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Regulation of naı̈ve fetal T-cell migration by the chemokines Exodus-2 and Exodus-3
Immunology Letters 69 (1999) 269 – 273
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Regulation of naı¨ve fetal T-cell migration by the chemokines Exodus-2 and Exodus-3 Kent Christopherson II a, Zacharie Brahmi b, Robert Hromas a,* a
Department of Biochemistry/Molecular Biology and Internal Medicine, Indiana Uni6ersity Cancer Center, R4 -202, 1044 West Walnut Street, Indianapolis, IN 46202, USA b Department of Medicine and Microbiology/Immunology, Indiana Uni6ersity Medical Center, RI-0615, 702 Barnhill Dri6e, Indianapolis, IN 46202, USA Received 22 March 1999; accepted 12 April 1999
Abstract We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3a, Exodus-2/6Ckine/ SLC/TCA4, and Exodus-3/Mip-3b/CKb11/ELC. These chemokines share an amino terminal AspCysCysLeu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naı¨ve human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4 + cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+ . Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue. © 1999 Elsevier Science B.V. All rights reserved. Keywords: CC Chemokine; Exodus-1/LARC/Mip-3a; Exodus-2/6Ckine/SLC/TCA4; Exodus-3/Mip-3b/CKb11/ELC; Naı¨ve fetal T-cell migration; T-cell ontogeny; Cord blood; CD45RA; CD45RO; CD44
1. Introduction Chemokines are a family of homologous proteins that direct leukocyte migration and activation to inflammatory stimulus [1 – 5]. The chemokine family can be divided into two groups depending on where the first two cysteines are located. The CXC family (also referred to as alpha) has a single amino acid between the initial two cysteines. If the first two cysteines are adjacent to each other, they are classified in the CC family (beta). Most CXC members are located in a gene cluster at human chromosome 4q12-21, whereas most CC members are in a cluster at human chromosome 17q11-32 [6–8]. Chemokines act through seven membrane-spanning G-protein-coupled receptors [9– 11]. Chemokines mediate inflammatory tissue destruc* Corresponding author. Tel.: +1-317-2743589; fax: + 1-3172740396. E-mail address: [email protected] (R. Hromas)
tion in a wide variety of human diseases, such as rheumatoid arthritis, myocardial infarction, and adult respiratory distress syndrome [12–18]. We and other workers have shown that some chemokines, especially members of the CC family, such as MIP-1 alpha, are able to decrease the proliferative status of myeloid progenitors at low concentrations [19–22]. This effect occurs in a primitive hematopoietic progenitor. The proliferative inhibition of marrow progenitors by some chemokines may protect the progenitors from cell cycle-specific chemotherapeutic cytotoxicity (reviewed in Ref. [22]). Several reports have shown that the CC chemokines MIP-1 alpha, MIP-1 beta, and RANTES inhibit HIV-1 production in newly infected peripheral blood mononuclear cells [23–25]. The HIV co-receptors (with CD4) are chemokine receptors, suggesting a mechanism for the inhibition of HIV infection by some chemokines [26–28].
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K. Christopherson et al. / Immunology Letters 69 (1999) 269–273
We and other workers recently cloned a sub-family of three novel CC chemokines that share a unique (among all chemokines) amino terminal AspCysCysLeu sequence. These chemokines were termed Exodus-1 (also known as Mip-3a and LARC) [29 – 31], Exodus-2 (also known as 6-Ckine and SLC) [32 – 34] and Exodus-3 (also known as Mip-3b, CKb-11, TCA4, and ELC) [30,35,36] for their ability to preferentially regulate the migration of adult T cells. Although the events that direct movement of adult circulating T cells are being elucidated, the regulation of naı¨ve fetal T-cell migration is less well understood. In this study, we investigated the ability of this subfamily of chemokines to control the migration of naive cord blood T cells as compared with adult peripheral blood T cells. We found that Exodus-2 and -3 were potent regulators of naive cord blood T-cell chemotaxis.
2. Materials and methods
2.1. Chemokines All chemokines were purchased commercially from R&D systems (Minneapolis, MN), reconstituted, and stored according to the manufacturer’s instructions.
All chemotaxis data represent the average of three experiments, each done in triplicate. Comparisons were made using the Student’s t-test.
2.3. Flow cytometry T cells in the upper and lower chambers were stained by monoclonal antibodies (mAbs) to fluorochrome-conjugated CD3 (all antibodies from Caltag Laboratories Burlingame, CA), and counted by FACscan. CD44+, CD45RA+ , CD45RO+ , CD4+, and CD8+ subtypes of T cells were quantitated in the upper versus lower chambers with fluorescent mAbs to CD44, CD45RA, CD45RO, CD4, CD8, and CD3, and analyzed by FACscan. Harvested cells from both the upper and lower chambers were washed in phosphate buffered saline (PBS)/1% BSA and resuspended in 100 ml PBS/ BSA. One microgram of the appropriate antibody was added, mixed, and the cells incubated at 4°C in the dark for 30 min. The cells were then washed twice in PBS/BSA and fixed in PBS/1% paraformaldehyde for FACscan analysis at 488 nm. Ten thousand events were accumulated for each analysis.
3. Results
2.2. Chemotaxis assays
3.1. Stimulation of adult peripheral blood T-cell subset chemotaxis
Chemotaxis assays were performed as previously described with minor modifications [32]. Half-a-million normal human fetal cord or adult peripheral blood low density non-adherent leukocytes in 200 ml of RPMI media supplemented with 0.5% bovine serum albumen (BSA) were added to the upper chamber of a Costar transwell (6.5 mm diameter, 5 mm pore size, polycarbonate membrane). The T-cell subset percentage of the input cell population was measured by flow cytometry, as described later. Chemokines were added to 1 ml of the identical media in the lower chamber. Total cell migration was obtained by counting the cells in the lower chamber versus the cells remaining in the upper chamber after 3 h incubation at 37oC, 5% CO2. Cells were then harvested by washing the upper and lower sides of the transwell membrane, and adding the wash to the respective upper or lower media. Chemokines placed only in the upper chamber abrogate migration to the lower chamber. This was done as a checkerboard control assay for each distinct chemotaxis assay. In those transwells that contained chemokine placed only in the lower chamber, the percent of cell migration was calculated by dividing the number of the cells in the lower chamber by the total cell input and then subtracting random transwell migration to the lower chamber without chemokine presence.
When successive concentrations of Exodus-1, -2, and -3 chemokines are used as stimulation for adult peripheral blood total T-cell migration, there is a dose-dependent increase in chemotaxis (Fig. 1a) for all three chemokines, as previously described. This was statistically significant using Student’s t test for all concentrations tested for all three chemokines [29,32]. Exodus-2 proved to be the most effective chemoattractant for adult T cells. Of the input cells, 31.79 0.2% (SEM) migrated across the transwell membrane at the most effective concentration of Exodus-2 (400 ng/ml) compared with 16.3 9 1.4% for Exodus-1 and 24.7 91.3% for Exodus-3 at the same concentration. Flow cytometric analysis of the migration of T-cell subsets was performed to assess if there were differential effects of these three chemokines on specific types of T cells. The composition of the migrated cell population was compared with the total cell population of the adult peripheral blood (Fig. 2). Adult peripheral blood CD4+ T cells (PB-CD4 + ) showed more migration than CD8+ T cells (PB-CD8+ ) for all three chemokines (Fig. 3). Of adult CD4+ T cells, 1093.3, 20.39 5.7, and 18.29 4.6% migrated in response to Exodus-1, -2, and -3, respectively at 200 ng/ml; whereas 4.5921, 8.69 4.0, and 7.4 9 3.2% of CD8 + T cells responded to Exodus-1, -2, and -3, respectively at 200 ng/ml. It is
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Fig. 1. (a) Response in chemotaxis to successive concentrations of Exodus-1, -2, and -3 chemokines used as stimulation for normal adult peripheral blood T-cell migration. (b) Response in chemotaxis to successive concentrations of Exodus-1, -2, and -3 chemokines used as stimulation for normal fetal cord blood T-cell migration.
also of note that within the CD4+ T-cell population of both peripheral blood and cord blood, Exodus-2 and -3 showed more chemotactic ability than Exodus-1 (Fig. 3). For CD4 + adult peripheral blood T-cell migration, Exodus-2 was twofold and Exodus-3 was 1.8-fold higher than that of Exodus-1. CD44+ T cells (PBCD44+ /3+) migrated more in response to Exodus-2 (17.491.1%) and Exodus-3 (18.490.5%) than to Exodus-1 (3.49 0.3%) at 200 ng/ml (Fig. 4). There was no difference between chemotactic effectiveness of all three Exodus chemokines within CD45RO+ adult T cells (PB-CD45RO +). Within CD45RA+ adult T cells (PB-CD45RA +), Exodus-2 (25.490.0%) and Exodus3 (20.9 90.2%) showed a greater ability than Exodus-1 (10.39 0.7%) to chemoattract at 200 ng/ml (Fig. 5).
3.2. Stimulation of cord blood T-cell subset chemotaxis When Exodus-1, -2, and -3 were tested for stimulation of cord blood T-cell chemotaxis, statistically significant dose-dependent activation of chemotaxis was seen for Exodus-2 and -3, but not for Exodus-1 (Fig. 1b). Exodus-2 and -3 were equally potent chemoattractants for cord blood T cells. Of input cord blood total T cells, 28.79 2.9% migrated across the transwell membrane in response to 400 ng/ml of Exodus-2, and 269 2.8% migrated to 400 ng/ml of Exodus-3. These values are compared with a 8.0 94.4% migration in response to Exodus-1 at 400 ng/ml. Flow cytometry was used to analyze whether the Exodus chemokines had differential effects on cord blood fetal T-cell subsets (Figs. 3 – 5). There were several remarkable differences. CD44+ cord blood T cells (CB-CD44+/3+ ) were preferentially chemoattracted across the transwell membrane by Exodus-2 at 50.89 0.6%, Exodus-3 at 50.794.2%, and Exodus-1 at 6.99 1.9% at 200 ng/ml (Fig. 4). For both Exodus-2 and -3, CD4+ cord blood T cells (CB-CD4+) were preferen-
tially stimulated to migrate as compared with CD8+ T cells (Fig. 3). Of CD4+ cells, 259 4.9% migrated in response to 200 ng/ml of Exodus-2 and 30.895.8 in response to 200 ng/ml of Exodus-3. Only 9.79 5.1% of CD8+ T cells migrated for Exodus-2 and 13.2 97% for Exodus-3 at 200 ng/ml. As was mentioned earlier, within the CD4+ T-cell population of both peripheral blood and cord blood cells, Exodus-2 and -3 showed a greater chemotactic ability than Exodus-1 (Fig. 3). Exodus-2 had 5.5-fold and Exodus-3 had 6.6-fold the CD4+ cord blood T-cells migration resulting from Exodus-1. Essentially, all of the cord blood T cells were CD45RA+ (CB-CD45RA+ ), whereas none were CD45RO+ (CB-CD45RO+ ) (Fig. 2). These CD45RA+ naive cord blood T cells were chemoattracted well by Exodus-2 (30.59 2.2%) and Exodus-3 (34.29 2.9%), but not by Exodus-1 (6.29 0.0%) at 200 ng/ml (Fig. 5).
Fig. 2. Composition of the total cell population of normal human fetal cord blood (CB) or normal human adult peripheral blood (PB) low-density non-adherent leukocytes.
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Fig. 3. Migration of CD4 + and CD8 + normal human adult peripheral blood (PB) T cells, and migration of CD4 + and CD8 + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
Fig. 5. Migration of CD45RO+ and CD45RA+ normal human adult peripheral blood (PB) T cells and migration of CD45RA + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
4. Discussion
all cord blood T-cells were CD45RA + , indicating that they were naı¨ve. Cord blood CD4+ , CD44+, and CD45RA+ T cells were attracted at much greater efficiency by Exodus-2 and -3 than Exodus-1. CD4+ cord blood T cells were preferentially stimulated to migrate compared with CD8+ in response to Exodus2 and -3. It is clear from these data that Exodus-2 and -3 preferentially chemoattract both cord and peripheral CD4+ /CD45RA+ T cells. Exodus-2 and -3 stimulated the migration of cord blood CD44+ T cells 2.3-fold better than peripheral blood CD44+ T cells, and stimulated the migration of cord blood CD45RA+ T cells 1.2- and 1.6-fold (respectively) that of adult peripheral blood CD45RA+ T cells. There is evidence that Exodus-2 and -3 play a role in T-cell adhesion to high endothelial venules in lymph nodes, and may be critical in adult T-cell trafficking through nodes [34]. It appears that Exodus-2 is most highly expressed in these high endothelial venules in lymph nodes [34]. It has also been reported that Exodus-2 has a higher selectivity for recruiting adult naı¨ve T lymphocytes than memory T lymphocytes in a parallel plate flow chamber [38]. Recently, mice with decreased expression of Exodus-2 have been shown to have abnormal migration of T cells into mature lymph node [39]. Taken together with the data presented in this paper, this indicates that Exodus-2 and -3 are very important mediators of naive cord blood fetal T-cell migration into nodal tissue for initial antigen presentation. However, it is clear that Exodus-1 preferentially attracts adult memory T cells as compared with naı¨ve fetal T cells. This study provides evidence that immature, naive fetal T cells can respond to chemotactic stimuli, such as chemokines. They also do not require an activation or other maturation event to respond to migratory regulation. It is possible that Exodus-2 and -3 may play a role
The three Exodus chemokines represent a sub-family of CC chemokines that share structural and functional characteristics. They all preferentially chemoattract T cells [32,34,37]. It has been recently shown that Exodus2 and -3 stimulate T-cell adhesion to L-selectin, and probably mediate T-cell trafficking through lymph nodes from the peripheral blood [34,37]. This study investigated whether these chemokines also could play a role in regulating the migration of naive fetal cord blood T cells. We found that these chemokines stimulated cord blood T-cell chemotaxis to a slightly greater extent than adult peripheral blood T cells. Exodus-2 and -3 preferentially attract CD4+ , CD44+, and CD45RA+ adult peripheral blood T cells, whereas Exodus-1 preferentially attracts CD45RO+ adult peripheral blood T cells. Essentially
Fig. 4. Migration of CD44+ normal human adult peripheral blood (PB) T cells and migration of CD44 + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
K. Christopherson et al. / Immunology Letters 69 (1999) 269–273
in fetal T-cell ontogeny, or in T-cell maturation to antigen responding cells in secondary lymphoid tissue. We propose that homologous recombination knockouts of the Exodus-2 and -3 genes may be especially interesting to assess whether there are any phenotypic T-cell maturation defects in such knock-outs.
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Regulation of naı¨ve fetal T-cell migration by the chemokines Exodus-2 and Exodus-3 Kent Christopherson II a, Zacharie Brahmi b, Robert Hromas a,* a
Department of Biochemistry/Molecular Biology and Internal Medicine, Indiana Uni6ersity Cancer Center, R4 -202, 1044 West Walnut Street, Indianapolis, IN 46202, USA b Department of Medicine and Microbiology/Immunology, Indiana Uni6ersity Medical Center, RI-0615, 702 Barnhill Dri6e, Indianapolis, IN 46202, USA Received 22 March 1999; accepted 12 April 1999
Abstract We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3a, Exodus-2/6Ckine/ SLC/TCA4, and Exodus-3/Mip-3b/CKb11/ELC. These chemokines share an amino terminal AspCysCysLeu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naı¨ve human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4 + cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+ . Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue. © 1999 Elsevier Science B.V. All rights reserved. Keywords: CC Chemokine; Exodus-1/LARC/Mip-3a; Exodus-2/6Ckine/SLC/TCA4; Exodus-3/Mip-3b/CKb11/ELC; Naı¨ve fetal T-cell migration; T-cell ontogeny; Cord blood; CD45RA; CD45RO; CD44
1. Introduction Chemokines are a family of homologous proteins that direct leukocyte migration and activation to inflammatory stimulus [1 – 5]. The chemokine family can be divided into two groups depending on where the first two cysteines are located. The CXC family (also referred to as alpha) has a single amino acid between the initial two cysteines. If the first two cysteines are adjacent to each other, they are classified in the CC family (beta). Most CXC members are located in a gene cluster at human chromosome 4q12-21, whereas most CC members are in a cluster at human chromosome 17q11-32 [6–8]. Chemokines act through seven membrane-spanning G-protein-coupled receptors [9– 11]. Chemokines mediate inflammatory tissue destruc* Corresponding author. Tel.: +1-317-2743589; fax: + 1-3172740396. E-mail address: [email protected] (R. Hromas)
tion in a wide variety of human diseases, such as rheumatoid arthritis, myocardial infarction, and adult respiratory distress syndrome [12–18]. We and other workers have shown that some chemokines, especially members of the CC family, such as MIP-1 alpha, are able to decrease the proliferative status of myeloid progenitors at low concentrations [19–22]. This effect occurs in a primitive hematopoietic progenitor. The proliferative inhibition of marrow progenitors by some chemokines may protect the progenitors from cell cycle-specific chemotherapeutic cytotoxicity (reviewed in Ref. [22]). Several reports have shown that the CC chemokines MIP-1 alpha, MIP-1 beta, and RANTES inhibit HIV-1 production in newly infected peripheral blood mononuclear cells [23–25]. The HIV co-receptors (with CD4) are chemokine receptors, suggesting a mechanism for the inhibition of HIV infection by some chemokines [26–28].
0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 9 9 ) 0 0 0 9 9 - 1
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We and other workers recently cloned a sub-family of three novel CC chemokines that share a unique (among all chemokines) amino terminal AspCysCysLeu sequence. These chemokines were termed Exodus-1 (also known as Mip-3a and LARC) [29 – 31], Exodus-2 (also known as 6-Ckine and SLC) [32 – 34] and Exodus-3 (also known as Mip-3b, CKb-11, TCA4, and ELC) [30,35,36] for their ability to preferentially regulate the migration of adult T cells. Although the events that direct movement of adult circulating T cells are being elucidated, the regulation of naı¨ve fetal T-cell migration is less well understood. In this study, we investigated the ability of this subfamily of chemokines to control the migration of naive cord blood T cells as compared with adult peripheral blood T cells. We found that Exodus-2 and -3 were potent regulators of naive cord blood T-cell chemotaxis.
2. Materials and methods
2.1. Chemokines All chemokines were purchased commercially from R&D systems (Minneapolis, MN), reconstituted, and stored according to the manufacturer’s instructions.
All chemotaxis data represent the average of three experiments, each done in triplicate. Comparisons were made using the Student’s t-test.
2.3. Flow cytometry T cells in the upper and lower chambers were stained by monoclonal antibodies (mAbs) to fluorochrome-conjugated CD3 (all antibodies from Caltag Laboratories Burlingame, CA), and counted by FACscan. CD44+, CD45RA+ , CD45RO+ , CD4+, and CD8+ subtypes of T cells were quantitated in the upper versus lower chambers with fluorescent mAbs to CD44, CD45RA, CD45RO, CD4, CD8, and CD3, and analyzed by FACscan. Harvested cells from both the upper and lower chambers were washed in phosphate buffered saline (PBS)/1% BSA and resuspended in 100 ml PBS/ BSA. One microgram of the appropriate antibody was added, mixed, and the cells incubated at 4°C in the dark for 30 min. The cells were then washed twice in PBS/BSA and fixed in PBS/1% paraformaldehyde for FACscan analysis at 488 nm. Ten thousand events were accumulated for each analysis.
3. Results
2.2. Chemotaxis assays
3.1. Stimulation of adult peripheral blood T-cell subset chemotaxis
Chemotaxis assays were performed as previously described with minor modifications [32]. Half-a-million normal human fetal cord or adult peripheral blood low density non-adherent leukocytes in 200 ml of RPMI media supplemented with 0.5% bovine serum albumen (BSA) were added to the upper chamber of a Costar transwell (6.5 mm diameter, 5 mm pore size, polycarbonate membrane). The T-cell subset percentage of the input cell population was measured by flow cytometry, as described later. Chemokines were added to 1 ml of the identical media in the lower chamber. Total cell migration was obtained by counting the cells in the lower chamber versus the cells remaining in the upper chamber after 3 h incubation at 37oC, 5% CO2. Cells were then harvested by washing the upper and lower sides of the transwell membrane, and adding the wash to the respective upper or lower media. Chemokines placed only in the upper chamber abrogate migration to the lower chamber. This was done as a checkerboard control assay for each distinct chemotaxis assay. In those transwells that contained chemokine placed only in the lower chamber, the percent of cell migration was calculated by dividing the number of the cells in the lower chamber by the total cell input and then subtracting random transwell migration to the lower chamber without chemokine presence.
When successive concentrations of Exodus-1, -2, and -3 chemokines are used as stimulation for adult peripheral blood total T-cell migration, there is a dose-dependent increase in chemotaxis (Fig. 1a) for all three chemokines, as previously described. This was statistically significant using Student’s t test for all concentrations tested for all three chemokines [29,32]. Exodus-2 proved to be the most effective chemoattractant for adult T cells. Of the input cells, 31.79 0.2% (SEM) migrated across the transwell membrane at the most effective concentration of Exodus-2 (400 ng/ml) compared with 16.3 9 1.4% for Exodus-1 and 24.7 91.3% for Exodus-3 at the same concentration. Flow cytometric analysis of the migration of T-cell subsets was performed to assess if there were differential effects of these three chemokines on specific types of T cells. The composition of the migrated cell population was compared with the total cell population of the adult peripheral blood (Fig. 2). Adult peripheral blood CD4+ T cells (PB-CD4 + ) showed more migration than CD8+ T cells (PB-CD8+ ) for all three chemokines (Fig. 3). Of adult CD4+ T cells, 1093.3, 20.39 5.7, and 18.29 4.6% migrated in response to Exodus-1, -2, and -3, respectively at 200 ng/ml; whereas 4.5921, 8.69 4.0, and 7.4 9 3.2% of CD8 + T cells responded to Exodus-1, -2, and -3, respectively at 200 ng/ml. It is
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Fig. 1. (a) Response in chemotaxis to successive concentrations of Exodus-1, -2, and -3 chemokines used as stimulation for normal adult peripheral blood T-cell migration. (b) Response in chemotaxis to successive concentrations of Exodus-1, -2, and -3 chemokines used as stimulation for normal fetal cord blood T-cell migration.
also of note that within the CD4+ T-cell population of both peripheral blood and cord blood, Exodus-2 and -3 showed more chemotactic ability than Exodus-1 (Fig. 3). For CD4 + adult peripheral blood T-cell migration, Exodus-2 was twofold and Exodus-3 was 1.8-fold higher than that of Exodus-1. CD44+ T cells (PBCD44+ /3+) migrated more in response to Exodus-2 (17.491.1%) and Exodus-3 (18.490.5%) than to Exodus-1 (3.49 0.3%) at 200 ng/ml (Fig. 4). There was no difference between chemotactic effectiveness of all three Exodus chemokines within CD45RO+ adult T cells (PB-CD45RO +). Within CD45RA+ adult T cells (PB-CD45RA +), Exodus-2 (25.490.0%) and Exodus3 (20.9 90.2%) showed a greater ability than Exodus-1 (10.39 0.7%) to chemoattract at 200 ng/ml (Fig. 5).
3.2. Stimulation of cord blood T-cell subset chemotaxis When Exodus-1, -2, and -3 were tested for stimulation of cord blood T-cell chemotaxis, statistically significant dose-dependent activation of chemotaxis was seen for Exodus-2 and -3, but not for Exodus-1 (Fig. 1b). Exodus-2 and -3 were equally potent chemoattractants for cord blood T cells. Of input cord blood total T cells, 28.79 2.9% migrated across the transwell membrane in response to 400 ng/ml of Exodus-2, and 269 2.8% migrated to 400 ng/ml of Exodus-3. These values are compared with a 8.0 94.4% migration in response to Exodus-1 at 400 ng/ml. Flow cytometry was used to analyze whether the Exodus chemokines had differential effects on cord blood fetal T-cell subsets (Figs. 3 – 5). There were several remarkable differences. CD44+ cord blood T cells (CB-CD44+/3+ ) were preferentially chemoattracted across the transwell membrane by Exodus-2 at 50.89 0.6%, Exodus-3 at 50.794.2%, and Exodus-1 at 6.99 1.9% at 200 ng/ml (Fig. 4). For both Exodus-2 and -3, CD4+ cord blood T cells (CB-CD4+) were preferen-
tially stimulated to migrate as compared with CD8+ T cells (Fig. 3). Of CD4+ cells, 259 4.9% migrated in response to 200 ng/ml of Exodus-2 and 30.895.8 in response to 200 ng/ml of Exodus-3. Only 9.79 5.1% of CD8+ T cells migrated for Exodus-2 and 13.2 97% for Exodus-3 at 200 ng/ml. As was mentioned earlier, within the CD4+ T-cell population of both peripheral blood and cord blood cells, Exodus-2 and -3 showed a greater chemotactic ability than Exodus-1 (Fig. 3). Exodus-2 had 5.5-fold and Exodus-3 had 6.6-fold the CD4+ cord blood T-cells migration resulting from Exodus-1. Essentially, all of the cord blood T cells were CD45RA+ (CB-CD45RA+ ), whereas none were CD45RO+ (CB-CD45RO+ ) (Fig. 2). These CD45RA+ naive cord blood T cells were chemoattracted well by Exodus-2 (30.59 2.2%) and Exodus-3 (34.29 2.9%), but not by Exodus-1 (6.29 0.0%) at 200 ng/ml (Fig. 5).
Fig. 2. Composition of the total cell population of normal human fetal cord blood (CB) or normal human adult peripheral blood (PB) low-density non-adherent leukocytes.
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Fig. 3. Migration of CD4 + and CD8 + normal human adult peripheral blood (PB) T cells, and migration of CD4 + and CD8 + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
Fig. 5. Migration of CD45RO+ and CD45RA+ normal human adult peripheral blood (PB) T cells and migration of CD45RA + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
4. Discussion
all cord blood T-cells were CD45RA + , indicating that they were naı¨ve. Cord blood CD4+ , CD44+, and CD45RA+ T cells were attracted at much greater efficiency by Exodus-2 and -3 than Exodus-1. CD4+ cord blood T cells were preferentially stimulated to migrate compared with CD8+ in response to Exodus2 and -3. It is clear from these data that Exodus-2 and -3 preferentially chemoattract both cord and peripheral CD4+ /CD45RA+ T cells. Exodus-2 and -3 stimulated the migration of cord blood CD44+ T cells 2.3-fold better than peripheral blood CD44+ T cells, and stimulated the migration of cord blood CD45RA+ T cells 1.2- and 1.6-fold (respectively) that of adult peripheral blood CD45RA+ T cells. There is evidence that Exodus-2 and -3 play a role in T-cell adhesion to high endothelial venules in lymph nodes, and may be critical in adult T-cell trafficking through nodes [34]. It appears that Exodus-2 is most highly expressed in these high endothelial venules in lymph nodes [34]. It has also been reported that Exodus-2 has a higher selectivity for recruiting adult naı¨ve T lymphocytes than memory T lymphocytes in a parallel plate flow chamber [38]. Recently, mice with decreased expression of Exodus-2 have been shown to have abnormal migration of T cells into mature lymph node [39]. Taken together with the data presented in this paper, this indicates that Exodus-2 and -3 are very important mediators of naive cord blood fetal T-cell migration into nodal tissue for initial antigen presentation. However, it is clear that Exodus-1 preferentially attracts adult memory T cells as compared with naı¨ve fetal T cells. This study provides evidence that immature, naive fetal T cells can respond to chemotactic stimuli, such as chemokines. They also do not require an activation or other maturation event to respond to migratory regulation. It is possible that Exodus-2 and -3 may play a role
The three Exodus chemokines represent a sub-family of CC chemokines that share structural and functional characteristics. They all preferentially chemoattract T cells [32,34,37]. It has been recently shown that Exodus2 and -3 stimulate T-cell adhesion to L-selectin, and probably mediate T-cell trafficking through lymph nodes from the peripheral blood [34,37]. This study investigated whether these chemokines also could play a role in regulating the migration of naive fetal cord blood T cells. We found that these chemokines stimulated cord blood T-cell chemotaxis to a slightly greater extent than adult peripheral blood T cells. Exodus-2 and -3 preferentially attract CD4+ , CD44+, and CD45RA+ adult peripheral blood T cells, whereas Exodus-1 preferentially attracts CD45RO+ adult peripheral blood T cells. Essentially
Fig. 4. Migration of CD44+ normal human adult peripheral blood (PB) T cells and migration of CD44 + normal human fetal cord blood (CB) T cells across a transwell membrane in response to 200 ng/ml of Exodus-1, -2, or -3.
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in fetal T-cell ontogeny, or in T-cell maturation to antigen responding cells in secondary lymphoid tissue. We propose that homologous recombination knockouts of the Exodus-2 and -3 genes may be especially interesting to assess whether there are any phenotypic T-cell maturation defects in such knock-outs.
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