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Originally published online as doi:10.1189/jlb.0305161 on May 27, 2005

Published online before print May 27, 2005
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(Journal of Leukocyte Biology. 2005;78:442-452.)
© 2005 by Society for Leukocyte Biology

Multiple pathways of amino terminal processing produce two truncated variants of RANTES/CCL5

Jean K. Lim*,{dagger}, Jennifer M. Burns{ddagger}, Wuyuan Lu* and Anthony L. DeVico*,1

* Institute of Human Virology, University of Maryland Biotechnology Institute, and
{dagger} Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore; and
{ddagger} Chemocentryx, Inc., Mt. View, California

1 Correspondence: Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, 725 W. Lombard Street, Baltimore, MD 21201. E-mail: devico{at}umbi.umd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CC chemokine regulated on activation, normal T cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) is expressed by macrophages, endothelial cells, keratinocytes, and T cells during a wide variety of immune responses. Post-translational proteolysis is expected to play an important role in regulating such broad-based expression; however, the rates and modes of RANTES processing by primary cell systems remain poorly understood. Here, we show that peripheral blood mononuclear cells (PBMC) secrete RANTES as an intact molecule that is subject to three post-translational processing pathways. One occurs in the presence of serum or plasma and rapidly generates a RANTES variant lacking two N-terminal residues (3–68 RANTES). Such processing is mainly attributable to soluble CD26. A second pathway, which is evident in the absence of serum or plasma, generates 3–68 RANTES in concert with the expression of cell-surface CD26. The third pathway is unique and generates a novel variant lacking three N-terminal residues (4–68 RANTES). This variant binds CC chemokine receptor 5, exhibits reduced chemotactic and human immunodeficiency virus (HIV)-suppressive activity compared with 1–68 and 3–68 RANTES, and is generated by an unidentified enzyme associated with monocytes and neutrophils. Overall, these results indicate that the production of RANTES by primary cells is regulated by multiple processing pathways which produce two variants with different functional properties. Such findings have important implications for understanding the immunological and HIV-suppressive activities of native RANTES.

Key Words: chemokines • CD26 • protease • PBMC


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines constitute a family of chemoattractant cytokines that stimulate and regulate the migration of lymphocytes, granulocytes, and monocytes during inflammatory immune responses. These molecules deliver signals via coordinated interactions with seven transmembrane-spanning G protein-coupled cell-surface receptors [1 ] and cell-surface proteoglycans [2 ]. Chemokine–receptor interactions are often redundant, as ligands may bind to multiple receptors, and certain receptors respond to more than one chemokine [3 ]. Two chemokine receptors, CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4), are also used by human immunodeficiency virus type 1 (HIV-1) as primary coreceptors for viral entry [4 , 5 ].

RANTES/CCL5 is a CC chemokine that binds to several chemokine receptors including CCR1, CCR3, CCR5, and Duffy antigen receptor for chemokines [6 , 7 ]. It is expressed by macrophages, endothelial cells, keratinocytes, and T cells and plays a key role in inflammation, cell recruitment, and T cell activation. It is also an antiviral agent that potently suppresses the entry of CCR5-tropic HIV-1 strains by occupying and down-regulating CCR5 [8 9 10 ].

It is reasonable to expect that such broad expression relies on post-translational proteolytic processing as a means for modulating immunological activity and receptor binding. Numerous studies have shown that chemokines are subject to proteolytic activity, which removes a small number of residues from the N- and/or C-terminus of the protein and thereby enhances or diminishes biological activity, expands or restrict receptor specificity, or converts a receptor agonist into an antagonist [11 12 13 14 15 16 17 18 19 20 21 22 23 ]. Furthermore, studies with stromal-derived factor 1 (SDF-1)/CXC chemokine ligand 12 (CXCL12) have shown that chemokines can be subject to multiple and overlapping processing pathways that occur simultaneously [23 ].

Previous studies have shown that a significant fraction of RANTES isolated from cell cultures is missing two N-terminal residues [24 , 25 ]. Biochemical studies indicate that this truncated form, designated 3–68 RANTES, is generated by the serine protease CD26/dipeptidyl peptidases IV [26 ]. Compared with unprocessed RANTES, the 3–68 variant is a selective ligand for CCR5 [27 , 28 ], a more potent suppressor of R5 HIV-1 [29 ], and a less-efficient inducer of monocyte or eosinophil migration [29 , 30 ]. However, it is not known whether or to what extent RANTES is subjected to proteolytic processing as it is secreted by activated cells. Further, the possibility of alternative processing pathways remains unexplored. Such information is critical for understanding the nature of RANTES function during immune responses.

To answer these questions, we used complementary immunochemical techniques to characterize the relationships between RANTES production and processing by cultures of peripheral blood mononuclear cells (PBMC), which are frequently used as sources of native chemokines [25 , 28 , 29 ] and soluble HIV suppressor activities [31 , 32 ]. These studies revealed three modes of post-translational processing that act concomitant with protein production to convert the majority of RANTES into two N-terminal variants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell preparation and culture
PBMC were obtained from healthy donors as described previously [33 ], activated with 2 µg/ml phytohemagglutinin (PHA; Murex Biotech Ltd., UK) and 10 ng/ml recombinant human interleukin (IL)-2 (R&D Systems, Minneapolis, MN) for 72 h, and cultured (1x106 cells/ml) in RPMI 1640 (Gibco-BRL, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS; Sigma Chemical Co., St. Louis, MO), human AB serum (Sigma Chemical Co.), human plasma, 5% serum-free HB101 (Irvine Scientific, Santa Ana, CA), or serum-free AIM V media (Invitrogen, Carlsbad, CA). All serum components were heat-inactivated at 56°C for 30 min.

CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells, and CD14+ cell subsets were recovered from fresh PBMC using the negative-selection cell-sorting kits (Miltenyi Biotec, Auburn, CA). Purity was assessed by flow cytometry and found to be consistently >90% for all cell types. In some experiments, the unwanted cell populations were recovered from the beads for further testing according to the manufacturer’s instructions.

Neutrophils were isolated from healthy donors using dextran sedimentation as described previously [34 ]. Briefly, fresh blood was collected in sodium heparin tubes from a healthy donor and mixed 1:1 with a solution of 3% dextran in 0.9% NaCl and kept at 22°C for 30 min. The neutrophil-rich upper layer was collected and centrifuged on Histopaque solution at 300 g for 30 min at 22°C. Residual erythrocytes in the neutrophil pellet were removed by hypotonic lysis. Neutrophil purity and viability were consistently >95%, as determined by microscopy and trypan blue dye exclusion, respectively.

Synthesis of 3–68 and 4–68 RANTES
Synthetic 3–68 RANTES and 4–68 RANTES were prepared by solid-phase peptide synthesis in combination with native chemical ligation, according to methods published previously [35 , 36 ]. Purification of the ligation products was essentially as described previously [37 ]. The purified protein was further characterized by analytical high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry to ascertain purity and the formation of disulfide bonds.

Production of rabbit anti-RANTES mono-specific antibody
Rabbit serum was raised against a 12-amino acid repeat peptide (SPYSSPYSSPYS) corresponding to the four N-terminal residues in the 1–68 RANTES sequence. The keyhole limpet hemocyanin-conjugated peptide (300 µg) was given intramuscularly to New Zealand white rabbits in Freund’s complete adjuvant. After 4 weeks, the rabbits were boosted five times at 2-week intervals with 300 µg peptide in Freund’s incomplete adjuvant. Sera were collected and tested for reactivity by direct enzyme-linked immunosorbent assay (ELISA) using published methods [38 ] with the immunizing peptide or 1–68 RANTES captured on the solid phase. Immunoglobulin (Ig) from immunoreactive serum was affinity-purified using activated sepharose 4B (Amersham Pharmacia, Little Chalfont, UK) conjugated to the immunizing peptide using published methods [39 ]. The eluted, mono-specific antibody was designated N-RANTES Ig.

RANTES ELISA
An assay that specifically detects the unprocessed 1–68 RANTES (designated N-RANTES ELISA) was constructed by adsorbing 200 ng anti-RANTES monoclonal antibody MAB678 (R&D Systems) to 96-well microtiter plate wells (Dynex Technologies, Chantilly, VA) for 16 h at 4°C. All subsequent steps were conducted at 22°C. Nonspecific sites were blocked for 1 h with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 5% dry milk (blocking buffer). After washing with PBS containing 0.1% Tween 20 (wash buffer), test samples or chemokine standard (100 µl/well) were diluted appropriately and incubated for 1 h. After washing, N-RANTES Ig (1 µg/ml in blocking buffer) was added for 1 h. Peroxidase-conjugated goat anti-rabbit Ig (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was then added (125 ng/ml in blocking buffer) for 30 min. The amount of bound Ig was detected by reaction to tetramethylbenzidine (BioFX Laboratories, Owings Mills, MD), and the resulting absorbance was measured at 450 nm. To determine RANTES concentrations in culture supernatants, the absorbance values were compared with a standard curve generated with serial concentrations of recombinant RANTES (starting at 500 pM) in the appropriate buffer or culture media.

A commercial RANTES ELISA system (designated here as the Standard ELISA) was used with MAB678 as a capture antibody and biotinylated anti-RANTES antibody BAF278 (R&D Systems) for detection. The assays were carried out according to the manufacturer’s instructions. The percentage of unprocessed RANTES in a sample was determined by calculating the mean N-RANTES ELISA concentrations/mean Standard RANTES concentrations x 100.

Mass Spectrometry (MS)
Surface-enhanced laser desorption and ionization (SELDI) MS (Ciphergen Biosystems, UK) was performed with PS10 protein chips. MAB678 (R&D Systems) or mouse IgG1 isotype control (Sigma Chemical Co.) was spotted on the chips (2 µg/spot in PBS) and incubated overnight at 4°C in a humid chamber. Active sites were blocked by the addition of 1 M ethanolamine in PBS, pH 8, for 1 h at 22°C. Culture supernatants were incubated with each spot overnight at 4°C, using a fitted bioprocessor unit (Ciphergen Biosystems). After washing with PBS containing 0.1% Triton-X 100, the samples were crystallized by the addition of 1 µl/spot saturated cyano-4-hydroxycinnamic acid (Sigma Chemical Co.) in 50% acetonitrile in HPLC grade water. Bovine ubiquitin (Sigma Chemical Co.) with a molecular weight of 8564.0 Da was added as an internal standard and analyzed using the Ciphergen Series PBS II reader and software.

Depletion of serum CD26
Heat-inactivated (56°C, 30 min) human AB serum (Sigma Chemical Co.) was treated with phycoerythrin (PE)-conjugated anti-CD26 antibody (Becton Dickinson, San Jose, CA) or PE-conjugated IgG1 isotype control and anti-PE immunomagnetic beads (Miltenyi Biotec) for 18 h at 4°C. The treated serum was then passed over a type LS+ magnetic cell sorter separation column (Miltenyi Biotec) to capture the PE-labeled antibody-antigen complexes. After washing the beads, the magnetically bound material was eluted according to the manufacturer’s instructions. Depletion of CD26 from serum was assessed by human soluble CD26 (sCD26) ELISA (Bender MedSystems, Burlingame, CA).

Detection of cell-surface CD26
Cells were incubated in PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide with PE-conjugated anti-CD26 antibody or isotype control (Becton Dickinson), according to the manufacturer’s protocol. After 1 h at 4°C, cells were washed and resuspended in PBS with 2% paraformaldahyde. Cell-surface expression of CD26 was analyzed using flow cytometry. Mean fluorescence intensity (MFI) of CD26-positive cells over isotype-control staining was determined by Cell Quest software analysis.

Ligand binding assays
Synthetic test chemokines were incubated with CCR5-NSO (mouse myeloma cell line) cells followed by the addition of 125I-macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}; 0.05 nM) for 3 h at 4°C in 25 mM HEPES, pH 7.1, containing 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and 0.2% BSA. Following incubation, reactions were aspirated onto polyethylenimine-treated GF/B glass filters (Packard, Downers Grove, IL) using a cell harvester (Packard Downers Grove, IL) and washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.1). Scintillant (MicroScint 10, Packard) was added to the wells, and the filters were counted in a Packard Topcount scintillation counter. Background signals were determined using radiolabeled chemokine in the absence of cells.

Chemotaxis assays
Stimulated PBMC were harvested and resuspended in RPMI with 0.5% BSA. Chemotaxis was measured in 96-well ChemoTx-disposable chambers with 3 µm pores (Neuro Probe, Gaithersburg, MD). Chemokines were placed in the lower chamber and covered with a filter. Cells were then added to the upper chamber, and the apparatus was placed in a 37°C incubator for 4 h. Cells that had migrated to the lower chamber were counted using a hemocytometer or lysed in the presence of CyQuant (Molecular Probes, Eugene, OR) dye, and fluorescence emission was measured at 535 nm.

HIV suppression assays
Stimulated PBMC from a healthy donor were infected with HIV-1BaL as described previously [40 ] and treated with serial concentrations of test chemokine. Fresh chemokine was added at day 3, and the assays were terminated at day 7. Infection was measured by p24 ELISA (New England Nuclear, Boston, MA) in the culture supernatants. Percent inhibition of infection was calculated relative to control assays performed in the absence of chemokine. Inhibition curves were generated and used to calculate a 50% inhibitory concentration (IC50), which denotes the highest concentration needed to cause greater than or equal to 50% inhibition of infection


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Assays for processed and unprocessed RANTES
Quantitative analysis of RANTES production and processing by primary cells requires detection assays capable of distinguishing between the unprocessed and processed RANTES variants. Accordingly, we raised mono-specific rabbit antibodies against the four N-terminal residues of 1–68 RANTES, which were expected to react only with the unprocessed chemokine. As shown in Figure 1A , an ELISA (designated N-RANTES ELISA), constructed with the rabbit antibody, failed to detect the 3–68 variant of RANTES but detected the unprocessed 1–68 molecule with a level of sensitivity comparable with a commercial ELISA (designated Standard ELISA), which detected 1–68 and 3–68 forms with equal sensitivity (Fig. 1B) . Thus, the two ELISAs permitted us to quantify the amount of RANTES with an unprocessed N terminus versus the total RANTES pool in a test sample.



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  		Figure 1. N-RANTES ELISA specifically detects the unprocessed (1–68) form of RANTES. Serial concentrations of 1–68 ({blacksquare}) or 3–68 RANTES ({blacktriangleup}) were added to duplicate assay wells with antibody MAB678 adsorbed to the solid phase. The captured chemokine was detected with rabbit N-RANTES Ig (A) or a commercially available anti-RANTES antibody BAF278 (B), which reacts with 1–68 and 3–68 RANTES. The bound antibodies were detected as described in Materials and Methods. The mean replicate values obtained from one of four experiments are shown; bars indicate standard deviation.

 
The limitation of the ELISA approach was its inability to reveal the positions and identities of residues removed by processing. To obtain this information, a SELDI MS method was developed that used an anti-RANTES antibody-coated chip to selectively capture RANTES from solution. This technique allowed us to evaluate the nature of proteolytic cleavage based on the apparent masses of proteins desorbed from the chip.

RANTES production and processing by stimulated PBMC
The two RANTES immunoassay formats were used to characterize the production and processing of RANTES by PBMC cultured in medium supplemented with FBS. PBMC were activated with PHA and placed in a series of parallel culture wells. Every 24 h, a separate well was harvested for analyses. This approach avoided perturbations of the cultures that might have been introduced by sequential sample collection. A representative experiment with cells from a single donor is shown in Figure 2A . Roughly the same amount of RANTES was detected by the N-RANTES and Standard ELISAs in samples collected at 6 h post-activation. These results indicated that most of the chemokine molecules possessed unprocessed N-termini. However, discordant RANTES values were obtained for samples collected at later times. Although the total RANTES concentration (measured in the Standard ELISA) steadily increased over time, the amount of unprocessed chemokine (measured in the N-RANTES ELISA) stayed relatively constant. Consequently, by 72 h after activation, only 22.6% of the total RANTES pool remained unprocessed. The same results were obtained in additional experiments with cells from 21 donors (Fig. 2B) , although the total RANTES production varied considerably (ranging from 0.2 and 2 nM; data not shown). In each case, only a minority of the total RANTES pool (28.8±6.8%) in the 72-h samples was unprocessed.



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  		Figure 2. RANTES is processed as it accumulates in PBMC cultures. (A) PBMC from a single donor were activated with PHA and cultured for 72 h in medium containing FBS. Culture supernatants were collected at the specified times and analyzed in Standard and N-RANTES ELISAs to quantify total ({blacksquare}) and 1–68 ({blacktriangleup}) RANTES, respectively. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed RANTES was determined from these values for each sample and plotted versus time. (B) The same experiment as in A was repeated with PBMC from 21 healthy donors. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed RANTES was determined from these values for each sample and plotted versus time. Bars indicate the standard deviation between percentage values amongst all 21 donors. (C) SELDI analysis of supernatants obtained from a representative experiment with cells from a single donor. The observed mass values [Daltons (Da)] and RANTES variant assignments are shown. None of the indicated species were recovered from chips spotted with isotype-control IgG1 (data not shown).

 
SELDI analyses showed that the samples contained two RANTES variants (Fig. 2C) . One exhibited a mass of 7847 ± 1 Da, which corresponded with the expected mass of 1–68 RANTES (7847.0 Da). The second exhibited a mass of 7663 ± 1 Da, which matched the predicted mass of a 3–68 RANTES molecule missing an N-terminal Ser1-Pro2 dipeptide (7662.8 Da). Notably, the signal for 1–68 RANTES diminished over time, whereas the 3–68 signal became more pronounced. In some experiments, the observed masses of the desorbed proteins were 16 Da higher then the expected masses of 1–68 and 3–68 RANTES (observed masses were 7863±2 Da and 7678 Da, respectively). We attribute this difference to the occasional oxidization of the methionine residue at position 67 in the RANTES sequence, as was reported previously [41 ].

The presence of 3–68 RANTES indicated that the secreted chemokine had been cleaved by CD26. In agreement, a heat-stable [42 , 43 ] and the soluable form of this enzyme (sCD26) found in serum and plasma [44 ] was shown to process recombinant SDF-1 [23 ]. To test this possibility, recombinant 1–68 RANTES was added to cell-free reagent media supplemented with heat-inactivated FBS, human AB serum, or human plasma. Control assays were performed in which the chemokine was added to serum-free RPMI supplemented with HB101 or AIM V medium. Aliquots of treated material were recovered at different incubation times and evaluated by the Standard and N-RANTES ELISAs. As shown in Figure 3A , the percentage of unprocessed RANTES steadily decreased over time, as calculated from the ELISA values. SELDI analysis confirmed that the chemokine was converted to the 3–68 variant in the presence of bovine or human serum and human plasma (data not shown). In comparison, 1–68 RANTES remained intact when added to serum-free RPMI containing HB101 supplement. Only a minor decrease in the percentage of 1–68 RANTES was observed after a 72-h incubation in AIM V medium.



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  		Figure 3. RANTES is processed in cell-free culture medium by enzymes in serum and plasma supplements. (A) Recombinant 1–68 RANTES was added to RPMI supplemented with 10% FBS ({blacksquare}), human AB serum ({blacktriangleup}), human plasma ({blacktriangledown}), and HB101 ({square}) or to serum-free AIM V medium ({circ}). In all cases, the final chemokine concentration was 2 nM. The mixtures were incubated at 37°C for the specified times and then analyzed in Standard and N-RANTES ELISAs. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed 1–68 RANTES was determined from these values for each sample and plotted versus time. Error bars indicate standard deviation between two separate experiments. (B) Human AB serum was depleted of CD26 using PE-conjugated anti-CD26 antibody/anti-PE beads. Recombinant 1–68 RANTES was then added to the depleted serum or the material eluted from the beads (Eluate) at a final concentration of 2 nM. Mock depletion experiments were carried out with a PE-conjugated IgG1 isotype-control Ig. After 8 h at 37°C, the reaction mixtures were analyzed in Standard and N-RANTES ELISAs to quantify total (solid bars) and unprocessed 1–68 RANTES (shaded bars), respectively. The mean values from duplicate assays are shown. The mean duplicate values obtained from one representative experiment of two are shown; bars indicate standard deviation.

 
To confirm that the serum-associated processing was mediated by sCD26, human AB serum was selectively depleted of the enzyme by treatment with PE-labeled anti-human CD26 antibody followed by the addition of magnetic particles coated with anti-PE antibodies. Mock depletion experiments were carried out with a PE-labeled IgG1 isotype control antibody. Analyses by CD26 ELISA showed that >95% of the sCD26 was removed from the serum by the anti-CD26 antibody/anti-PE beads, whereas mock depletion did not alter the serum sCD26 concentration (data not shown). The various fractions were then treated with recombinant 1–68 RANTES (2 nM final concentration) for 8 h and analyzed by ELISA. As shown in Figure 3B , N-terminal processing of RANTES was evident in the mock-depleted serum. The concentration of RANTES detected by the N-RANTES ELISA was much lower than the total amount detected in the Standard ELISA, which matched the starting concentration of chemokine. In contrast, the N-RANTES and Standard ELISAs detected the same amounts of RANTES in the sCD26-depleted serum. This indicated that the anti-CD26 antibody/anti-PE beads had removed the processing activity. In accordance, N-terminal processing occurred in the fraction eluted from the anti-CD26 beads, which was expected to contain sCD26. In this case, the amount of chemokine measured by the N-RANTES ELISA was roughly 50% of the amount detected by the Standard ELISA. No such proteolytic activity was evident in the mock bead eluate, as the RANTES concentrations detected in the two ELISAs were the same.

RANTES processing under serum-free conditions
To examine whether cell-associated enzymes are capable of processing RANTES, PBMC from 10 healthy donors were stimulated with PHA and cultured separately in serum-free medium formulations (AIM V or RPMI supplemented with HB101). Samples of culture medium were collected at different times and analyzed by ELISA. In all cultures, the amount of RANTES detected by the N-RANTES ELISA was roughly 50% of the total measured in the Standard ELISA (Fig. 4A ) at 24 h post-activation. At 72 h post-activation, the proportion of unprocessed RANTES ranged between 30% and 60% of the total RANTES in the sample, depending on the donor. These data indicated that the chemokine was subject to N-terminal modification under serum-free conditions.



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  		Figure 4. RANTES is processed by activated lymphocytes under serum-free conditions. (A) PBMC from 10 healthy donors were activated with PHA and cultured in serum-free conditions. Culture supernatants were collected at the specified times and analyzed in the Standard and N-RANTES ELISAs. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed RANTES was determined from these values for each sample and plotted versus time. The mean percentage values determined for all donors are shown; bars indicate standard deviation. (B) Activated PBMC from a single donor were cultured in HB101-supplemented RPMI media for 6 days. The medium was completely replaced after 48 h, after which the cells were maintained in IL-2-supplemented media. Samples were collected at the specified times and analyzed in Standard and N-RANTES ELISAs. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed RANTES was determined from these values for each sample and plotted versus time (solid line). Cells were stained with anti-CD26 antibody prior to stimulation with PHA (time 0) or at the specified times after stimulation. The MFI values measured by flow cytometry are shown (dashed line). (C) SELDI MS was performed on the same supernatant samples analyzed by ELISA. The observed mass values (Daltons) and variant assignments are shown. Experiments carried out with cells from three different donors produced similar results. Samples were tested in parallel on IgG1-coated chips and show no significant peaks above background (data not shown).

 
To further explore this possibility, PBMC were activated with PHA and placed in a series of parallel culture wells containing RPMI supplemented with serum-free HB101. After 48 h, the cells were washed and placed in fresh medium for an additional 4 days. Every 24 h, an entire well was harvested to obtain samples for analyses. Representative results from a single-donor culture are shown in Figure 4 . As shown in Figure 4B (solid line), RANTES was secreted into the culture medium within the first 48 h post-activation. However, the chemokine concentration measured in the N-RANTES ELISA was roughly 50% of what was determined in the Standard ELISA.

RANTES secretion persisted after the medium change (Fig. 4B) . At 72 h post-activation (24 h after the medium change), the ELISAs indicated that roughly 90% the chemokine was unprocessed. However, the proportion of unprocessed RANTES in the secreted pool steadily decreased over time. At 144 h post-activation, less than 25% of the total RANTES was unprocessed.

SELDI analyses (Fig. 4C) of the samples taken after the medium change revealed the presence of one truncated form with the expected mass (7663±1 Da) of 3–68 RANTES. The signal for this variant became more pronounced over time, in agreement with the temporal decrease in chemokine concentration indicated by the N-RANTES ELISA. Notably, flow cytometric analyses of the cells after the medium change (Fig. 4B , dashed line) revealed an increasing frequency of CD26+ cells over time, in accordance with the progressive intensification of the SELDI signal for 3–68 RANTES.

The same analyses of supernatants collected before the medium change revealed the presence of unprocessed RANTES, traces of the 3–68 variant, and a third species with an apparent mass of 7515 ± 1 Da. This value matched the theoretical mass of RANTES missing the N-terminal Ser1-Pro2-Tyr3 tripeptide (7499.6 Da). Accordingly, the third species was designated 4–68 RANTES. Notably, this putative variant was not detected in samples taken after the media change.

Monocytes and neutrophils generate 4–68 RANTES
To identify the source of the activity that generated 4–68 RANTES, fresh PBMC were sorted into populations enriched for CD4+ T cells, CD8+ T cells, B cells, NK cells, and CD14+ cells [monocytes, macrophages, and/or monocyte-derived dendritic cells (DCs)]. The various subsets were then cultured in RPMI supplemented with HB101 in the absence of PHA or IL-2. Recombinant 1–68 RANTES (5 nM final concentration) was added to the cultures and then evaluated after 24 h by SELDI analysis for evidence of N-terminal processing.

As shown in Figure 5A , 1–68 RANTES was partially converted into the 4–68 variant after exposure to cultures of unfractionated PBMC. No significant processing was evident in cultures of CD4+ T cells, CD8+ T cells, B cells, or NK cells. However, the 1–68 RANTES was converted extensively into 4–68 RANTES in cultures of CD14+-enriched cells. Conversely, CD14+-depleted PBMC, which contained <2% CD14+ cells, generated only trace amounts of 4–68 RANTES (Fig. 5B) .



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  		Figure 5. RANTES is processed into a 4–68 variant by an enzyme associated with CD14+ cells. (A) Recombinant 1–68 RANTES was incubated with unfractionated PBMC or enriched populations of CD4+ T cells, CD8+ T cells, B cells, NK cells, or CD14+ cells for 24 h at 37°C in HB101-supplemented media in the absence of mitogen or growth factors. Supernatants were then analyzed by SELDI for RANTES processing. The observed mass values (in Daltons) and variant assignments are shown. Data are from a representative experiment with cells from a single donor. The experiment was repeated with cells from three different donors. (B) SELDI analysis was performed to compare CD14+ cell-enriched verses CD14+-depleted PBMC. The observed mass values (in Daltons) and variant assignments are shown. Data are from a representative experiment with cells from a single donor. The experiment was repeated with cells from three different donors.

 
Neutrophils were also tested for the capacity to process RANTES, as they were shown to be a source of enzymes capable of processing chemokines [21 , 45 ]. Isolated neutrophils were cultured at different densities in RPMI containing HB101 or 10% human AB serum. Recombinant 1–68 RANTES (5 nM final concentration) was then added to the cultures and analyzed by ELISA and SELDI after 18 h. Control assays were carried out in cell-free medium. A representative experiment with cells from a single donor is shown in Figure 6 . As shown in Figure 6A , less than 40% of the total RANTES was unprocessed in the absence of cells when the medium contained serum. SELDI confirmed that the RANTES was processed exclusively into the 3–68 variant (Fig. 6B) . This was consistent with the presence of sCD26 in the serum (Fig. 3) . No processing was evident in cell-free HB101-supplemented media (Fig. 6A and 6B) .



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  		Figure 6. RANTES is processed into a 4–68 variant by an enzyme associated with neutrophils. (A) 1–68 RANTES was incubated in medium supplemented with serum-free HB101 ({blacksquare}) or with 10% human AB serum ({blacktriangleup}) in the absence or presence of different concentrations of neutrophils. After 18 h, culture media were harvested and analyzed in the Standard and N-RANTES ELISAs. Assays were performed in duplicate and used to calculate mean ELISA values. The percentage of unprocessed RANTES was determined as described in Materials and Methods and is shown. (B) Culture samples were tested in parallel by SELDI. The observed mass values (in Daltons) and variant assignments are shown. Data are from a representative experiment with cells from a single donor. The experiment was repeated with cells from a different donor and yielded the same results.

 
In the presence of neutrophils, there was an overall decrease in the amount of unprocessed RANTES compared with cell-free media (Fig. 6A) . SELDI analyses of the HB101-supplemented cultures revealed that RANTES was processed exclusively into the 4–68 variant. Moreover, the intensity of the 4–68 RANTES signal was proportional to the neutrophil concentration (Fig. 6B , upper panels). In cultures supplemented with 10% human serum, 4–68 RANTES was apparent along with the 3–68 variant (Fig. 6B , lower panels) generated by serum enzyme. Again, the relative intensity of the 4–68 RANTES signal was proportional to cell concentration.

Properties and biological functions of 4–68 RANTES
The ability of the 4–68 variant to bind CCR5 was tested in a competition-binding assay using 125I-MIP-1{alpha} as ligand and CCR5-expressing NSO cells as targets. MIP-1{alpha}, MIP-1ß, and the 1–68 and 3–68 RANTES variants were tested in parallel. Competition-binding curves were plotted for each chemokine and used to calculate 50% effective concentrations (EC50). As shown in Figure 7A , 4–68 RANTES exhibited an EC50 of 283.1 ± 84.6 pM, which was seven to 10 times higher than the values measured for 1–68 and 3–68 RANTES (29.1±4.0 pM and 41.2±19.2 pM, respectively). However, the EC50 for 4–68 RANTES was comparable with those calculated for MIP-1{alpha} (311.4±214.9 pM) and MIP-1ß (410.1±90.5 pM).



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  		Figure 7. Functional analysis of the 4–68 RANTES variant. (A) 1–68 ({blacktriangledown}), 3–68 ({blacktriangleup}), or 4–68 RANTES ({blacksquare}) were compared for receptor interactions using a competition-binding assay. NSO cells expressing CCR5 were incubated at 4°C with serial concentrations of RANTES and 125I-MIP-1{alpha} at a limiting concentration (0.05 nM). The cells were washed and placed in a scintillation counter to determine amounts of bound 125I-MIP-1{alpha}. The data points shown represent mean counts per minute (CPM) obtained in quadruplicate assays; bars indicate standard deviation. (B) PHA-activated PBMC were infected with HIV-1BaL in the presence of serial concentrations of 1–68 RANTES ({blacktriangledown}), 3–68 RANTES ({blacktriangleup}), or 4–68 RANTES ({blacksquare}). Virus replication was determined on day 7 by HIV-1 p24 ELISA. All assays were performed in quadruplicate; bars indicate standard deviation. The experiment was repeated three times and produced similar results. (C) Serial concentrations of 1–68, 3–68, and 4–68 RANTES and MIP-1ß were tested at the indicated concentrations in chemotaxis assays using cultured lymphocytes suspended in serum-free Hanks’ balanced salt solution medium. Medium without chemokine was tested as a control. The migrated cells were counted using a hemocytometer. Chemotactic index was determined by dividing the number of cells migrated in the presence of chemokine by the number of cells migrated in the absence of chemokine. Results were confirmed using a Cyquant Dye quantification method (data not shown). One representative experiment of five is shown; standard deviations are shown with bars.

 
In accordance, 4–68 RANTES inhibited R5 HIV-1 infection of PBMC (Fig. 7B) . However, the IC50 of 4–68 RANTES (9.921±0.933 nM) was roughly 10 times higher than values calculated for the 1–68 and 3–68 variants (0.985±0.282 nM and 0.888±0.175 nM, respectively). A CXCR4-specific ligand SDF-1/CXCL12 showed no antiviral effect against the R5 virus (data not shown).

Similarly, the 4–68 RANTES was a less-potent stimulator of lymphocyte chemotaxis than the other variants (Fig. 7C) . The concentrations needed for maximum effect were 100 nM for 4–68 RANTES, 10 nM for 1–68 RANTES, and 1 nM for 3–68 RANTES. However, the 4–68 RANTES activity was comparable with that of MIP-1ß.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Relationships among rates of protein expression, proteolytic processing kinetics, and alternative cleavage pathways are likely to provide a key means for regulating the functions of many chemokines during an immune response. The goal of this study was to define relationships between the production and processing of RANTES by activated PBMC in vitro. Our experiments revealed three overlapping modes of processing that occur while the chemokine is being released by the cells.

The first was apparent in PHA-stimulated PBMC cultures containing serum or plasma. Under these conditions, RANTES was specifically processed into the 3–68-truncated variant as it was secreted. The rate of processing was consistent among different donors (Fig. 2B) , although the total amount of chemokine varied considerably between donors. We conclude that this processing was mediated by serum- or plasma-derived sCD26, as recombinant 1–68 RANTES was converted to the 3–68 variant in cell-free medium containing serum but not in serum-free media (Fig. 3A) . Moreover, treatment of serum-containing medium with anti-CD26 antibody-coated beads abrogated RANTES processing. Conversely, the material recovered from the beads processed RANTES into the 3–68 variant (Fig. 3B) . Thus, like SDF-1 [23 ], RANTES appears to be subject to efficient processing by serum sCD26. Our data suggest that it is quite extensive; in the in vitro system, peak production levels primarily yielded 3–68 RANTES. These findings suggest that 1–68 RANTES may be a relatively minor component of the total RANTES pool secreted by circulating cells.

The second processing mode also yielded 3–68 RANTES but under serum-free conditions. It is highly likely that cell-surface CD26 was responsible for such processing, as surface expression of the enzyme exhibited an inverse relationship with the amount of unprocessed RANTES detected by ELISA (Fig. 4B) . Further, there was a direct relationship between increases in CD26 expression and the 3–68 RANTES SELDI signal over time. However, a small percentage of unstimulated PBMC was also positive for cell-surface CD26 but did not generate 3–68 RANTES (Fig. 4B , dashed line at time 0). Similarly, unstimulated CD4+ T cells failed to generate 3–68 RANTES, although 10–60% of the population stained positive for CD26, depending on the donor (data not shown). It is possible that a threshold level of surface CD26 expression is needed to produce enough 3–68 RANTES to be detected in our assays. Alternatively, a specific molecular form of cell-surface CD26 may be required. The existence of CD26 variants was indicated by findings that antigenic changes occur in the enzyme after T cell activation [46 ].

The third processing mode was entirely unique and produced a novel variant, which we designated 4–68 RANTES in accordance with its apparent mass. A comparison of cell subsets revealed that the CD14+ cell subset (Fig. 5) and neutrophils (Fig. 6) generate this variant. The production of 4–68 RANTES did not depend on cell activation (Fig. 5) . However, the 4–68 variant was not detected in PBMC cultures 72 h post-activation (Fig. 4C) . This probably reflected the temporal loss of the CD14+ cell subset that occurs in PHA-activated PBMC cultures [47 ]. In accordance, flow cytometric analysis failed to detect CD14+ cells in the PBMC cultures by 48 h post-activation (data not shown). We expect that monocytes comprise a majority of the CD14+ cells we tested, although we cannot eliminate the possibility that macrophages and DCs were also components of the population.

It is important that neutrophils generated the 4–68 variant in serum-free and serum-supplemented conditions (Fig. 6) , although in the latter case, higher cell concentrations were needed. In contrast, 4–68 RANTES was not apparent in PBMC cultures (Fig. 2C) or CD14+-enriched cultures (data not shown) when serum was present. One potential explanation for these differences is that 4–68 RANTES is produced by an enzyme that competes with sCD26 for 1–68 RANTES substrate. Thus, production of 3–68 RANTES might be favored under conditions where sCD26 is more abundant than the alternate cell-associated enzyme. Conversely, production of the 4–68 variant might occur when neutrophil/CD14+ cell densities are high (Fig. 6) and when expression of the alternate enzyme(s) is enhanced.

An alternative explanation is that protease inhibitors in serum or plasma target the enzyme(s) that produces 4–68 RANTES. In this regard, we cannot eliminate the possibility that RANTES is processed into the 4–68 variant through multiple enzymatic activities that are cell type-specific. Thus, a CD14+ cell-associated enzyme could be sensitive to soluble protease inhibitors that do not affect a neutrophil-associated enzyme.

In any case, our findings that 4–68 RANTES is generated in the presence of human serum strongly indicates the existence of an alternative processing pathway that is relevant to natural immune processes. Notably, certain diseases are associated with significant decreases in the amount and/or enzymatic activity of serum CD26, including systemic lupus erythematosus, rheumatoid arthritis, and HIV infection, which might ultimately favor the production of the 4–68 variant [26 ]. Further, RANTES may be inaccessible to sCD26 under certain subcellular conditions but available to the monocyte/neutrophil-associated enzyme.

The processing of RANTES into the 4–68 RANTES did not abrogate its ability to bind to CCR5. The 4–68 variant competed with radiolabeled MIP-1{alpha} for CCR5 binding with an EC50, that was appoximately seven- to tenfold higher than either 1–68 or 3–68 RANTES but roughly equivalent to MIP-1ß and MIP-1{alpha} (Fig. 7A) . However, the 4–68 variant was less potent than 1–68 or 3–68 RANTES in stimulating lymphocyte chemotaxis or inhibiting HIV infection (Fig. 7B and 7C) . These data demonstrate that CCR5 binding and activation are heavily determined by the nature of N-terminal processing and the resulting truncated variant. In accordance, we have determined that the 4–68 variant fails to stimulate calcium mobilization in THP-1 cells, which express CCR1 and CCR3 [48 ], whereas 1–68 RANTES and MIP-1{alpha} were active (data not shown) as previously shown [49 ]. Further, 4–68 RANTES did not desensitize cells to 1–68 RANTES (data not shown). This suggests that 4–68 RANTES is unable to bind CCR1 or CCR3 and/or to induce a functional response.

Taken together, our results indicate that RANTES function is modulated by at least two processing pathways during the course of an immune response. It is likely that the production of 3–68 versus 4–68 RANTES will be determined by the local concentrations of proteases and protease inhibitors, the surrounding and infiltrating cell types, and the processing kinetics of the enzymes. Overall, circumstances where the unprocessed chemokine predominates may be rare, as sCD26, cell-surface CD26, and/or the unidentified CD14+ cell/neutrophil-associated enzymes are capable of processing RANTES into truncated variants during the chemokine production phase.

These findings also identify an important caveat for in vitro analyses of native chemokines derived from primary cell sources. Based on the results presented here, RANTES molecules harvested from serum-supplemented PBMC cultures at peak accumulation levels are likely to be 3–68 variants that bind specifically to CCR5. This must be considered when comparisons of native versus recombinant or synthetic 1–68 RANTES are made. Similarly, studies that examine the antiviral or immunological properties of exogenous RANTES in long-term cultures must consider that processing may occur during the course of the experiment, thus changing the properties of the reagent over time. In this regard, a number of RANTES analogs with N-terminal modifications are being explored as antiviral agents [50 51 52 ]. It will be important to determine whether these analogs are substrates for proteases that digest native RANTES.


   ACKNOWLEDGEMENTS
 
This work was supported by Grants RO1 HL063647 and RO1 HD39108 to A. L. D. The authors thank Drs. Nicolas Ambulos and Patricia Campbell of the University of Maryland Biopolymer Core Facility for assistance with SELDI MS.

Received March 19, 2005; revised April 29, 2005; accepted May 2, 2005.


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 DISCUSSION
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I Tikhonov, C. Deetz, R Paca, S Berg, V Lukyanenko, J. Lim, and C. Pauza
Human V{gamma}2V{delta}2 T cells contain cytoplasmic RANTES
Int. Immunol., August 1, 2006; 18(8): 1243 - 1251.
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