Natural Truncation of the Chemokine MIP-1β/CCL4 Affects Receptor Specificity but Not Anti-HIV-1 Activity
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m203077200
ISSN1083-351X
AutoresEnnan Guan, Jinhai Wang, Gregory Roderiquez, Michael A. Norcross,
Tópico(s)Immune Cell Function and Interaction
ResumoActivated lymphocytes synthesize and secrete substantial amounts of the β-chemokines macrophage inflammatory protein (MIP)-1α/CCL3 and MIP-1β/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1β secreted by activated human peripheral blood lymphocytes (MIP-1β(3–69)) lacks the two NH2-terminal amino acids of the full-length protein. This truncated form of MIP-1β has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1β(3–69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1β(3–69) showed that the truncated protein not only shares the ability of intact MIP-1β to induce Ca2+ signaling through CCR5, but unlike the full-length protein, it also triggers a Ca2+ response via CCR1 and CCR2b. These results demonstrate that NH2-terminally truncated MIP-1β functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses. Activated lymphocytes synthesize and secrete substantial amounts of the β-chemokines macrophage inflammatory protein (MIP)-1α/CCL3 and MIP-1β/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1β secreted by activated human peripheral blood lymphocytes (MIP-1β(3–69)) lacks the two NH2-terminal amino acids of the full-length protein. This truncated form of MIP-1β has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1β(3–69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1β(3–69) showed that the truncated protein not only shares the ability of intact MIP-1β to induce Ca2+ signaling through CCR5, but unlike the full-length protein, it also triggers a Ca2+ response via CCR1 and CCR2b. These results demonstrate that NH2-terminally truncated MIP-1β functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses. macrophage inflammatory protein human immunodeficiency virus type 1 regulated on activation, normal T expressed and secreted interleukin phytohemagglutinin peripheral blood lymphocyte macrophage chemoattractant protein matrix-assisted laser desorption ionization and time-of-flight stromal cell-derived factor-1 interferon-γ-inducible protein 10 macrophage-derived chemokine T helper Human macrophage inflammatory protein-1β (MIP-1β)1 is a proinflammatory chemokine that both promotes leukocyte accumulation in various inflammatory conditions and contributes to protective immunity against human immunodeficiency virus type 1 (HIV-1) (1Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar, 2Verani A. Scarlatti G. Comar M. Tresoldi E. Polo S. Giacca M. Lusso P. Siccardi A.G. Vercelli D. J. Exp. Med. 1997; 185: 805-816Crossref PubMed Scopus (147) Google Scholar, 3Zagury D. Lachgar A. Chams V. Fall L.S. Bernard J. Zagury J.F. Bizzini B. Gringeri A. Santagostino E. Rappaport J. Feldman M. O'Brien S.J. Burny A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3857-3861Crossref PubMed Scopus (188) Google Scholar, 4Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar, 5Garzino-Demo A. Moss R.B. Margolick J.B. Cleghorn F. Sill A. Blattner W.A. Cocchi F. Carlo D.J. DeVico A.L. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11986-11991Crossref PubMed Scopus (131) Google Scholar). Studies of recombinant MIP-1β(1–69) have demonstrated that this full-length protein induces intracellular Ca2+ signaling, exerts its chemotactic activity, and inhibits HIV-1 infection through interaction with the chemokine receptor CCR5.The specificity of chemokines for certain leukocyte subtypes depends on the expression by the latter of ligand-specific G protein-coupled receptors. Chemokines inhibit HIV-1 infection by blocking or down-regulating the receptors CCR5 or CXCR4, which also serve as coreceptors for the entry of HIV-1 (6Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3622) Google Scholar, 7Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2437) Google Scholar, 8Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M., Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. 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Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar), and MIP-1α, MIP-1β, and RANTES/CCL5 (regulated on activation, normal T expressed and secreted) are the major anti-HIV-1 β-chemokines secreted by human peripheral T lymphocytes stimulated by the combination of interleukin (IL)-2 and either phytohemagglutinin (PHA) or IL-12 (4Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar, 12Cocchi F. DeVico A.L. Yarchoan R. Redfield R. Cleghorn F. Blattner W.A. Garzino-Demo A. Colombini-Hatch S. Margolis D. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 97: 13812-13817Crossref Scopus (110) Google Scholar). Recent studies have suggested that, among normal human peripheral blood lymphocytes (PBLs), perforin-low memory CD8+ T cells are the predominant producers of MIP-1β (13Kamin-Lewis R. Abdelwahab S.F. Trang C. Baker A. DeVico A.L. Gallo R.C. Lewis G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9283-9288Crossref PubMed Scopus (23) Google Scholar). We have shown previously that activated human PBLs secrete MIP-1β in a complex with MIP-1α (14Guan E. Wang J. Norcross M.A. J. Biol. Chem. 2001; 276: 12404-12409Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To date, however, the biological function of naturally produced MIP-1β has remained unclear.We have now purified MIP-1β from the culture supernatant of stimulated human PBLs by affinity chromatography and used this material to study the biological activity of native MIP-1β. Our results demonstrate that the endogenous form of MIP-1β, which lacks the first two amino acid residues of the encoded protein, is active at CCR1 and CCR2 in addition to CCR5 and retains anti-HIV-1 activity.DISCUSSIONWe have characterized the structure and function of the native form of MIP-1β that is secreted by activated human PBLs in vitro. MIP-1β was dissociated from its complex with MIP-1α under acidic conditions and then purified by affinity chromatography with a MIP-1β-specific monoclonal antibody. Mass spectrometric analysis of the purified protein revealed it to be a truncated form lacking the two NH2-terminal amino acids. The removal of the Ala-Pro dipeptide from the NH2 terminus of MIP-1β did not impair its biological activities mediated by CCR5; through its interaction with this receptor, the truncated protein thus induced down-modulation of the surface expression of CCR5, increased the cytosolic-free Ca2+ concentration, and inhibited CCR5-mediated entry of HIV-1. Unexpectedly, however, MIP-1β(3–69), unlike the full-length protein, also triggered a cellular Ca2+ response mediated by CCR1 or CCR2b.MIP-1β is a multifunctional factor secreted by T lymphocytes and monocytes in a complex with MIP-1α. It is a specific ligand for CCR5 and induces the migration of CCR5-positive macrophages and lymphocytes into tissues or lymph nodes. Our demonstration that MIP-1β(3–69) also interacts functionally with CCR1 and CCR2b suggests that secretion of this truncated protein with MIP-1α might also result in the attraction of cells that express CCR1 or CCR2, such as monocytes, immature dendritic cells, and lymphocytes, possibly through heterodimer-induced interactions between multiple receptors on the cell surface. The truncated form of MIP-1β may also act on regulatory T cells (CD4+, CD25+), which are thought to respond primarily to MIP-1β (23Bystry R.S. Aluvihare V. Welch K.A. Kallikourdis M. Betz A.G. Nat. Immunol. 2002; 2: 1126-1132Crossref Scopus (405) Google Scholar). MIP-1β(3–69) appears to be produced only by activated T cells; it has not been detected in culture supernatants of monocytes or macrophages (14Guan E. Wang J. Norcross M.A. J. Biol. 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The NH2-terminal truncation of IL-8, ENA-78 (epithelial cell-derived neutrophil-activating protein), and MIP-1α results in enhanced proinflammatory activity, and that of MIP-1α/LD78β/CCL3 increases anti-HIV-1 activity (35Nufer O. Corbett M. Walz A. Biochemistry. 1999; 38: 636-642Crossref PubMed Scopus (74) Google Scholar, 36Struyf S. Menten P. Lenaerts J.P. Put W. D'Haese A., De Clercq E. Schols D. Proost P. Van Damme J. Eur. J. Immunol. 2001; 31: 2170-2178Crossref PubMed Scopus (89) Google Scholar).The enzyme responsible for MIP-1β truncation has not been identified. Although the presence of a proline in the second NH2-terminal position of MIP-1β suggests that DPPIV (CD26) is a candidate enzyme for this cleavage reaction, it has proved difficult to demonstrate processing of recombinant MIP-1β by DPPIV (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar). Other proteases implicated in chemokine processing include cathepsin G and matrix metalloproteinase, both of which have been shown to cleave and inactivate SDF-1 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 30Delgado M.B. Clark-Lewis I. Loetscher P. Langen H. Thelen M. Baggiolini M. Wolf M. Eur. J. Immunol. 2001; 31: 699-707Crossref PubMed Scopus (134) Google Scholar).Chemokine receptors and other molecules are differentially expressed by human T helper 1 (Th1) and Th2 cells. 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Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar), a ligand for CXCR3, results in loss of its activity at this latter receptor. In contrast, chemokines (such as eotaxin and MDC) that are ligands for receptors on Th2 cells (CCR4 and CCR3) are inactivated by CD26 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 25Struyf S. Proost P. Schols D., De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M., De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar). Under these conditions, the interaction of truncated MIP-1β and RANTES with CCR5 may constitute the predominant pathway for Th1 cell recruitment. Further studies are required to determine both the mechanism of MIP-1β truncation and the impact of the expanded chemokine receptor reactivity of MIP-1β(3–69) with regard to lymphocyte, monocyte, and dendritic cell migration and differentiation. Human macrophage inflammatory protein-1β (MIP-1β)1 is a proinflammatory chemokine that both promotes leukocyte accumulation in various inflammatory conditions and contributes to protective immunity against human immunodeficiency virus type 1 (HIV-1) (1Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar, 2Verani A. Scarlatti G. Comar M. Tresoldi E. Polo S. Giacca M. Lusso P. Siccardi A.G. Vercelli D. J. Exp. Med. 1997; 185: 805-816Crossref PubMed Scopus (147) Google Scholar, 3Zagury D. Lachgar A. Chams V. Fall L.S. Bernard J. Zagury J.F. Bizzini B. Gringeri A. Santagostino E. Rappaport J. Feldman M. O'Brien S.J. Burny A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3857-3861Crossref PubMed Scopus (188) Google Scholar, 4Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar, 5Garzino-Demo A. Moss R.B. Margolick J.B. Cleghorn F. Sill A. Blattner W.A. Cocchi F. Carlo D.J. DeVico A.L. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11986-11991Crossref PubMed Scopus (131) Google Scholar). Studies of recombinant MIP-1β(1–69) have demonstrated that this full-length protein induces intracellular Ca2+ signaling, exerts its chemotactic activity, and inhibits HIV-1 infection through interaction with the chemokine receptor CCR5. The specificity of chemokines for certain leukocyte subtypes depends on the expression by the latter of ligand-specific G protein-coupled receptors. Chemokines inhibit HIV-1 infection by blocking or down-regulating the receptors CCR5 or CXCR4, which also serve as coreceptors for the entry of HIV-1 (6Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3622) Google Scholar, 7Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2437) Google Scholar, 8Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M., Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3189) Google Scholar, 9Dragic T. Litwin V. Allaway G.P. Martin S.R. Huang Y. Nagashima K.A. Cayanan C. Maddon P.J. Koup R.A. Moore J.P. Paxton W.A. Nature. 1996; 381: 667-673Crossref PubMed Scopus (2811) Google Scholar, 10Doranz B.J. Rucker J., Yi, Y. Smyth R.J. Samson M. Peiper S.C. Parmentier M. Collman R.G. Doms R.W. Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1684) Google Scholar, 11Choe H. Farzan M. Sun Y. Sullivan N. Rollins B. Ponath P.D., Wu, L. Mackay C.R. LaRosa G. Newman W. Gerard N. Gerard C. Sodroski J. Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). HIV-1-specific CD8+ T cell lines have been shown to secrete β-chemokines (1Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2621) Google Scholar), and MIP-1α, MIP-1β, and RANTES/CCL5 (regulated on activation, normal T expressed and secreted) are the major anti-HIV-1 β-chemokines secreted by human peripheral T lymphocytes stimulated by the combination of interleukin (IL)-2 and either phytohemagglutinin (PHA) or IL-12 (4Wang J. Guan E. Roderiquez G. Norcross M.A. J. Immunol. 1999; 163: 5763-5769PubMed Google Scholar, 12Cocchi F. DeVico A.L. Yarchoan R. Redfield R. Cleghorn F. Blattner W.A. Garzino-Demo A. Colombini-Hatch S. Margolis D. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 97: 13812-13817Crossref Scopus (110) Google Scholar). Recent studies have suggested that, among normal human peripheral blood lymphocytes (PBLs), perforin-low memory CD8+ T cells are the predominant producers of MIP-1β (13Kamin-Lewis R. Abdelwahab S.F. Trang C. Baker A. DeVico A.L. Gallo R.C. Lewis G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9283-9288Crossref PubMed Scopus (23) Google Scholar). We have shown previously that activated human PBLs secrete MIP-1β in a complex with MIP-1α (14Guan E. Wang J. Norcross M.A. J. Biol. Chem. 2001; 276: 12404-12409Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To date, however, the biological function of naturally produced MIP-1β has remained unclear. We have now purified MIP-1β from the culture supernatant of stimulated human PBLs by affinity chromatography and used this material to study the biological activity of native MIP-1β. Our results demonstrate that the endogenous form of MIP-1β, which lacks the first two amino acid residues of the encoded protein, is active at CCR1 and CCR2 in addition to CCR5 and retains anti-HIV-1 activity. DISCUSSIONWe have characterized the structure and function of the native form of MIP-1β that is secreted by activated human PBLs in vitro. MIP-1β was dissociated from its complex with MIP-1α under acidic conditions and then purified by affinity chromatography with a MIP-1β-specific monoclonal antibody. Mass spectrometric analysis of the purified protein revealed it to be a truncated form lacking the two NH2-terminal amino acids. The removal of the Ala-Pro dipeptide from the NH2 terminus of MIP-1β did not impair its biological activities mediated by CCR5; through its interaction with this receptor, the truncated protein thus induced down-modulation of the surface expression of CCR5, increased the cytosolic-free Ca2+ concentration, and inhibited CCR5-mediated entry of HIV-1. Unexpectedly, however, MIP-1β(3–69), unlike the full-length protein, also triggered a cellular Ca2+ response mediated by CCR1 or CCR2b.MIP-1β is a multifunctional factor secreted by T lymphocytes and monocytes in a complex with MIP-1α. It is a specific ligand for CCR5 and induces the migration of CCR5-positive macrophages and lymphocytes into tissues or lymph nodes. Our demonstration that MIP-1β(3–69) also interacts functionally with CCR1 and CCR2b suggests that secretion of this truncated protein with MIP-1α might also result in the attraction of cells that express CCR1 or CCR2, such as monocytes, immature dendritic cells, and lymphocytes, possibly through heterodimer-induced interactions between multiple receptors on the cell surface. The truncated form of MIP-1β may also act on regulatory T cells (CD4+, CD25+), which are thought to respond primarily to MIP-1β (23Bystry R.S. Aluvihare V. Welch K.A. Kallikourdis M. Betz A.G. Nat. Immunol. 2002; 2: 1126-1132Crossref Scopus (405) Google Scholar). MIP-1β(3–69) appears to be produced only by activated T cells; it has not been detected in culture supernatants of monocytes or macrophages (14Guan E. Wang J. Norcross M.A. J. Biol. Chem. 2001; 276: 12404-12409Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).The NH2 termini of chemokines are important for receptor binding, activation, and specificity (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 25Struyf S. Proost P. Schols D., De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M., De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar, 26Proost P. Struyf S. Schols D. Opdenakker G. Sozzani S. Allavena P. Mantovani A. Augustyns K. Bal G. Haemers A. Lambeir A.M. Scharpe S. Van Damme J. De Meester I. J. Biol. Chem. 1999; 274: 3988-3993Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 27Xin X. Shioda T. Kato A. Liu H. Sakai Y. Nagai Y. FEBS Lett. 1999; 457: 219-222Crossref PubMed Scopus (24) Google Scholar, 28Laurence J.S. LiWang A.C. LiWang P.J. Biochemistry. 1998; 37: 9346-9354Crossref PubMed Scopus (36) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 30Delgado M.B. Clark-Lewis I. Loetscher P. Langen H. Thelen M. Baggiolini M. Wolf M. Eur. J. Immunol. 2001; 31: 699-707Crossref PubMed Scopus (134) Google Scholar, 31Proost P. Menten P. Struyf S. Schutyser E., De Meester I. Van Damme J. Blood. 2001; 96: 1674-1680Crossref Google Scholar, 32Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E., Hu, H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar). Relatively small changes in the NH2-terminal amino acid sequence thus markedly affect the biological activity of chemokines. Members of both the CC and CXC subfamilies of chemokines occur naturally as post-translationally modified proteins. We and others have shown that the dipeptidyl peptidase IV (DPPIV) (CD26) plays an important role in the processing of chemokines that contain a penultimate alanine or proline residue at the NH2 termini and that such processing influences the functional properties of chemokines including RANTES, stromal cell-derived factor-1 (SDF-1)/CXCL12, eotaxin/CCL11, IP-10/CXCL10 (interferon-γ-inducible protein 10), granulocyte chemotactic protein/CXCL6, macrophage-derived chemokine (MDC)/CCL22, and MIP-1α(P)/LD78β (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 28Laurence J.S. LiWang A.C. LiWang P.J. Biochemistry. 1998; 37: 9346-9354Crossref PubMed Scopus (36) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 33Struyf S., De Meester I. Scharpe S. Lenaerts J.P. Menten P. Wang J.M. Proost P. Van Damme J. Eur. J. Immunol. 1998; 28: 1262-1271Crossref PubMed Scopus (109) Google Scholar, 34Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar, 35Nufer O. Corbett M. Walz A. Biochemistry. 1999; 38: 636-642Crossref PubMed Scopus (74) Google Scholar, 36Struyf S. Menten P. Lenaerts J.P. Put W. D'Haese A., De Clercq E. Schols D. Proost P. Van Damme J. Eur. J. Immunol. 2001; 31: 2170-2178Crossref PubMed Scopus (89) Google Scholar, 37Muller A. Trabandt A. Gloeckner-Hofmann K. Seitzer U. Csernok E. Schonermarck U. Feller A.C. Gross W.L. J. Pathol. 2002; 192: 113-120Crossref Scopus (91) Google Scholar). The removal of the NH2-terminal dipeptide from RANTES, SDF-1α, or MDC results in reduced receptor binding and signaling and consequent impairment of chemotactic responses. Such truncation of RANTES also results in a loss of the ability to signal through CCR1, in contrast to the situation with MIP-1β, which gains activity at CCR1. RANTES(3–68), like MIP-1β(3–69), retains activity at CCR5 (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar). Amino-terminal modification of other chemokines has been shown to result in gain of function. The NH2-terminal truncation of IL-8, ENA-78 (epithelial cell-derived neutrophil-activating protein), and MIP-1α results in enhanced proinflammatory activity, and that of MIP-1α/LD78β/CCL3 increases anti-HIV-1 activity (35Nufer O. Corbett M. Walz A. Biochemistry. 1999; 38: 636-642Crossref PubMed Scopus (74) Google Scholar, 36Struyf S. Menten P. Lenaerts J.P. Put W. D'Haese A., De Clercq E. Schols D. Proost P. Van Damme J. Eur. J. Immunol. 2001; 31: 2170-2178Crossref PubMed Scopus (89) Google Scholar).The enzyme responsible for MIP-1β truncation has not been identified. Although the presence of a proline in the second NH2-terminal position of MIP-1β suggests that DPPIV (CD26) is a candidate enzyme for this cleavage reaction, it has proved difficult to demonstrate processing of recombinant MIP-1β by DPPIV (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar). Other proteases implicated in chemokine processing include cathepsin G and matrix metalloproteinase, both of which have been shown to cleave and inactivate SDF-1 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 30Delgado M.B. Clark-Lewis I. Loetscher P. Langen H. Thelen M. Baggiolini M. Wolf M. Eur. J. Immunol. 2001; 31: 699-707Crossref PubMed Scopus (134) Google Scholar).Chemokine receptors and other molecules are differentially expressed by human T helper 1 (Th1) and Th2 cells. Th1 cell-associated molecules include CD26, interferon-γ, lymphocyte activation gene-3 (LAG-3), and the chemokine receptors CCR5 and CXCR3, whereas Th2 cell-associated molecules include the chemokine receptors CCR3, CCR4, and CCR8 as well as CD62L and CD30. The expression of CD26 correlates with the production of interferon-γ in Th1 cell-associated granulomatous disorders and in inflamed synovia in individuals with rheumatoid arthritis (37Muller A. Trabandt A. Gloeckner-Hofmann K. Seitzer U. Csernok E. Schonermarck U. Feller A.C. Gross W.L. J. Pathol. 2002; 192: 113-120Crossref Scopus (91) Google Scholar, 38Gerli R. Muscat C. Bertotto A. Bistoni O. Agea E. Tognellini R. Fiorucci G. Cesarotti M. Bombardieri S. Clin. Immunol. Immunopathol. 1996; 80: 31-37Crossref PubMed Scopus (46) Google Scholar, 39Cordero O.J. Salgado F.J. Mera-Varela A. Nogueira M. Rheumatol. Int. 2002; 21: 69-74Crossref Scopus (73) Google Scholar, 40Cuchacovich M. Gatica H. Pizzo S.V. Gonzalez-Gronow M. Clin. Exp. Rheumatol. 2002; 19: 673-680Google Scholar). CD26 is also associated with the production of Th1 cytokines in CD4+ T cell clones (41De Meester I.A. Kestens L.L. Vanham G.L. Vanhoof G.C. Vingerhoets J.H. Gigase P.L. Scharpe S.L. J. Leukocyte Biol. 1995; 58: 325-330Crossref PubMed Scopus (25) Google Scholar). The differential expression of chemokine receptors by human Th1 and Th2 cells underlies the differential migration of these cells in response to various chemokines. Chemokines are thus important contributors to polarized Th1 cell- or Th2 cell-mediated immune responses. Truncation of the CCR5-specific chemokines RANTES and MIP-1β, possibly mediated by CD26 expressed on activated lymphocytes, does not affect their activities at CCR5, whereas cleavage of IP-10 (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar), a ligand for CXCR3, results in loss of its activity at this latter receptor. In contrast, chemokines (such as eotaxin and MDC) that are ligands for receptors on Th2 cells (CCR4 and CCR3) are inactivated by CD26 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 25Struyf S. Proost P. Schols D., De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M., De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar). Under these conditions, the interaction of truncated MIP-1β and RANTES with CCR5 may constitute the predominant pathway for Th1 cell recruitment. Further studies are required to determine both the mechanism of MIP-1β truncation and the impact of the expanded chemokine receptor reactivity of MIP-1β(3–69) with regard to lymphocyte, monocyte, and dendritic cell migration and differentiation. We have characterized the structure and function of the native form of MIP-1β that is secreted by activated human PBLs in vitro. MIP-1β was dissociated from its complex with MIP-1α under acidic conditions and then purified by affinity chromatography with a MIP-1β-specific monoclonal antibody. Mass spectrometric analysis of the purified protein revealed it to be a truncated form lacking the two NH2-terminal amino acids. The removal of the Ala-Pro dipeptide from the NH2 terminus of MIP-1β did not impair its biological activities mediated by CCR5; through its interaction with this receptor, the truncated protein thus induced down-modulation of the surface expression of CCR5, increased the cytosolic-free Ca2+ concentration, and inhibited CCR5-mediated entry of HIV-1. Unexpectedly, however, MIP-1β(3–69), unlike the full-length protein, also triggered a cellular Ca2+ response mediated by CCR1 or CCR2b. MIP-1β is a multifunctional factor secreted by T lymphocytes and monocytes in a complex with MIP-1α. It is a specific ligand for CCR5 and induces the migration of CCR5-positive macrophages and lymphocytes into tissues or lymph nodes. Our demonstration that MIP-1β(3–69) also interacts functionally with CCR1 and CCR2b suggests that secretion of this truncated protein with MIP-1α might also result in the attraction of cells that express CCR1 or CCR2, such as monocytes, immature dendritic cells, and lymphocytes, possibly through heterodimer-induced interactions between multiple receptors on the cell surface. The truncated form of MIP-1β may also act on regulatory T cells (CD4+, CD25+), which are thought to respond primarily to MIP-1β (23Bystry R.S. Aluvihare V. Welch K.A. Kallikourdis M. Betz A.G. Nat. Immunol. 2002; 2: 1126-1132Crossref Scopus (405) Google Scholar). MIP-1β(3–69) appears to be produced only by activated T cells; it has not been detected in culture supernatants of monocytes or macrophages (14Guan E. Wang J. Norcross M.A. J. Biol. Chem. 2001; 276: 12404-12409Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The NH2 termini of chemokines are important for receptor binding, activation, and specificity (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 25Struyf S. Proost P. Schols D., De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M., De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar, 26Proost P. Struyf S. Schols D. Opdenakker G. Sozzani S. Allavena P. Mantovani A. Augustyns K. Bal G. Haemers A. Lambeir A.M. Scharpe S. Van Damme J. De Meester I. J. Biol. Chem. 1999; 274: 3988-3993Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 27Xin X. Shioda T. Kato A. Liu H. Sakai Y. Nagai Y. FEBS Lett. 1999; 457: 219-222Crossref PubMed Scopus (24) Google Scholar, 28Laurence J.S. LiWang A.C. LiWang P.J. Biochemistry. 1998; 37: 9346-9354Crossref PubMed Scopus (36) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 30Delgado M.B. Clark-Lewis I. Loetscher P. Langen H. Thelen M. Baggiolini M. Wolf M. Eur. J. Immunol. 2001; 31: 699-707Crossref PubMed Scopus (134) Google Scholar, 31Proost P. Menten P. Struyf S. Schutyser E., De Meester I. Van Damme J. Blood. 2001; 96: 1674-1680Crossref Google Scholar, 32Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E., Hu, H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (159) Google Scholar). Relatively small changes in the NH2-terminal amino acid sequence thus markedly affect the biological activity of chemokines. Members of both the CC and CXC subfamilies of chemokines occur naturally as post-translationally modified proteins. We and others have shown that the dipeptidyl peptidase IV (DPPIV) (CD26) plays an important role in the processing of chemokines that contain a penultimate alanine or proline residue at the NH2 termini and that such processing influences the functional properties of chemokines including RANTES, stromal cell-derived factor-1 (SDF-1)/CXCL12, eotaxin/CCL11, IP-10/CXCL10 (interferon-γ-inducible protein 10), granulocyte chemotactic protein/CXCL6, macrophage-derived chemokine (MDC)/CCL22, and MIP-1α(P)/LD78β (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar, 28Laurence J.S. LiWang A.C. LiWang P.J. Biochemistry. 1998; 37: 9346-9354Crossref PubMed Scopus (36) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 33Struyf S., De Meester I. Scharpe S. Lenaerts J.P. Menten P. Wang J.M. Proost P. Van Damme J. Eur. J. Immunol. 1998; 28: 1262-1271Crossref PubMed Scopus (109) Google Scholar, 34Ohtsuki T. Hosono O. Kobayashi H. Munakata Y. Souta A. Shioda T. Morimoto C. FEBS Lett. 1998; 431: 236-240Crossref PubMed Scopus (57) Google Scholar, 35Nufer O. Corbett M. Walz A. Biochemistry. 1999; 38: 636-642Crossref PubMed Scopus (74) Google Scholar, 36Struyf S. Menten P. Lenaerts J.P. Put W. D'Haese A., De Clercq E. Schols D. Proost P. Van Damme J. Eur. J. Immunol. 2001; 31: 2170-2178Crossref PubMed Scopus (89) Google Scholar, 37Muller A. Trabandt A. Gloeckner-Hofmann K. Seitzer U. Csernok E. Schonermarck U. Feller A.C. Gross W.L. J. Pathol. 2002; 192: 113-120Crossref Scopus (91) Google Scholar). The removal of the NH2-terminal dipeptide from RANTES, SDF-1α, or MDC results in reduced receptor binding and signaling and consequent impairment of chemotactic responses. Such truncation of RANTES also results in a loss of the ability to signal through CCR1, in contrast to the situation with MIP-1β, which gains activity at CCR1. RANTES(3–68), like MIP-1β(3–69), retains activity at CCR5 (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar). Amino-terminal modification of other chemokines has been shown to result in gain of function. The NH2-terminal truncation of IL-8, ENA-78 (epithelial cell-derived neutrophil-activating protein), and MIP-1α results in enhanced proinflammatory activity, and that of MIP-1α/LD78β/CCL3 increases anti-HIV-1 activity (35Nufer O. Corbett M. Walz A. Biochemistry. 1999; 38: 636-642Crossref PubMed Scopus (74) Google Scholar, 36Struyf S. Menten P. Lenaerts J.P. Put W. D'Haese A., De Clercq E. Schols D. Proost P. Van Damme J. Eur. J. Immunol. 2001; 31: 2170-2178Crossref PubMed Scopus (89) Google Scholar). The enzyme responsible for MIP-1β truncation has not been identified. Although the presence of a proline in the second NH2-terminal position of MIP-1β suggests that DPPIV (CD26) is a candidate enzyme for this cleavage reaction, it has proved difficult to demonstrate processing of recombinant MIP-1β by DPPIV (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar). Other proteases implicated in chemokine processing include cathepsin G and matrix metalloproteinase, both of which have been shown to cleave and inactivate SDF-1 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 29McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 30Delgado M.B. Clark-Lewis I. Loetscher P. Langen H. Thelen M. Baggiolini M. Wolf M. Eur. J. Immunol. 2001; 31: 699-707Crossref PubMed Scopus (134) Google Scholar). Chemokine receptors and other molecules are differentially expressed by human T helper 1 (Th1) and Th2 cells. Th1 cell-associated molecules include CD26, interferon-γ, lymphocyte activation gene-3 (LAG-3), and the chemokine receptors CCR5 and CXCR3, whereas Th2 cell-associated molecules include the chemokine receptors CCR3, CCR4, and CCR8 as well as CD62L and CD30. The expression of CD26 correlates with the production of interferon-γ in Th1 cell-associated granulomatous disorders and in inflamed synovia in individuals with rheumatoid arthritis (37Muller A. Trabandt A. Gloeckner-Hofmann K. Seitzer U. Csernok E. Schonermarck U. Feller A.C. Gross W.L. J. Pathol. 2002; 192: 113-120Crossref Scopus (91) Google Scholar, 38Gerli R. Muscat C. Bertotto A. Bistoni O. Agea E. Tognellini R. Fiorucci G. Cesarotti M. Bombardieri S. Clin. Immunol. Immunopathol. 1996; 80: 31-37Crossref PubMed Scopus (46) Google Scholar, 39Cordero O.J. Salgado F.J. Mera-Varela A. Nogueira M. Rheumatol. Int. 2002; 21: 69-74Crossref Scopus (73) Google Scholar, 40Cuchacovich M. Gatica H. Pizzo S.V. Gonzalez-Gronow M. Clin. Exp. Rheumatol. 2002; 19: 673-680Google Scholar). CD26 is also associated with the production of Th1 cytokines in CD4+ T cell clones (41De Meester I.A. Kestens L.L. Vanham G.L. Vanhoof G.C. Vingerhoets J.H. Gigase P.L. Scharpe S.L. J. Leukocyte Biol. 1995; 58: 325-330Crossref PubMed Scopus (25) Google Scholar). The differential expression of chemokine receptors by human Th1 and Th2 cells underlies the differential migration of these cells in response to various chemokines. Chemokines are thus important contributors to polarized Th1 cell- or Th2 cell-mediated immune responses. Truncation of the CCR5-specific chemokines RANTES and MIP-1β, possibly mediated by CD26 expressed on activated lymphocytes, does not affect their activities at CCR5, whereas cleavage of IP-10 (17Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (317) Google Scholar), a ligand for CXCR3, results in loss of its activity at this latter receptor. In contrast, chemokines (such as eotaxin and MDC) that are ligands for receptors on Th2 cells (CCR4 and CCR3) are inactivated by CD26 (24Proost P. Struyf S. Schols D. Durinx C. Wuyts A. Lenaerts J.P., De Clercq E., De Meester I. Van Damme J. FEBS Lett. 1998; 432: 73-76Crossref PubMed Scopus (179) Google Scholar, 25Struyf S. Proost P. Schols D., De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M., De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar). Under these conditions, the interaction of truncated MIP-1β and RANTES with CCR5 may constitute the predominant pathway for Th1 cell recruitment. Further studies are required to determine both the mechanism of MIP-1β truncation and the impact of the expanded chemokine receptor reactivity of MIP-1β(3–69) with regard to lymphocyte, monocyte, and dendritic cell migration and differentiation. We thank K. Fields for the preparation of human PBLs and monocytes. CCR5, CCR1, and CCR2b transfectants were obtained through the AIDS Research and Reference Reagent Program of the Division of AIDS, NIAID, National Institutes of Health.
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