Identification of Human Macrophage Inflammatory Proteins 1alpha and 1beta as a Native Secreted Heterodimer*

Ennan GuanDagger, Jinhai Wang, and Michael A. NorcrossDagger

From the Laboratory of Gene Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 17, 2000, and in revised form, January 12, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta ) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH2-terminal truncated MIP-1alpha (residues 5-70) together with either intact MIP-1beta or NH2-terminal truncated MIP-1beta (residues 3-69), respectively. The secreted MIP-1alpha /beta heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH2-terminal truncated MIP-1alpha and full-length MIP-1beta in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1alpha /beta may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines constitute a family of small secreted proteins that were initially characterized on the basis of their chemotactic effects on a variety of leukocytes (1, 2). They are produced locally in tissues and interact with selective G protein-coupled receptors expressed on the leukocyte surface. Chemokines also regulate leukocyte maturation, the trafficking and homing of lymphocytes, and the development of lymphoid tissues. Human immunodeficiency virus-type 1 (HIV-1)1 also targets chemokine receptors during entry into cells, and certain chemokines act as HIV-1 suppressive factors (3-5).

To date, at least 50 chemokines have been identified. Despite its large size, the chemokine family is remarkably homogeneous. Chemokines are divided into two subfamilies, alpha (CXC) and beta (CC), on the basis of conserved cysteine residues. Four conserved cysteines form two essential disulfide bonds, Cys1-Cys3 and Cys2-Cys4, in all chemokines. The three-dimensional structures of three alpha  chemokines (interleukin (IL)-8, growth-related oncogene-alpha , and platelet factor 4) and four beta  chemokines (macrophage inflammatory protein (MIP)-1alpha , MIP-1beta , RANTES (regulated on activation normal T cell expressed), and macrophage chemoattractant protein-1 (MCP-1)) have been determined either by multidimensional nuclear magnetic resonance (NMR) or by x-ray crystallography (6-11). These studies have revealed that chemokines possess a short NH2-terminal domain preceding the first cysteine, a backbone that comprises three antiparallel beta  strands and a COOH-terminal alpha -helix. Whereas the backbone exhibits a well ordered structure, the structure of the NH2 terminus is disordered. The similarity in the three-dimensional structures of the chemokine monomers is consistent with the marked sequence homology of these proteins.

The quaternary structures of alpha  and beta  chemokines, however, differ markedly from each other, and the dimer interfaces are formed by distinct sets of residues. Whereas the IL-8 dimer is globular, the homodimers formed by MIP-1alpha , MIP-1beta , and RANTES are cylindrical (6, 7, 11, 12). Calculation of the solvation-free energies of dimerization and analysis of hydrophobic clusters of amino acids suggest that the formation and stabilization of the two different types of dimers result from the burial of hydrophobic residues and that the distinct quaternary structures of alpha  and beta  chemokine dimers are preserved throughout the two subfamilies. The biological existence and significance of dimeric forms of chemokines, especially that of chemokine heterodimers, have remained unclear.

Human T lymphocytes and monocytes release anti-HIV-1 chemokines, predominantly the beta  chemokines MIP-1alpha , MIP-1beta , and RANTES (3, 4, 13), in response to stimulation with mitogens, cytokines, or bacterial pathogens. We now show that human monocytes stimulated by lipopolysaccharide, human peripheral blood lymphocytes (PBLs) stimulated by cytokines (IL-2 and IL-12), or phytohemagglutinin produce a chemokine heterodimer containing MIP-1alpha and MIP-1beta .


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Recombinant human MIP-1alpha (full-length), RANTES, MDC, MCP-1, and IP-10 were obtained from Peprotech (Rocky Hill, NJ); recombinant human MIP-1beta was from Sigma; and recombinant human (-4)MIP-1alpha and antigen affinity purified antibodies to chemokines were from R&D Systems (Minneapolis, MN).

Cell Culture-- Human PBLs and monocytes were isolated by elutriation from normal blood donors and cultured as described previously (13, 14). PBLs were cultured under 5% CO2 at 37 °C in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 150 units/ml IL-2 (R&D Systems), and 10 ng/ml IL-12 (kindly provided by Genetics Institute, Boston, MA) or 2 µg/mo PHA (sigma). Monocytes were cultured in macrophage serum-free medium (Life Technologies, Inc.) supplemented with 1 µg/ml LPS (Sigma). Cells were plated at a density of 1 × 106 cells/ml in 6-well plates.

Immunoprecipitation and Immunoblot Analysis-- Cell culture supernatants were incubated at 4 °C for 2 h with antibodies to anti-MIP-1alpha or anti-MIP-1beta , and then for an additional 2 h, they were incubated with protein G-Sepharose beads (Amersham Pharmacia Biotech). The beads were washed three times with phosphate-buffered saline containing 0.3% Triton X-100, after which proteins were eluted with 25 mM Tris-glycine, pH 3.0, subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions on a 4-20% gradient gel in the presence of Tricine, and transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 2 h at room temperature with Tris-buffered saline containing 3% bovine serum albumin and then exposed to either anti-MIP-1alpha or anti-MIP1beta . Immune complexes were detected with alkaline phosphatase-conjugated goat antibodies to mouse immunoglobulin G, alkaline phosphatase substrate, and Lumi-Phos TM 530 (Roche Molecular Biochemicals).

Mass Spectrometry-- Culture supernatants were subjected to immunoprecipitation with anti-MIP-1alpha or anti-MIP-1beta . The molecular size of precipitated proteins was determined by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry (PerSeptive Biosystems, Boston, MA). alpha -Cyano-4-hydroxycinnamic acid (Sigma) and recombinant MIP-1alpha or MIP-1beta were used as matrix and internal standards, respectively. For some experiments, cells were cultured in the presence of monensin (GolgiStop, Pharmingen, San Diego, CA) for 10 h. They were then washed and lysed for 1 h on ice at a density of 1 × 108 cells/ml in a solution containing 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml E64, and 40 µg/ml bestatin (Roche Molecular Biochemicals). After centrifugation of lysates at 6700 × g for 15 min, the resulting supernatants were subjected to immunoprecipitation with either anti-MIP-1alpha or anti-MIP-1beta for mass spectrometric analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a Heterodimer of MIP-1alpha /beta in Culture Supernatants of Human Monocytes and PBLs-- Supernatants were collected from cultures of either LPS-stimulated monocytes or PBLs stimulated with IL-2 and IL-12 after incubation for 1 or 6 days, respectively, times that correspond to peak chemokine secretion (data not shown). Enzyme-linked immunosorbent assays revealed that the culture supernatants contained large amounts of MIP-1alpha and MIP-1beta but only a low concentration of RANTES (data not shown) (13). Immunoprecipitation of culture supernatants from both cell types with either anti-MIP-1alpha or anti-MIP-1beta followed by immunoblot analysis with each of these antibodies revealed that each immunoprecipitate contained both MIP-1alpha and MIP-1beta (Fig. 1A). The antigen affinity purified antibodies to each MIP-1 protein were specific and did not cross-react with the other isoform on Western blot analysis (Fig. 1B) or with immunoprecipitation followed by mass spectrometric analysis (Fig. 2B).



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Fig. 1.   Coimmunoprecipitation of MIP-1alpha and MIP-1beta from culture supernatants of monocytes and PBLs. A, culture supernatants of either monocytes stimulated with LPS for 1 day or PBLs stimulated with IL-2 and IL-12 for 6 days were subjected to immunoprecipitation (IP) with anti-MIP-1alpha or anti-MIP-1beta . The resulting precipitates were then subjected to immunoblot analysis with anti-MIP-1alpha (left panel) or anti-MIP-1beta (right panel). Lanes 1 and 4 contain recombinant human (-4)MIP-1alpha (7.4 kDa) and recombinant human MIP-1beta (7.8 kDa), respectively, probed with the corresponding antibody. Data in this and subsequent figures are representative of at least three independent experiments. B, recombinant human MIP-1alpha and recombinant MIP-1beta probed with anti-MIP-1alpha or anti-MIP-1beta antibody.



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Fig. 2.   Mass spectrometric analysis of MIP-1alpha /beta complexes secreted by monocytes and PBLs. A, culture supernatants of either PBLs stimulated with IL-2 and IL-12 for 6 days (left panels) or monocytes stimulated with LPS for 1 day (right panels) were subjected to immunoprecipitation with anti-MIP-1alpha (upper panels) or anti-MIP-1beta (lower panels). The molecular mass of the precipitated proteins was then determined by mass spectrometry. B, mass spectrometric analysis of immunoprecipitation of recombinant MIP-1alpha and recombinant MIP-1beta with anti-MIP-1alpha or anti-MIP-1beta antibody.

To determine the molecular size of the chemokines precipitated by anti-MIP-1alpha or anti-MIP-1beta , we analyzed the precipitated proteins by mass spectrometry. The two antibodies precipitated identical complexes from the culture supernatant of LPS-stimulated monocytes (Fig. 2). These complexes consisted predominantly of two polypeptides with molecular masses of 7459 and 7826 Da, which correspond to MIP-1alpha lacking the four NH2-terminal residues ((-4)MIP-1alpha ) and intact MIP-1beta , respectively. The complexes immunoprecipitated by each of the two antibodies from the culture supernatant of IL-2- and IL-12-stimulated PBLs contained (-4)MIP-1alpha and a protein with a molecular mass of 7658 Da, which corresponds to MIP-1beta lacking the two NH2-terminal residues ((-2)MIP-1beta ). Similar results were obtained with culture supernatants of PBLs stimulated with IL-2 and phytohemagglutinin (data not shown). Immunoprecipitates prepared from the monocyte and PBL culture supernatants with anti-RANTES or anti-MDC were also analyzed by mass spectrometry. However, this approach failed to detect either RANTES or MDC in the supernatants (data not shown). Thus, these data demonstrate that LPS-treated monocytes and PBLs stimulated with IL-2 and IL-12 release a MIP-1alpha /beta heterodimer into the culture supernatant.

MIP-1alpha and MIP-1beta Are Secreted Predominantly as a Heterodimer-- We next examined whether secreted MIP-1alpha and MIP-1beta were present in the culture supernatant of LPS-stimulated monocytes predominantly as a heterodimer, or whether they were also present as monomers. The supernatant was subjected to immunoprecipitation first with anti-MIP-1alpha and then either with anti-MIP-1beta or again with anti-MIP-1alpha . The various immunoprecipitates were then subjected to immunoblot analysis with anti-MIP-1alpha or anti-MIP-1beta (Fig. 3). Only a small amount of residual MIP-1alpha was detected in the second precipitate prepared with anti-MIP-1alpha , indicating that the first immunoprecipitation with this antibody efficiently removed most of the target antigen. The observation that the second precipitate prepared with anti-MIP-1beta contained little MIP-1beta indicated that most of this protein had been previously precipitated as a heterodimer with MIP-1alpha .



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Fig. 3.   Secretion of MIP-1alpha and MIP-1beta from monocytes predominantly as a heterodimer. Culture supernatant of monocytes stimulated with LPS for 1 day was subjected to immunoprecipitation (IP) first with anti-MIP-1alpha and then either with anti-MIP-1alpha again or with anti-MIP-1beta . The three precipitates were then subjected to immunoblot analysis with anti-MIP-1alpha or anti-MIP-1beta as indicated. Recombinant human MIP-1alpha (7.4 kDa) or MIP-1beta (7.8 kDa) were also probed with the corresponding antibodies.

MIP-1alpha /beta Heterodimer in the Golgi Complex-- We next investigated the kinetics and site of MIP-1alpha /beta dimerization. Immunoprecipitation and mass spectrometric analysis revealed the presence of the MIP-1alpha /beta heterodimer in the culture supernatants of monocytes or PBLs as early as 7-10 h after exposure to LPS or to IL-2 and IL-12, respectively (data not shown). To determine whether the heterodimerization of MIP-1alpha and MIP-1beta occurs intracellularly in the Golgi complex, we treated cells with monensin to prevent intracellular protein transport by inducing retention of proteins in the Golgi complex (15, 16). PBLs and monocytes were incubated with IL-2 and phytohemagluttinin A or with LPS, respectively, for 20 h, the last 10 h of which they were also exposed to monensin. Under these conditions, chemokines were produced primarily during the last 10 h of culture. The cells were then lysed and subjected to immunoprecipitation with anti-MIP-1alpha or anti-MIP-1beta , and the resulting precipitates were analyzed by mass spectrometry. The immunoprecipitates prepared from both PBLs (Fig. 4A) and monocytes (Fig. 4B) with either antibody consisting predominantly of heterodimers of (-4)MIP-1alpha and full-length MIP-1beta . The results revealed that MIP-1beta was processed from the intact form (7826 Da) detected in the Golgi complex and in the culture supernatant that was obtained after 1 day (data not shown) to a mixture of both the full-length protein and (-2)MIP-1beta (7658 Da) in the supernatant obtained after 3 days. After incubation of PBLs for 6 days, almost all of the MIP-1beta had been converted to the NH2-terminal truncated form. In contrast, analysis of the culture supernatants of monocytes obtained after 1 day (Fig. 4B) or 3 days (data not shown) did not reveal proteolytic processing of MIP-1beta . Given that monensin only blocks protein transport to the post-Golgi compartment from the trans-Golgi network, these data indicate that MIP-1alpha /beta heterodimers probably form in the endoplasmic reticulum or cis-Golgi. Other experiments using brefeldin A to disrupt the Golgi complex also revealed the presence of heterodimers in cell lysates (data not shown) and suggest that heterodimers are formed in a pre-Golgi compartment. They also suggest that the proteolytic processing of MIP-1beta released by PBLs may occur at a late or post-Golgi step during secretion. The MIP-1alpha /beta heterodimer was also detected in immunoprecipitates prepared from both PBL and monocyte lysates without monensin treatment (Fig. 4C), indicating that MIP-1alpha /beta dimerization occurs under normal conditions.



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Fig. 4.   Intracellular formation of the MIP-1alpha /beta heterodimer. PBLs (A) or monocytes (B) were stimulated with IL-2 and PHA or with LPS, respectively, for 10 h and then incubated for an additional 10 h in the presence or absence of monensin (C). The cells were then either lysed or incubated further in the absence of monensin. Cell lysates, as well as culture supernatants obtained after incubation of monocytes for 1 day or PBLs for 3 or 6 days, were subjected to immunoprecipitation with anti-MIP-1alpha or anti-MIP-1beta as indicated, and the resulting precipitates were analyzed by mass spectrometry.

Dissociation of Native MIP-1alpha /beta Heterodimers under Acidic Conditions-- To investigate the nature of the interaction between MIP-1alpha and MIP-1beta , we adjusted the culture supernatants of stimulated PBLs or monocytes to pH 4.0 before immunoprecipitation with anti-MIP-1alpha or anti-MIP-1beta . Under these low pH conditions, each antibody precipitated only the corresponding antigen (Fig. 5). These results suggest that acidic conditions induce the dissociation of the MIP-1alpha /beta heterodimer and that electrostatic interactions among charged amino acids therefore are likely to contribute to formation of the dimer interface.



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Fig. 5.   Dissociation of the MIP-1alpha /beta heterodimer under low pH conditions. Culture supernatants of either PBLs stimulated with IL-2 and IL-12 for 6 days (left panels) or monocytes stimulated with LPS for 1 day (right panels) were adjusted to pH 7.0 or 4.0 and then subjected to immunoprecipitation with anti-MIP-1alpha or anti-MIP-1beta as indicated. The molecular mass of the precipitated proteins was then determined by mass spectrometry.

Specificity of MIP-1alpha /beta Heterodimerization-- Direct measurements of monomer-dimer equilibrium by NMR or sedimentation analysis have revealed that alpha  chemokines (IL-8, growth-related oncogene-alpha , neutrophil-activating protein-2, low affinity platelet factor 4, and PF4) and beta  chemokines (MCP-1, MCP-2, MCP-3, MIP-1alpha , MIP-1beta , RANTES, and I-309) (6-9, 17-20) are monomeric at physiological (nanomolar) concentrations. To examine the specificity of MIP-1alpha /beta heterodimerization at physiological concentrations, we mixed various combinations of recombinant chemokines in vitro at a 1:1 ratio and concentrations of 25-50 nM at physiological pH. We paired MIP-1alpha with MIP-1beta , RANTES, MDC, MCP-1, or IP-10. In parallel, we mixed RANTES with MIP-1beta , MDC, MCP-1, or IP-10; IP-10 was also tested for pairing with MCP-1. The various mixtures were then subjected to immunoprecipitation with corresponding specific antibodies, and the resulting precipitates were analyzed by mass spectrometry. Heterodimerization was detected only between MIP-1alpha and MIP-1beta and not with RANTES (Fig. 6) or with the other chemokines tested (data not shown), thus indicating that this interaction is highly specific. The antigen affinity purified antibodies to each MIP-1 protein were specific and did not cross-react with the other isoform (Fig. 6).



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Fig. 6.   Specificity of MIP-1alpha /beta heterodimerization in vitro. Recombinant (-4)MIP-1alpha {(7450 Da), MIP-1beta (7820 Da), and RANTES (7864 Da) dissolved in phosphate-buffered saline, pH 7.4, were mixed at final concentrations of 25 nM in the indicated combinations at room temperature and then subjected to immunoprecipitation with the indicated antibodies. The resulting precipitates were analyzed by mass spectrometry.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The possible existence and activity of native chemokine dimers have been controversial (21). The beta  chemokines MIP-1alpha , MIP-1beta , and RANTES tend to self-associate and thereby form homodimers, tetramers, or larger aggregates in vitro. This process is dynamic and reversible, but it has been thought that chemokine concentrations in vivo may be too low for the formation of such multimers to occur. However, high local concentrations of chemokines may occur in vivo under certain conditions, such as during platelet degranulation, inflammatory disease, and local accumulation of chemokines on cell membranes mediated by receptors or by glycosaminoglycans.

We have now identified a naturally occurring MIP-1alpha /beta heterodimer produced by activated monocytes and PBLs. Our data demonstrate that the MIP-1alpha /beta heterodimer forms in the endoplasmic reticulum or Golgi complex, and that these two chemokines are secreted in the form of the heterodimer. Furthermore, the combination of MIP-1alpha with MIP-1beta in vitro showed that these two chemokines indeed form heterodimers at physiological (nanomolar) concentrations.

Most studies on chemokine homodimerization have been performed in vitro, and the natural formation of either homodimers or heterodimers of chemokines has not been described previously. Native MIP-1 purified from LPS-stimulated mouse macrophage RAW 264.7 migrated on SDS-polyacrylamide gels as a doublet composed of peptides with similar physical characteristics. The NH2-terminal sequences of the two peptides identified them as MIP-1alpha and MIP-1beta (22). Although it was not shown that the co-purified mouse chemokines originally existed as a heterodimer, our data now suggest that these previous results might be explained by the formation of a native mouse MIP-1alpha /beta heterodimer.

The three-dimensional structures of both alpha  and beta  chemokines have been determined mostly with the molecules in the form of crystallized homodimers. Whereas the monomeric structures of both alpha  and beta  chemokines are highly similar, the dimeric structures of members of these two subfamilies, as typified by IL-8 (12, 20, 23) and MIP-1beta (9, 11, 20), respectively, differ markedly from each other with the dimer interfaces being formed by distinct sets of interacting residues. Whereas the IL-8 dimer is globular, the MIP-1beta dimer is cylindrical. The three-dimensional structure of the alpha  chemokine PF4 revealed it to be a tetramer composed of two dimers of the IL-8 type (18, 24). NMR revealed MIP-1beta as an end-on-end dimer with the dimer interface showing a large number of contacts between the two monomers. Other beta  chemokines, including RANTES, MIP-1alpha , MCP-1, and MCP-2, exhibit a similar homodimeric structure (6, 8, 17, 25). MCP-3 remains monomeric at concentrations of up to 20 mg/ml (7), whereas I-309, another beta  chemokine, was also shown to exist as a monomer at high concentrations during sedimentation (17).

A truncation mutant of MIP-1beta lacking the NH2-terminal five residues forms a dimer similar to that formed by the wild-type protein, whereas a mutant lacking the NH2-terminal eight residues exists only as a folded monomer (11). An MCP-1 mutant lacking the NH2-terminal eight amino acids exists predominantly as a monomer (7). IL-8 and melanocyte growth stimulating activity each dimerize by the formation of a central six-stranded beta  sheet, three strands of which are contributed by each subunit (26-30). The calculation of the solvation-free energies of dimerization and analysis of clusters of hydrophobic amino acids indicates that the formation and stabilization of the two main types of chemokine homodimers result from the burial of hydrophobic residues and that the distinct quaternary structures are preserved throughout the two subfamilies (12, 21). Such a scenario would explain the lack of receptor cross-binding and cross-reactivity, which is apparent between the alpha  and beta  chemokine subfamilies.

Most three-dimensional structural determinations have been performed at high concentrations (>1 mM) of chemokines with thermodynamics further strongly favoring dimerization. In contrast, physical data indicate that chemokines are fully dissociated into monomers at their normal physiological (nanomolar) concentrations. Dimer dissociation constants have been determined as 35 µM for RANTES, 33 µM for MCP-1, 58 µM for MCP-2, 40 nM for MIP-1beta , 18 µM for IL-8, 73 µM for melanocyte growth stimulating activity/growth-related oncogene-alpha , and 100 µM for neutrophil-activating protein-2. MIP-1alpha also tends to aggregate, but this process is reversible, and MIP-1alpha exists as a monomer under physiological conditions (6, 7, 9, 17-20).

Comparison of our present data with those of previous studies of chemokine homodimers indicates that substantial differences exist between MIP-1alpha /beta heterodimers and chemokine homodimers in terms of physical properties: (i) the formation of homodimers requires high concentrations of chemokines, whereas the MIP-1alpha /beta heterodimer forms at physiological concentrations of monomers; (ii) the dissociation of homodimers occurs under physiological conditions, whereas dissociation of MIP-1alpha /beta heterodimers is apparent only at low pH; (iii) homodimers have been detected only in solution under in vitro conditions, whereas MIP-1alpha /beta heterodimers are secreted from primary monocytes and PBLs; and (iv) the formation of the MIP-1alpha /beta heterodimer appears to be mediated by electrostatic interactions, whereas the formation of chemokine homodimers is thought to be mediated by hydrophobic interactions.

The existence of native chemokine homodimers remains to be demonstrated with the data having been obtained that are consistent or inconsistent with homodimers being the functionally active form of these proteins (12). Mutagenesis and cross-linking studies indicate that the active form of MCP-1 is a dimer (31). However, other studies have shown that IL-8 and MIP-1beta derivatives that do not dimerize are fully active (32, 33).

With regard to the functional role of MIP-1alpha /beta heterodimerization, it is possible that the formation of stable heterodimers protects these chemokines from enzymatic digestion and, thus, increases or stabilizes their activity. Preliminary data indicate that MIP-1alpha /beta heterodimer-containing mixtures have potent activity in inducing down-regulation of the CCR5 receptor.2 The formation of the MIP-1alpha /beta heterodimer under natural conditions may induce (possibly heterologous) receptor dimerization that may have an impact on intracellular signaling events, which contribute to CCR5 and possibly other chemokine receptor functions. Production of pure homogeneous preparations of heterodimers is required for further characterization of heterodimer activities and receptor binding properties.


    ACKNOWLEDGEMENTS

We thank K. Fields and V. Calvert for assistance with the isolation of human PBLs and monocytes and R. Boykins for help with mass spectrometry.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence may be addressed: Div. of Therapeutic Proteins, Ctr. for Biologics Evaluation and Research, FDA, NIH, Bldg. 29B, Rm. 4E12, HFM 535, 8800 Rockville Pike, Bethesda, MD 20892. Tel.: 301-827-0793; Fax: 301-480-3256; E-mail: guan@cber.fda.gov or norcross@cber.fda.gov.

Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M006327200

2 E. Guan, J. Wang, M. A. Norcross, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus-type 1; IL, interleukin; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed; MCP, macrophage chemoattractant protein; LPS, lipopolysaccharide; PBL, peripheral blood lymphocyte; MDC, macrophage-derived chemokine; IP-10, gamma interferon inducible protein-10; anti-, antibodies to; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PHA, phytohemagglutinin A.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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