Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities

Experimental Hematology 27 (1999) 1113–1123

Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities Hal E. Broxmeyer and Chang H. Kim Departments of Microbiology/Immunology and Medicine, and the Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, IN (Received 5 February 1999; accepted 23 February 1999)

The field of chemokine biology is a rapidly advancing one, with over 50 chemokines identified that mediate their effects through one or more of 16 different chemokine receptors. Chemokines, originally identified as chemotactic cytokines, manifest a number of functions, including modulation of blood cell production at the level of hematopoietic stem/progenitor cells and the directed movement of these early blood cells. This report reviews chemokines and chemokine/receptor activities mainly in the context of hematopoietic cell regulation and the numerous chemokines that manifest suppressive activity on proliferation of stem/progenitor cells. This is contrasted with the specificity of only a few chemokines for the chemotaxis of these early cells. The large number of chemokines with suppressive activity is hypothesized to reflect the different cell, tissue, and organ sites of production of these chemokines and the need to control stem/progenitor cell proliferation in different organ sites throughout the body. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Chemokines—Chemokine receptors—Stem/progenitor cells—Hematopoiesis—Chemotaxis

Introduction Chemokines are large family of molecules that have been implicated in the movement and production of blood cells [1–5]. They were originally named for their capacity to act as chemotactic cytokines for functionally mature blood cell types. There are currently more than 50 known chemokines, and these are presently classified into four different subgroups, distinguished by their position invariant cysteine (C) motifs near the N-terminal portion of the molecule. The two largest families are the CC and CXC (where X can be any amino acid) groups. One chemokine each has been

Offprint requests to: Hal E. Broxmeyer, Walther Oncology Center, Indiana University School of Medicine, 1044 W. Walnut Street, Room 302, Indianapolis, IN 46202-5254; E-mail: [email protected]

identified for the C and CX3C groups. In many cases, the same chemokine has been given a number of different names based on the near simultaneous identification of the same chemokine by different investigators. The numerous chemokines and their different designations can be overwhelming for those in the field and especially so for those more casually interested in the area. Table 1 lists many of the known chemokines grouped according to the receptors they bind, along with their commonly used abbreviations. An interesting characteristic of chemokines is the apparent redundancy in some of their actions. For example, MIP1a was identified as a suppressor molecule for colony forming unit-spleen (CFU-S) stem cells [6] and for multigrowth factor responsive multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells [7]. However, there are now at least 22 other chemokines crossing the CC, CXC, and C family subgroups that have been found to manifest a type of cellular suppressor activity similar to that of MIP-1a [8]. Redundancy is also noted in the chemotactic responsiveness of mature blood cell types, but not hematopoietic progenitor cells, to chemokines [1–5]. Redundancy in action may be a safeguard to ensure correct functioning and regulation of blood cell production. This review details some of the current knowledge of chemokine actions and postulates potential reasons for this noted redundancy in some of the actions of chemokines. Chemokines as regulators of hematopoiesis in vitro and in vivo One of the first hematopoietic regulatory activities for chemokines in general and for MIP-1a in particular was that of the enhancement of colony formation in vitro by CFU-GM and macrophage (CFU-M) progenitors, respectively stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) [7,9]. This was an effect on more mature subsets of progenitors, those that responded to stimulation by a single growth factor. It was, however, the suppressive ef-

0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(99)0 0 0 4 5 - 4

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Table 1. Chemokine receptors and the chemokines that bind them Receptor (A) CC Chemokine receptor CCR1

CCR2

CCR3

CCR4 CCR5

CCR6 CCR7 CCR8

CCR9*

CCR10 Unknown

(B) CXC chemokine receptor CXCR1 CXCR2

CXCR3

CXCR4

Chemokines Macrophage inflammatory protein (MIP)-1a Hemofiltrate CC chemokine (HCC)-2/novel CC chemokine (NCC)-3/MIP-5/leukotactin (Lkn)-1 Myeloid progenitor inhibitor factor (MPIF)-1/chemokine (CK) b–8/MIP-3 CKb–8–1, a splicing variant of CDb-8 Regulated on activation of normal T cell expressed and secreted (RANTES) Monocyte chemotactic protein (MCP)-3 HCC-1/NCC-2 Murine C10/MRP-1 Murine MIP-1a related protein (MRP)-2/CCF18/MIP-1g MCP-1/monocyte chemotactic and activating factor (MCAF) MCP-2/HC14 MCP-3 MCP-4/NCC-1/CKb-10 Murine MCP-5 MCP-4/NCC-1/CKb-10 HCC-2/NCC-3/MIP-5/Lkn-1 Eotaxin-2/MPIF-2/CKb-6 Eotaxin-1 RANTES MCP-2 MCP-3 Thymus and activation-regulated chemokine (TARC) Macrophage-derived chemokine (MDC)/stimulated T-cell chemoattractant protein-1 (STCP)-1/ABCD-1 MIP-1a MIP-1b RANTES Exodus-1/MIP-3a/liver and activation-regulated chemokine (LARC) CKb-11/EBV-induced gene 1-ligand chemokine (ELC)/MIP-3b/Exodus-3 Secondary lymphoid tissue chemokine (SLC)/6Ckine/Exodus-2/T-cell activation gene (TCA) 4 I-309/murine TCA 3 TARC MIP-1b MIP-1a MIP-1b RANTES MCP-1 to MCP-5 Eotaxin-1 Thymus expressed chemokine (TECK) NCC-4/liver expressed chemokine (LEC)/HCC-4/lymphocyte and monocyte chemoattractant (LMC) Dendritic cell-derived C-C chemokine (DC-CK) 1/pulmonary and activation-regulated chemokine (PARC)/ alternative macrophage activation-associated CC chemokine (AMAC)-1/MIP-4 Interleukin (IL)-8/neutrophil activating peptide (NAP)-1 Granulocyte chemoattractant protein (GCP)-2 IL-8/NAP-1 GCP-2 Growth-related oncogene (GRO)-b/melanoma growth stimulatory activity (MGSA)-b/MIP-2a Epithelial-derived neutrophil attractant (ENA)-78/LPS-induced CXC chemokine (LIX) GRO-a/MGSA-a NAP-2/connective tissue activating peptide-III (CTAF III)/leukocyte-derived growth factor (LDGF)/platelet basic protein (PBP) GRO-g/MIP-2b g-Interferon-inducible protein (IP)-10/cytokine responsive gene-2 (crg)-2/mob-1 Monocyte induced by g-interferon (MIG) Murine SLC/6Ckine/Exodus-2/TCA4 Stromal cell-derived chemokine (SDF)-1/pre-B-cell growth-stimulating factor (PBSF)/TPA repressed gene 1 (TPAR1) (Continued)

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Table 1. Continued Receptor CXCR5 Unknown (C) CX3C chemokine receptor CX3CR1 (D) C chemokine receptor CR1

Chemokines B-lymphocyte chemoattractant (BLC)/B-cell attracting chemokine (BCA)-1 Platelet factor (PF) 4 Fractalkine/neurotactin Lymphotactin/single cysteine motif (SCM)-1/activation-induced T-cell-derived and chemokine-related (ATAC)

Most of the chemokines listed in this table are human proteins. The mouse chemokines are less well-studied in terms of the receptor(s) they bind. *Chemokines binding to CCR9 do not initiate calcium flux (signaling). / - separation of different names for the same chemokine; () - abbreviation for chemokine.

fects of MIP-1a that overshadowed the enhancing effects of this molecule [6,7,9–18]. In vivo administration of MIP-1a to mice resulted in suppression, rather than enhancement, of hematopoiesis [15,19–23]. The suppressive effects of MIP1a in vitro were direct acting on the stem/progenitor cells as determined by studies of purified populations of cells [7,11] and at the level of single stem/progenitor cells [14] growing in defined medium. The actions of MIP-1a seemed to be mediated by the monomeric form of the molecule [12, 13,20], effects initiated during the DNA synthesis (S) phase of the cell cycle [12,13]. Injection of MIP-1a into mice resulted in dose-dependent, time-related, and reversible suppression of the cycling status and absolute numbers of bone marrow and spleen stem/progenitor cells [13,19,20]. MIP1a and an analogue, BB10010 [23], were noted to have some myeloprotective effects in mice subjected to single chemotherapeutic drugs such as hydroxyurea or cytosine arabinoside [21–23]. This set the stage for phase I/II testing of BB10010 in the context of countering the myelotoxicity associated with chemotherapy [24–27]. Although BB10010 was found to be safe, and a number of the myelosuppressive effects of MIP-1a and BB10010 noted in preclinical studies in mice were verified in the phase I portion of the human clinical trial [25,27], BB10010 had little or no myeloprotective effects on the subsequently administered chemotherapy. It was of interest that patients receiving MIP-1a, while having their progenitor cells in a slow- or noncycling state and manifesting a decreased frequency of progenitors, had normal nucleated cellularity and numbers of morphologically recognizable proliferating cells. This, in part, may reflect the decreased apoptosis of the mature cells noted in patients receiving BB10010 [25]. MIP-1a has been shown to have apoptosis-inducing action on immature sets of progenitor cells, an activity heightened with such cells from mice deleted in the Fanconi anemia complement C group gene [28]. Thus, if MIP-1a/BB10010 is to be more useful as a myeloprotective effector molecule, it may be necessary to use it in combination with other molecules. In this setting, a number of other chemokines that have been tested have now been demonstrated to suppress progenitor cell proliferation in vitro to at least the same degree as MIP-1a [8,11,13,14, 29–41]. Suppressive CC chemokines in the context of inhi-

bition of myeloid progenitor cells stimulated to proliferate by combinations of growth factors such as erythropoietin, interleukin 3 (IL-3), GM-CSF, and steel factor include MIP1a, MCP-1, MCP-4/CKb-10, LKN-1/MIP-5/HCC2, Exodus-1/LARC/MIP-3a/CKb-4, Exodus2/6Ckine/SLC/CKb-9, CKb-11/ELC/MIP-3b/Exodus 3, MIP-4, CKb-7, MPIF-1/ CKb-8 and its splicing variant CKb-8-1, MPIF-2/Eotaxin-2/ CKb-6, I-309, TECK, MRP-1/C10, MRP2/CCF18/MIP-1g, and LMC/HCC-4/NCC-4/LEC. CXC chemokines suppressive in the same context include GRO-b/MIP-2a, IL-8/ NAP-1, GCP-2, platelet factor 4 (PF4), IP-10, ENA-78, and Mig. The one member of the C chemokine family, lymphotactin, is also suppressive. Those CC chemokines without suppressive activity include MIP-1b, RANTES, MCP-2, MCP-3, Eotaxin-1, MCIF/HCC-1/CKb-1, TARC, and MDC. Those CXC chemokines without suppressive activity include GRO-a, GRO-g/MIP-2b, NAP-2, and SDF-1/PBSF. However, GRO-a has been reported to have a suppressive effect on IL-3–dependent proliferation of the murine 32D cell line [39]. The one member of the CX3C chemokine family, fractalkine/neurotactin, is without suppressive activity. Many of the above mentioned inhibitory chemokines in vitro have also been found to be active in myelosuppression in vivo when exogenously administered to mice [13,42]. Because the suppressive chemokines can act in synergy with each other [11,31] and with other cytokines such as macrophage stimulating protein and vascular endothelial growth factor [43] to inhibit progenitor cell proliferation in vitro at concentrations below which any chemokine can suppress by itself, and because this suppressive synergy has also been noted for many of these chemokines after administration to mice [42], the design of in vivo preclinical mouse myeloprotective assays using chemokines in combination could prove to be of potential clinical usefulness. PF4 and IL-8 have shown some myeloprotective activity in mice [44,45]. MIP-1a has been found to be an important ingredient in maintenance of cellular growth in long-term marrow cultures [46,47]; however, it is not clear how this effect is being manifested. Also, Eotaxin has enhancing activity on myelopoiesis [48,49], but how this is mediated remains to be determined.

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Chemokine/chemokine receptor interactions Chemokine receptors are seven transmembrane G-protein– linked receptors [1–5]. Ten CC chemokine receptors, designated CCR1 to CCR10, have been identified. Five CXC chemokine receptors, designated CXCR1 to CXCR5, have been identified. One C chemokine receptor, XCR1, and one CX3C chemokine receptor, CX3CR1, have been identified. As is apparent from Table 1, there is redundancy in the chemokines that bind which receptors. For example, some receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, and CCR9 bind more than one chemokine. Of the CC receptors, only CCR6 and CCR10 have been shown thus far to bind only one chemokine. Although CCR9 binds many chemokines, it appears to be a nonsignaling receptor. CXCR1, CXCR2, and CXCR3 bind a number of chemokines, whereas CXCR4 and CXCR5 have thus far been shown to bind only one chemokine each. An added complication in terms of interpreting how chemokines function is that some chemokines are known to bind to more than one chemokine receptor. Thus, MIP-1a binds CCR1, CCR5, and CCR9. MIP-1b binds CCR5, CCR8, and CCR9. RANTES binds CCR1, CCR3, CCR5, and CCR9. IL-8 and GCP-2 bind both CXCR1 and CXCR2. There is also some evidence that, in the murine system, the CC chemokine Exodus 2/6Ckine/SLC/CKb-9 can bind both the CCR7 and the CXCR3 receptors [50], making this the first chemokine shown to bind receptors in more than one chemokine subfamily. Making sense out of all these interactions will most likely require the analysis of mice in which the genes for one or more chemokine receptors have been “knocked out”/ functionally deleted. Analysis of mice in which the gene for a single cytokine receptor has been functionally deleted (2/2) has already produced interesting information. The use of CCR1 (2/2) mice has demonstrated abnormalities in trafficking of myeloid progenitor cells from marrow to spleen, especially in response to bacterial lipopolysaccharide [51]. Bone marrow progenitor cells from CCR1 (2/2) mice were as sensitive as these cells from wildtype littermate controls (1/1) to inhibition by MIP-1a, but the MIP-1a enhancing activity on mature CFU-GM and CFU-M progenitors stimulated to proliferate by GM-CSF or M-CSF was lost with CCR1 (2/2) cells, suggesting that whereas CCR1 is not a dominant receptor for MIP-1a inhibition, it is a dominant receptor for MIP-1a enhancement of proliferation [52]. MIP-1a/BB10010 is a modest but rapid agent for mobilizing stem/progenitor cells to the blood [23,25,53], and CCR1 is apparently a dominant receptor for this activity of MIP-1a [52]. The use of CCR2 (2/2) mice has shown that CCR2 is a dominant receptor for the myelosuppressive effects of MCP-1 and its murine analog JE [54,55]. The cycling status of myeloid progenitors in bone marrow of CCR2 (2/2) mice was greatly increased over that of the wild-type littermate CCR2 (1/1) mice, but there was no difference in the absolute numbers of progenitors or nucleated cells in mar-

row or spleen, an effect apparently counterbalanced by the enhanced programmed cell death (apoptosis) seen in progenitor cells from the marrow of CCR2 (2/2) compared to CCR2 (1/1) mice [55] . Thus, CCR2 is involved in the proliferation and apoptosis of progenitor cells. The use of CXCR2 (2/2), also called murine IL-8 receptor homologue (2/2), mice has highlighted the role of CXCR2 in negative regulation [56]. CXCR2 (2/2) marrow progenitors are insensitive to inhibition by IL-8 and murine MIP-2 (a CXC chemokine that also binds CXCR2). Moreover, myelopoiesis is greatly enhanced in CXCR2 (2/2) mice, a biologic effect noted in mice bred and raised under normal environment conditions, but not seen in mice kept under germ-free conditions. This suggests that CXCR2 is involved in mediating negative regulation but that this is an environmentally inducible effect. It will take the evaluation of many more chemokine receptor deleted mice to further unravel which chemokine receptors act as dominant effector molecules for which functions and through which particular chemokines. Further understanding of the roles chemokine receptors play will no doubt benefit from studies in mice in which more than one chemokine receptor has been functionally deleted. A small amount of information is available for the intracellular signal transduction events mediating the actions of chemokines [1–5,57], especially on inhibition of stem/progenitor cells [58]. What is known is based mainly on studies using growth factor-dependent cell lines. It is possible that although the suppressive actions of many of the different chemokines occurs through different chemokine receptors, some of the intracellular events mediating these effects are the same. A systematic approach to elucidating these similar candidate intracellular signals through the use of mice in which some of the genes for these intracellular proteins are functionally deleted may shed light on this question. Such studies are currently under way in the author’s laboratory utilizing mice deleted in the transcription factors Stat4, Stat6, and BCL-6, in the protein tyrosine phosphatases SHP-1 and SHIP, and in other intracellular molecules.

Possible reasons for redundancy in chemokine suppression of myelopoiesis Philosophically, one would wonder why so many different chemokines should have the capacity to elicit the same endpoint, suppression of stem/progenitor cell proliferation. Redundancy is a phenomenon noted with many biologic systems, but the chemokine system seems particularly evident in this context. Of course, not all chemokines may be physiologically relevant, although all those chemokines that we have tested that can suppress progenitor cell proliferation in vitro cause suppression in vivo when administered to mice [13,19,20,42]. Thus, the possibility that all these suppressive chemokines may be active in the normal functioning of blood cell production remains an open possibility. Some

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Table 2. Expression of chemokines Chemokine BLC/BCA-1* C10/MRP-1 CKb-11/ELC/MIP-3b* DC-CK1/PARC/MIP-4/AMAC-1* ENA-78/LIX

Eotaxin-1

Eotaxin-2/MPIF-/CKb-6 Exodus-1/MIP-3a/LARC Fractalkine/neurotactin GCP-2 GRO-a/MGSA-a GRO-b/MGSA-b GRO-g/MIP-2b HCC-1/HCC-3/NCC-2* HCC-2/NCC-3/MIP-5/Lkn-1 HCC-4/NCC-4/LEC//LMC I-309/TCA3 IL-8/NAP-1 IP-10/crg-2/mob-1

I-TAC Lymphotactin/SCM-1a/ATAC MCP-1/MCAF MCP-2/HC14 MCP-3 MCP-4/NCC-1/CKb-10 mMCP-5 MDC/STCP-1/ABCD-1* MIG MIP-1a MIP-1b

Chemokine-expression sites, expressing cells, and conditions Follicles and germinal centers (B-cell zones) of lymphoid tissues: spleen, lymph nodes, payer’s patch, appendix; stomach, liver, [60,61] IL-4–activated mouse macrophages [62]; GM-CSF–activated 32D cells and bone marrow cells [63] Thymus, lymph node, appendix, small and large intestines, lung, kidney, stomach, trachea [64,65]; dendritic cells [66]; induced in bone marrow stromal cells [67] Dendritic cells, tonsil (germinal centers and T-cell area) [68]; lung, lymph nodes, thymus, appendix [69] Intestinal epithelium in inflammatory bowel disease [70]; mast cells [71]; epithelial cells of the intestinal mucosa of patients with Crohn’s disease, ulcerative colitis, and acute appendicitis [72]; enterocytes [73]; heart, lung, spleen, liver during acute endotoxemia [74] Lungs of naive and allergen-sensitized guinea pigs [75]; lung, intestines, stomach, spleen, liver, heart, thymus, testes, and kidney [76], tumor cells, IFN-g–activated endothelial cells [77]; type I alveolar epithelial cells [78]; small intestine and colon [79]; human dermal fibroblasts [80] Activated monocytes, activated T cells, absent in most tissues [39] Lung, liver, colon, small intestine, testis, prostate, thymus, spleen, appendix, PBL [64]; upregulated by TNF-a or LPS in lymphocytes and monocytes [29] Heart, brain, lung, skeletal muscle, kidney, pancreas, activated endothelial cells [81,82] Human osteosarcoma cells (MG-63), induced in fibroblasts by IL-1b [83]; induced in uterine endometrium by IFN-tau [85]; epithelial cells [85] Monocytes, bronchoalveolar macrophages, neutrophils, endothelial cells, airway epithelial cells, fibroblasts, keratinocytes [86–89]; melanoma [90] Melanoma [90]; monocytes, neutrophils, endothelial cells, mammary epithelial cells, fibroblasts [86] Melanoma [90]; monocytes, neutrophils, endothelial cells, mammary epithelial cells, fibroblasts [86] Present in blood plasma; expressed in spleen, liver, gut, muscle, and bone marrow [91] Gut and liver [92] IL-10–activated monocytes [93]; liver [94] Activated T cells, activated monocytes [95] Heart, lung, PBL [96]; monocytes, neutrophils, T cells, endothelial cells, fibroblasts, astrocytes, chondrocytes, keratinocytes, mesangial cells, smooth muscle, tumor cell lines (summarized in [97]) Lung, PBL, thymus, spleen [96]; IFN-g–activated monocytic cells [98]; induced in heart, liver, lung, ovary, spleen, uterus during inflammation [99]; induced during adult respiratory distress syndrome (ARDS)-like microvascular lung injury elicited by infusion of IL-2 [100] IFN-g and IL-1-activated astrocytes and monocytes, pancreas, lung, thymus, and spleen [101] Spleen, activated CD81 T cells [102-105] Pancreas, kidney, skeletal muscle, liver, lung, placenta, heart, small and large intestines, thymus, spleen, prostate, testis, ovary [96] Osteosarcoma cells (MG-63) [106], activated PBL [107] Monocytes, osteosarcoma, activated U937 promonocytic cells by PMA [106,108] Heart, placenta, lung, muscle, kidney, pancreas, thymus, prostate, testis, ovary, small and large intestines, endothelial cells [109] Lung, heart, lymph node, breast, salivary gland, elicited peritoneal macrophages, activated macrophage cell line (RAW264.7) [110] Macrophage, dendritic cells, thymus, lymph node, appendix [111,112]; activated splenic B lymphocytes and dendritic cells [113] IFN-g–activated monocytic cells [114]; induced in heart, liver, lung, ovary, spleen, uterus during inflammation [99] Activated lymphocytes [115]; activated monocytic cells (HL-60 and U937) with phorbol 12-myristate 13-acetate (PMA); human glioma cell line U105MG, fibroblasts [116] Liver, lung, placenta, heart, PBL, small and large intestines, thymus, spleen [96]; monocytes, T cells, B cells, fibroblasts [117,118] (Continued)

chemokines may be active in some situations and not others. For example, MCP-1, but not MIP-1a, has been implicated as the endogenous chemokine that cooperates with transforming growth factor-b to inhibit cycling of primitive normal progenitors in long term culture [59]. Another possibility, entirely hypothetical at this time, is that the redundancy

in action may be explained by the constitutive or induced cellular and tissue sites of production of these chemokines. Hematopoietic stem and progenitor cells, although found mainly in the marrow of adults, is also present in a number of different organs. Stem and progenitor cells traffic to different organs during embryonic and fetal life and can move

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Table 2. Continued Chemokine MPIF-1/CKb–8/MIP-3 MIP-related protein (MRP)-2/ CCF18/MIP-1g NAP-2/CTAP III/LDGF/PBP PF4 RANTES SDF-1/PBSF/TPAR1* SLC/6Ckine/exodus-2/TCA4* TARC TECK

Chemokine-expression sites, expressing cells, and conditions Liver, lung, myeloid cell lines (HL-60 and THP-1) [39]; induced in monocytes by IL-1b and IFN-g, human liver and gastrointestinal tract [119] Macrophage cell lines (P388D1, RAW 264.7), monocyte cell lines (WEHI3) [32] Induced in activated T cells [120]; megakaryocyte [121] Platelet a granule [122] Activated T cells [123]; lung, PBL, colon, small intestines, ovary, prostate, thymus, spleen [96]; platelet [124] Constitutive expression in brain, heart, lung, kidney, bone marrow, thymus, liver, spleen, pancreas, prostate, ovary, small and large intestines [124,125]; the surrounding cells of germinal center [127] Heart, pancreas, spleen, thymus, small and large intestines, spleen, lymph nodes, appendix, thyroid, trachea, high endothelial venules of lymph nodes, in the T cell areas of secondary lymphoid tissues [128–130] Lung, colon, small intestine, thymus, induced in PHA-activated PBMC [96] Thymus, small intestine, thymic dendritic cells [131]

*Designates chemokines expressed constitutively, and which act as a basic factor for trafficking of progenitor cells or naive lymphocytes. Those chemokines not designed by an asterick are induced on inflammation and mediate inflammatory reactions. Underlined chemokines have been shown to have suppressive activity on stem/progenitor cells stimulated to proliferate by combinations of growth factors.

from organ to organ during adult life, especially in stressinduced states such as during infection. As shown in Table 2, different chemokines are produced by different cells and in different tissues [29,32,39,60–131]. The known myelosuppressive chemokines are underlined in Table 2. In some cases this production is constitutive; in other cases this production can be induced. Perhaps the redundancy in chemokine suppression is the means by which hematopoietic stem and progenitor cell proliferation is kept in check in organs in which this proliferation is not needed or warranted during either steady-state and/or induced blood cell production.

Lack of redundancy in chemokine-directed movement of hematopoietic progenitor cells In contrast to the large number of chemokines that can suppress myelopoiesis and cause the directed movement of mature blood cell types such as neutrophils, T and B lymphocytes, and monocytes (macrophages) [1–5], only three chemokines have thus far been reported that can direct the movement of myeloid progenitor cells. SDF-1 was first implicated in the movement of these cells during fetal life through studies in SDF-1 (2/2) mice [132], results confirmed through studies in SDF-1 receptor, CXCR4, (2/2) mice [133]. SDF-1 was found to cause chemotaxis in vitro of CFU-GEMM, BFU-E, CFU-GM, and CFU-MK [8,134– 137], although, like MIP-1a, by itself SDF-1 is a relatively weak stem/progenitor cell mobilizer (H E Broxmeyer, unpublished data). Movement/homing to certain organs also requires integrin-mediated adhesion to extracellular matrix components such as fibronectin. This adhesion is activated by certain cytokines such as GM-CSF, steel factor, IL-3, and thrombopoietin [138–141]. Of potential relevance, SDF-1 blocks/decreases growth factor-induced, integrinmediated adhesion of progenitor cells to fibronectin [142].

It is currently not known if SDF-1 can cause chemotaxis of the early subsets of stem cells with long-term marrow repopulating capacity. It was subsequently shown that CKb11/ELC/MIP-3b/ Exodus 3 [67] and Exodus 2/SLC/6Ckine/ CKb-9 [143], which both bind the CCR7 receptor, also can cause chemotaxis of myeloid progenitors. However, in this case, it is mainly the more mature macrophage progenitors (CFU-M, which respond to stimulation by M-CSF) that chemotax in response to CKb-11 and Exodus 2 [67,143]. It may be of relevance that this display of chemotactic responsiveness of more immature cells (such as CFU-GEMM, BFU-E, and CFU-GM) to SDF-1 and a mature progenitor (CFU-M) to CKb-11 and Exodus 2 closely follows the chemotactic responsiveness of different T-lymphocyte subsets to these chemokines [144,145]. Thus, it is mainly the more immature CD82CD42 and CD81CD41 thymocytes that are chemoattracted by SDF-1, and the more mature CD81 or CD41 thymocytes that chemotax in response to CKb-11 and Exodus 2. Screening of a large number of other chemokines has failed to identify other chemokines that can chemotax myeloid progenitor cells. Chemokines like MIP1a [23,25,53] and IL-8 [146], which can rapidly mobilize myeloid progenitor cells from marrow to blood, do not have chemotatic activity for these cells [67,134,135]. There is the intriguing possibility that newly discovered chemokines may have specificities for chemotaxis of other stem/progenitor cells types, such as the long-term marrow repopulating stem cell or the granulocyte progenitor cell, CFU-G. Information on stem/progenitor cell chemotaxis by chemokines would be of potential clinical usefulness, to direct limiting numbers of these cells to their target tissue sites during transplantation. Work is currently in progress in the author’s laboratory to evaluate stem/progenitor and more mature cell trafficking in mice overexpressing SDF-1 and CKb-11 in the whole animal and, more specifically, in the thymus.

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The future for chemokines New chemokines are still being discovered, and these will probably be found to be of use in a number of functionally relevant ways that could be of clinical utility. Additionally, modification of chemokines for more efficacious use is an on-going project of many laboratories. Switching of amino acid motifs between different chemokines, such as IL-8 and PF4, has already identified muteins with enhanced myelosuppressive activity in vitro and in vivo [147]. As more is learned about chemokine receptors and their functional motifs through site-directed mutagenesis and domain swapping, chemokines or peptides with chemokine-like activities will potentially be able to be designed to specifically modulate one functional activity as opposed to another. A family of molecules with so many members mediating their effects through a large number of receptors is likely high up on a list of ligands/receptors that can mediate physiologically and pathologically relevant events. We look forward to the further unraveling of information and an enhanced understanding of this fascinating area of research. Acknowledgments We thank Cynthia Booth and Becki Miller for typing the manuscript. Studies cited from the laboratory of H.E.B. were supported by U.S. Public Health Service Grants R01 HL 56416, R01 DK 53674, and T32 DK 07519.

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