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Macrophage-Inflammatory Protein-1α Receptor Expression on Normal and Chronic Myeloid Leukemia CD34+ Cells This information is current as Sian E. Nicholls, Guy Lucas, Gerard J. Graham, Nigel H. of September 26, 2021. Russell, Rachel Mottram, Anthony D. Whetton and Anne-Marie Buckle J Immunol 1999; 162:6191-6199; ; http://www.jimmunol.org/content/162/10/6191 Downloaded from References This article cites 41 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/162/10/6191.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Macrophage-Inflammatory Protein-1a Receptor Expression on Normal and Chronic Myeloid Leukemia CD341 Cells1 Sian E. Nicholls,* Guy Lucas,† Gerard J. Graham,‡ Nigel H. Russell,§ Rachel Mottram,* Anthony D. Whetton,* and Anne-Marie Buckle2* We have assessed expression of MIP-1a binding sites on the surface of CD341 cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD341 (15.7 6 6.2% in NBM and 9 6 4% in CML), which has specific macrophage-inflammatory protein-1a (MIP-1a) cell surface binding sites. Further pheno- typic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD341 Thy1 stem cells. However, more than 80% of methanol-fixed CD341 cells do bind MIP-1a, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface 1 1 expression by cytokine stimulation. The percentage of these CD34 , MIP-1a-R cells present in the CD34 compartment of NBM Downloaded from is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1a receptor implicated in HIV infection, CCR5, revealed that very few CD341 cells expressed these receptors and that expression was confined to the CD341 Thy2 progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1a are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5. The Journal of Immunology, 1999, 162: 6191–6199. http://www.jimmunol.org/ acrophage-inflammatory protein-1a (MIP-1a)3 is a accessory cells. Effects of MIP-1a on more primitive stem cell member of the chemokine family of cytokines that populations have also been described both in inhibition of stem M have been shown to elicit a wide variety of effects on cell proliferation (6) and in maintenance of the stem cell compart- cells of the immune system, including adhesion, chemoattraction, ment under conditions promoting differentiation (7–9). However, and stimulation of Ig synthesis, thereby defining chemokines as since stem cells have a requirement for stromal feeder cells, it is important mediators of the inflammatory response (1). not clear whether the action of MIP-1a is directly on the stem In addition to these shared chemokine activities, MIP-1a has cells, or an indirect effect via the stroma. by guest on September 26, 2021 been reported to act as an inhibitor of the proliferation of murine Chemokines are divided into four groups, C, CC, CXC, and hemopoietic stem cells both in vivo and in vitro (2, 3); indeed CX3C, based on the number and arrangement of conserved cys- MIP-1a was originally described as a murine stem cell inhibitor teines (10, 11). MIP-1a is a CC chemokine, for which multiple and has subsequently been shown to inhibit epidermal stem cells human G protein-coupled seven-membrane-spanning receptors a (4). Although both inhibitory and stimulatory effects of MIP-1 on have been cloned, and termed CCR (12). These receptors have the proliferation of human stem cells have been reported according complex patterns of ligand binding, whereby they display both a to the specific cytokine conditions of the assay (3), MIP-1 is specificity and promiscuity. For example, human chemokine re- known to inhibit the formation of methylcellulose colonies from ceptors CCR1 and CCR5 bind MIP-1a, but also bind other mem- highly purified progenitors (5). Clonogenic studies suggest that bers of the CC group, such as MIP-1b and RANTES. Furthermore, a MIP-1 is acting directly on progenitors rather than indirectly via CCR1 additionally binds monocyte-chemotactic protein 3 (12, 13), whereas CCR5 binds monocyte-chemotactic protein 2 (14). It has also been shown that cross-desensitization can occur among the *Leukemia Research Fund Cellular Development Unit, University of Manchester chemokine receptors, although the underlying mechanisms of re- Institute of Science and Technology (UMIST), Manchester, United Kingdom; †De- partment of Clinical Hematology, Manchester Royal Infirmary, Manchester, United ceptor internalization and signal transduction are not well charac- Kingdom; ‡The Beatson Institute for Cancer Research, Glasgow, United Kingdom; terized (15, 16). Recently, an additional high affinity receptor for § and Department of Haematology, Nottingham City Hospital, Nottingham, United a Kingdom MIP-1 , known as D6, has been identified in the mouse (17) and Received for publication September 16, 1998. Accepted for publication March in humans (18). There is currently much interest in understanding 1, 1998. how specific receptor engagement relates to individual responses The costs of publication of this article were defrayed in part by the payment of page such as chemotaxis or growth inhibition, although there is now charges. This article must therefore be hereby marked advertisement in accordance emerging evidence in the murine system that some MIP-1a recep- with 18 U.S.C. Section 1734 solely to indicate this fact. tors such as CCR1 and CCR4 may be related to inflammation, 1 This work was funded by the Leukaemia Research Fund (U.K.). whereas others such as CCR5 and the novel receptor, D6, may be 2 Address correspondence and reprint requests to Dr. Anne-Marie Buckle, Leukaemia important in the control of proliferation (17). The recent finding Research Fund Cellular Development Unit, Department of Biomolecular Sciences, UMIST, Sackville Street, Manchester, U.K. M60 1QD. E-mail address: that several chemokine receptors may act with CD4 as coreceptors [email protected] for HIV-1 infection suggests that these proteins may play a role in 3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; APC, controlling the progression of AIDS (19). CCR5, CCR2b, and to a allophycocyanin; bMIP, biotinylated MIP; CML, chronic myeloid leukemia; HI, heat- inactivated; MNC, mononuclear cell; NBM, normal bone marrow; NCS, newborn calf lesser extent CCR3 have been shown to serve as coreceptors for serum; PI, propidium iodide; SAv, streptavidin. HIV infection of macrophage-tropic HIV-1 (20–22), whereas Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 6192 MIP-1a RECEPTORS ON CD341 CELLS CXCR4 functions as a coreceptor for T cell-trophic HIV strains CCR5 were kindly provided by Dr. Charles Mackay (Leukosite, Cam- (23). The detection of CD4 expression on some CD341 cells (24) bridge, MA) and were used in conjunction with FITC anti-mouse (Phar- has raised the possibility that these cells may be a target for HIV-1 Mingen). infection. Although it is not clear whether primitive stem cells do Labeling for MIP-1a binding sites indeed constitute a potential reservoir for HIV-1, the detection by PCR of the CXCR4 receptor suggests that they may be a target for T MNC, prelabeled with anti-CD34 APC and anti-Thy-1 PE, were assayed for the presence of MIP-1a binding sites using the method provided with cell-trophic strains (25), although no conclusions could be drawn from a Fluorokine kit purchased from R&D Systems. Briefly, cells were incu- the PCR data generated in this study about CCR5 expression (25). bated with a biotinylated human rMIP-1a at 4°C for 60 min. The cells were Since cancer chemotherapy is dose limited by the damage in- then incubated (without washing) with streptavidin-fluorescein (SAv- flicted on proliferating cells within the bone marrow, members of FITC). The kit includes a negative control Ab (a soybean trypsin inhibitor protein that has been biotinylated to the same extent as the MIP-1a) and a the chemokine family are under consideration in strategies aimed MIP-1a-blocking Ab (a polyclonal goat IgG anti-human MIP-1a Ab). The at the chemoprotection and mobilization of normal bone marrow competable binding of the bMIP-1a was assessed using a 50-fold excess of (NBM) stem and progenitor cells (26). There is now evidence in unlabeled BB-10010 (British Biotech Phamaceuticals, Oxford, U.K.), diseases such as CML (6, 27, 28), acute myelogenous leukemia which is a well-characterized mutant of MIP-1a with a single amino acid a (29), and some forms of cancer (30) that chemokines may not inhibit substitution (33), or a 50-fold excess of the R&D Systems MIP-1 of the same composition as that which is biotinylated in the Fluorokine kit.

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