TS. Migration of Bone Marrow Cells to Thymus Supernatant BM cells were collected after migration to TS as previously described (Haar and Loor, 1981) with the fol- Labeling With H33342 Injection of Cells and Tissue Preparation Into the tail vein of each animal (1-4) x lo6H33342labeled MEBM or NEBM cells (the same number for each pair of animals) were injected in a volume of 0.1 -0.4 ml PBS into each of three to six mice for each time period specified. At 2, 6, and 16 hr after injection, recipient spleen, pelvic lymph nodes, distal ileum, liver, lung, and thymus were dissected free. The spleen was weighed immediately upon removal and half (weighed separately) was placed in 2 ml PBSBSA to prepare a cell suspension. The remaining spleen and other tissues were placed into specimen molds (Miles 4565) and COVered with embedding medium (Miles 4583) for 30 rnin in the dark a t 4°C prior to freezing. The tissues were snap-frozen for 2 min in isopentane cooled with liquid nitrogen and transferred to a cryostat microtome (Damon/IEC 3398), where they equilobrated to - 18°C for 1 hr in the dark. Tissue sections were cut at 16-pm thickness, mounted onto acid-cleaned glass slides, and stored in the dark a t 4°C. Tissues obtained 16 hr after injection of NEBM cells, incubated with TS prior to labeling, were prepared from two animals. Tissues from animals injected with unlabeled MEBM and NEBM cells were also prepared to serve as negative controls. In order t o verify the splenic architecture seen in unstained tissue sections, several adjacent sections were prepared for light microscopy with 0.1% methylene blue/ 7 16 K.G. KLUSSMANN AND J.L. HAAR toluidine bluelsodium borate after fixing with a 4% formaldehydelglutaraldehyde for 2 min. labeled cells were also observed t o verify the absence of autofluorescence by injected or resident cells. Counting and Antibody Labeling of Cells Localized in the Spleen RESULTS The percentage injected cells localized in the spleen was determined from cell suspensions of weighed spleen. Spleen halves were pressed gently through a fine-wire mesh washed with 5 ml PBS, leaving the capsule behind. Suspensions of the total cells obtained were prepared and counted exactly as were the BM cells. The number of H33342-labeled cells in each suspension was determined with a hemacytometer under epifluorescent illumination. The total number of cells that had homed to the spleen was calculated by multiplying the number of H33342-labeled cells counted in suspension by the ratio: weight of whole spleedweight of splenic half used for suspension; thus, the calculations are based on the assumption that injected cells are distributed evenly throughout both halves of the spleen. The total cells collected from the spleen 16 hr after injection were labeled with rhodamine-conjugatedmonoclonal anti-mouse Thy-1.2 antibody (a-Thy-1; Miles 63351). The collected cells were resuspended in Dulbecco's modified Eagle's medium with 5%added fetal calf serum (DMEM/FCS)to a concentration of 1 x lo6 cells per 1 ml. Equal volumes of cell suspensions and a 1:lOO dilution of a-Thy-1 in DMEMIFCS were incubated for 15 min at 4°C in the dark. Cells were washed twice with PBS and were counted in a hemacytometer. A total of 400 of each of H33342-positive MEBM and NEBM cells were examined for Thy-1. Positive controls were H33342labeled BM donor thymocytes treated identically to the splenic cells. Unlabeled thymocytes were observed for autofluorescence. Fluorescent Microscopy A Nikon microscope with a mercury vapor lamp and exciter and barrier filters for epi-illumination of H33342 (365-nm excitation and >420-nm barrier filters) and rhodamine (550-nm excitation and >580-nm barrier filters) was used throughout. Tissue sections were observed and photographed within 1hr of sectioning, and cell suspensions were counted immediately after preparation. Tissue from control animals injected with un- Epifluorescent microscopic examination of tissue sections revealed injected H33342-labeled MEBM and NEBM cells in the spleen, lymph nodes, liver, lung, Peyer's patches, and thymus at 2, 6, and 16 hr after injection. The H33342-labeled cells were clearly seen in each section as brightly fluorescent individual cells, which were best visualized in spleen tissue sections (Fig. 1 a-f) where characteristic differences between MEBM and NEBM cell localization was observed. In the spleen, red and white pulp areas were easily distinguished as light and dark areas, respectively, in unstained tissues under epifluorescent illumination (Fig. 1). At 2 hr after injection both MEBM and NEBM cells were located in the marginal zone and in the red pulp (Fig. l a , b). At 6 hr the MEBM cells were distributed in the marginal zone and the white pulp (Fig. lc), whereas the NEBM cells remained predominately in the marginal zone or red pulp (Fig. Id). By 16 hr after injection, MEBM cells were located within the white pulp (Fig. le), whereas NEBM cells were seen primarily in the marginal zone (Fig. 10. NEBM cells incubated with TS prior to injection were distributed identically to the NEBM cells at 16 hr. In order to confirm the localization described, adjacent splenic tissue sections stained for light microscopy (not shown) were examined, verifying that MEBM cells were located in the white pulp, frequently surrounding small central arterioles. Germinal centers contained few of either injected cell types. The distribution of the injected cells in other tissues (not shown) was generally sparse at all times examined, and no obvious differences in distribution patterns was noted. The greatest number of injected cells of either type per tissue section were observed, in decreasing order, in spleen, lung, liver, lymph nodes, Peyer's patches, and thymus. The total number of injected MEBM and NEBM cells was calculated for the spleen. As shown in Table 1,the greatest average number of injected cells was counted in the spleen a t 16 hr, although there was no statistical difference (based on a Student t test, P < 0.05)between the number of MEBM TABLE 1. Percentage of injected cells recovered from recipient spleen and migration indices' of MEBM cells injected' Time after injection 2hr 6hr 16 hr Cells recovered Cell type injected 6%* S.D.) MEBM NEBM MEBM NEBM MEBM NEBM 2.6 f 0.4 1.7 0.1 7.0 2 1.0 5.5 2 0.2 9.0 2 2.5 8.4 2.0 * * Migration index (5% f S.D.) of BM cells migrated to: TS IMDM 17.1 2 5.3" 3.7 2 1.6 13.7 2 5.8" 3.2 & 0.1 15.2 4.8 f 2.0 2 4.9" 'Migration index defined as percentage of cells applied to top well that are collected in bottom well of migration chamber. 'Abbreviations: MEBM, migration-enriched bone marrow; BM, bone marrow; TS, thymus supernatant; IMDM, Iscove's modified Dulbecco's medium (control); NEBM, nonenriched bone marrow. *Statistically significant difference (P< 0.05) between in vitro migration to TS vs. IMDM (Student t test). HOMING OF THYMUS ENRICHED BONE MARROW CELLS Fig. 1, Sections of spleen viewed under epi-illumination demonstrating localization differences between MEBM cells (a,c,e)and NEBM cell contmls at 2 hr (a,b), 6 hr (c,d), and 16 hr (e,D after injection into jrradiated recipient mice. Individual cells are brightly fluorescent. Note that MEBM cells are located predominately in the marginal zones at 2 hr,that MEBM cells have distributed into the RP and particularly the 717 marginal zones and white pulp at 6 hr; and that MEBM cells home almost exclusively within the white pulp at 16 hr. In contrast, NEBM cells remain predominately in the marginal zones or red pulp for the duration of the experiment. MZ, marginal zones; RP, red pulp; WP, white pulp. a-d, X 180 e,f, X 118. K.G. KLUSSMANN AND J.L. HAAR 718 and NEBM cells at any one time studied. Furthermore, there was no statistically significant difference in the number of either MEBM or NEBM cells at 6 or 16 hr but there were significantly more cells at those two times than at 2 hr. Based on these data (Table l),an average of 1.7-2.6% of injected NEBM or MEBM cells appeared in the spleen by 2 hr after intravenous injection, and the number of cells of either type increased to an average of 5.5-9.0% from 6 to 16 hr. Thus, by 2 hr there was neither a difference in the number of localized MEBM or NEBM cells nor a difference in their apparent distribution within the spleen (Fig. la,b). In contrast, there was was a distinct differencein the splenic localization between MEBM and NEBM cells by 6 hr (Fig. lc,d) and 16 hr (Fig. le,Q after injection although there was still no significant difference in the number of cells that had homed. Table 1also shows the migration indices of the MEBM cells injected for each time period evaluated. The migration index is a measure of the selectivity with which BM cells migrate in vitro, as previously reported (Haar and Loor, 1981). Overall, the number of BM cells migrated to TS was significantly more than those that migrated to media alone. The net difference between the percentage of cells that migrated t o TS and those that migrated to media alone is on the order of 10% and is assumed to reflect the actual percentage of BM cells that respond to TS. Their morphology has previously been described (Haar and Loor, 1981). Antibody labeling of splenic cell suspensions with aThy-1 a t 16 hr after injection demonstrated that all of the MEBM cells were negative for Thy-1, compared to 4.0 & 1.0% (mean percentage ? S.D.) NEBM cells that were positive. Control H33342-labeled Thy-1.2 mouse thymocytes were clearly double-labeled with a-Thy-1. The morphology of individual MEBM and NEBM cells was observed and showed the majority of cells to be mononuclear lymphoid cells. DISCUSSION The localization of H33342-labeledMEBM and NEBM cells in the spleen a t 2 hr after injection (Fig. la,b) indicates that both cell populations enter the spleen in the marginal zones, as suggested for mature B and T cells seen at a similar time after injection of H33342labeled lymphocytes (Brenan et al., 1985). The difference in distribution betwen the MEBM (Fig. lc) and NEBM (Fig. Id) cells at 6 hr after injection can be explained by a tendency of the MEBM cells to home towards T-cell-dependent regions of the spleen, in the splenic white pulp, whereas the NEBM cells tend to remain in the marginal zone or move into the red pulp, in a direction outward from the PALS. The selective homing behavior of MEBM cells is further evidenced at 16 hr aRer injection when the majority of cells are within the PALS (Fig. le). This same tendency to home selectively to and localize in the white pulp has been demonstrated for mature T cells at similar times by Brenan et al. (1985),although the percentage of injected mature T cells collected from spleen (23.2%) in their study is greater, and is maximal at 2 hr after injection, compared with an average of 9.0% MEBM cells collected here at 16 hr (Table 1). In contrast, the NEBM cells remain in the marginal zone or red pulp, although a few can be seen in the white pulp a t 16 hr. Since the MEBM cells are a subpopulation of NEBM cells and because NEBM cells are made up, in part, of mature (Thy-l-positive) lymphocytes, it might be expected that some NEBM cells appeared in the white pulp. A question that arises is whether the TS migration assay simply selects for a population of MEBM with an intrinsic homing capability or whether it also modifies the homing properties of the MEBM cells during their 90 min incubation with TS. It appears that TS alone is not sufficient to confer the homing ability observed for MEBM cells; NEBM cells incubated with TS prior to injection were distributed in the same manner as NEBM cells unexposed to TS. However, the potential direct influence of TS on the homing of MEBM cells remains uncertain. It is still possible that TS not only selects MEBM cells but also modifies their homing properties. ’ The homing ability of MEBM cells does not appear to correlate with the expression of Thy-1 antigen. The MEBM cells do not express it before (Haar and Loor, 1981) or up to 16 hr after injection. It may be that MEBM cells possess the marker in quantities insufficient to be demonstrated here. Thy-1 has been detected on up to 25-30% of murine BM cells (Berman and Basch, 1985) including prothymocytes. Basch and Berman (1982) showed that pretreatment of injected BM cells with monoclonal a-Thy-1 reduced the thymic repopulation in irradiated recipients, compared with no effect on repopulation with conventional a-Thy-1 antiserum (see also Boersma et al., 1981). The in vivo mechanisms by which MEBM cells home to T-cell areas in the spleen are presumed to be analogous to the incompletely understood mechanisms governing mature T cells. In the case of mature T cells, migration to lymph node and Peyer’s patch (reviewed in Rouse et al., 1984) occurs through high endothelial venules (HEV).But the spleen does not possess classical HEV (Kraal et al., 1983). Futhermore, the ability to to migrate through specialized vessels does not in itself account for T-cell and B-cell localization differences once the cells have entered lymphoid tissues. A more likely explanation (Rouse et al., 1984; Witmer and Steinman, 1984; Veerman and van Ewijk, 1975) involves direct cell contact between lymphocytes and interdigitating or follicular dendritic cells, respectively, in T-cell and Bcell areas once the cells enter lymphoid tissues. Another unresolved issue is the developmental age at which lymphocyte progenitors and subsets possess the ability to home. It has been shown (reviewed in Rouse et al., 1984) that immature BM precursors do not bind nearly as well to HEV as mature B and T cells, indicating that this ability is acquired through cell maturation. Furthermore, the homing abilities of mature T cells differ depending on the organ source and type of antigenic determinants expressed (Jonjic et al., 1987; Kraal et al., 1983; Rocha et al., 1983; Carroll and de Sousa, 1983; Kumararatne and MacLennon, 1981). In this study we demonstrate that MEBM cells, presumed to represent immature lymphoid cells, possess the ability to home to T-cell-dependent splenic areas. The observed migration of MEBM cells is an important finding; it shows that there is a Thy-l-negative BM cell population capable of selective homing to T-cell areas, at least in the spleen. And though there is evidence that functional T cells must first mature in the thymic microenvironment in order to repopulate lym- HOMING OF THYMUS ENRICHED BONE MARROW CELLS phocyte-depleted tissues (Le Gross et al.,1983; Boersma et al., 198l),our data suggest that specific localization to T-cell areas may not require physical interaction between thymic stromal and BM cells. Although we cannot rule out the possibility that MEBM cells have passed through the donor thymus prior to isolation from the BM, the absence of surface Thy-1 on MEBM cells, even after localizing in the spleen at 16 h r argues against such a conclusion. Our results add credence to the suggestion that MEBM cells represent prethymic cells resident in the BM. The migration indices of MEBM cells (Table 1)are in good agreement with previous work (Haar and Loor, 1981; Taubenberger and Haar, 1987a) that further characterizes the in vitro migration of MEBM cells. Since the original characterization of the in vitro migration assay, considerable work has demonstrated a selective migration of BM cells to thymic epithelial cell culture medium (Taubenberger and Haar, 1987a) which contains peptides (Taubenberger and Haar, 1987b) responsible for the chemoattraction. Other work (Haar et al., 1987) employing flow cytometry analysis has shown significantly more MEBM cells homing to the thymus in vivo although in the present study we were unable to observe a morphological difference in the distribution of MEBM and NEBM cells in thymic sections (not shown). The results presented here are the first description of H33342-labeled BM cells localized in irradiated recipients. The methods used were particularly useful for describing the profound morphological differences in in vivo homing observed between MEBM and NEBM cell populations, differences that were not reflected in the absolute numbers of homing cells. 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