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Departments of
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Molecular Virology and Host Defense,
Project Management,
Microbial Infectivity,
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Molecular Genetics, and
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Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426; and Departments of
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Protein Biochemistry,
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Oncology Research, and
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Immunology, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SK&F 107647 indirectly stimulates myeloid stem and progenitor cell
proliferation, elevates host defense effector cell respiratory burst
and antimicrobial activity in vivo, and enhances survival in animal
models of infectious diseases (8, 9, 10). Mechanistically,
the activities of SK&F 107647 are indirectly mediated by a soluble
factor produced by marrow stromal cells which synergizes with all
myeloid CSFs. We have identified, purified, and sequenced specific
murine and human stromal cell-derived hematopoietic synergistic factors
(HSF) induced by SK&F 107647, which mediate the in vivo effects of this
peptide. Murine HSF is an N-terminal 4-aa truncated form of KC (aa
5–72), a member of the C-X-C chemokine family of proteins (11, 12). The murine KC gene is homologous to a superfamily of
related human chemotactic heparin binding proteins, with high homology
to the GRO
, ß, 
gene family (13). Both heparin and
anti-GROß mAb completely neutralize all SK&F 107647-induced HSF
bioactivity in vitro and in vivo. Subsequently, we utilized heparin
binding and anti-GROß affinity chromatography to purify human HSF
from a marrow stromal cell line, TF274. Human HSF was identified as an
N-terminal truncated form of GROß 5–73(5–73) by amino acid sequence and
mass spectrometry. In this paper we use KC-T and GROß-T to represent
the specific 4-aa N-terminally truncated chemokines isolated from cell
supernatants after SK&F 107647 stimulation; and KC and GROß, to refer
to the full-length forms of these proteins. Recombinant KC-T and
GROß-T proteins were cloned and produced, which confirmed the
biological activities of the truncated chemokines produced in SK&F
107647-stimulated stromal cell cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine bone marrow stromal cell line, C6, was derived as previously described (14) and used as the source of SK&F 107647-induced synergistic activity. A transfected human stromal cell line, TF274, was utilized as the source of human SK&F 107647-induced synergistic activity. Briefly, the TF274 stromal cell line was derived from normal human bone marrow stimulated in vitro for 24 h with 0.003% Staph A, before transfection with HL-60 DNA and PzipTex (SV40 large T) used as an immortalizing agent, and delivered by electroporation. Clone TF274 was chosen based upon stromal cell morphology and responsiveness to SK&F 107647. Stromal cell lines were grown in either roller bottles (C6) or T-175 tissue culture flasks (TF274) and were treated with either 10 µg/ml (C6) or 100 µg/ml (TF274) SK&F 107647 under serum-free conditions. Cell-free supernatants were collected from treated cells after overnight stimulation. This process was repeated twice weekly. Cultures were washed extensively and refed with FCS-containing medium between treatment cycles.
CFU-GM assay
Murine bone marrow cells were harvested from 6- to 12-wk-old female C57BL/6 mice (Harlan Sprague-Dawley, Indianapolis, IN) and cultured as described (8). Cell supernatants from C6 or TF274 cells stimulated by SK&F 107647 (10–100 µg/ml) were collected, passed through 30,000 m.w. cut-off membranes (Centricon, Amicon, Beverly, MA), and tested for direct CFU-GM colony stimulating activity as well as for the presence of synergistic activity. Suboptimal concentrations of recombinant human M-CSF (Cellular Products, Buffalo, NY) were combined with dilutions of stromal cell supernatants, mouse serum, purification column fractions, or recombinant proteins and added to cultures of 7.5 x 104 murine bone marrow cells in semisolid agar. CFU-GM colonies (>50 cells) were enumerated microscopically at 7 days postincubation at 37°C in 7.5% CO2, 5% O2 in humidified air.
Protein purification and characterization
Amino acid sequence analysis. N-terminal amino acid sequence analysis was performed on an Applied Biosystems (San Jose, CA) model 470A gas-phase protein sequencer equipped with a Beckman (Fullerton, CA) 126/166 system for on-line phenylthiohydantoin analysis. Data were acquired using System Gold chromatography software (Beckman, Fullerton, CA). Samples were spotted directly onto Polybrene coated GF/C filters (Applied Biosystems) and standard Applied Biosystems sequencing cycles used.
Matrix-assisted laser desorption ionization mass spectrometry
(MALDI-MS).
MALDI-MS data on active fractions were obtained on a Perceptive
Biosystems (Cambridge, CA) Voyager RP laser desorption linear
time-of-flight mass spectrometer. Samples were prepared for analysis by
mixing analyte 1:2 with 3,5-dimethoxy-4-hydroxy-cinnamic acid (Aldrich,
Milwaukee, WI), prepared as a saturated solution in 33%
CH3CN/0.1% trifluoracetic acid. Horse
apomyoglobin (Sigma, St. Louis, MO) was used as an external calibrant
(MH+ 16952.5 Da). Desorption/ionization was
accomplished using a nitrogen laser (337 nm, 3-ns pulse). The
accelerating voltage in the ion source was 30kV. Spectra were averaged
over 
100 laser scans. Calibrations were conducted using a customized
version of IGOR Pro (WaveMetrics, Lake Oswego, OR).
Preparation of recombinant proteins
KC, KC-T. A 264-bp DNA fragment encoding aa 1–72 of KC was assembled from a partial cDNA clone and synthetic oligonucleotide linkers were used to complete the sequence. DNA fragments corresponding to the full-length mature (aa 1–72 with an added N-terminal methionine) and truncated (aa 5–72, with an N-terminal methionine) forms of murine KC were subcloned to the Escherichia coli expression vector pEA181kn, and expressed under control of the inducible PL promoter. Nalidixic acid induction of bacteria carrying the plasmid constructs resulted in the expression of the predicted proteins as detected by SDS-PAGE and Western blotting. Proteins were purified from the insoluble cell lysate pellet (50 mM Tris, pH 6). The pellets were dissolved in 2 M guanidine HCl (Sigma) and 20 mM DTT (Sigma). The reduced proteins were dialyzed and refolded in 50 mM Tris at pH 8.5 by air oxidation. Refolded proteins were purified by Vydac (Hesperia, CA) C18 reverse phase-HPLC. Expected m.w. and amino acid compositions were confirmed via N-terminal sequencing and MALDI-MS analysis.
GROß, GROß-T. Human full-length GROß coding sequences were amplified by PCR from plasmids containing cDNA sequences using a forward primer encoding for an NdeI site, a defined epitope tag (DET) site, an enterokinase cleavage site, and a reverse primer containing an XbaI site. These PCR fragments were subcloned into the E. coli PL-dependent expression vector pEAKn (pSKF301 derivative) between NdeI and XbaI sites. These polypeptides were expressed, separately, by chemical induction of the PL promoter in a lysogenic strain of E. coli containing the wild-type repressor gene (c I+) AR120. Recombinant GROß and GROß-T were expressed intracellularly in E. coli using nalidixic acid induction. To obtain the initiator methionine-free N-terminal chemokines and to simplify the purification, DET-1-DDDDK was constructed to the N terminus of chemokines. DET-1 is a 12-aa peptide tag for an anti-DET-1 mAb, and DDDDK introduced an enterokinase-specific cleavage site. The E. coli cell pellet was lysed in 50 mM Tris (pH 6.0) buffer, with chemokines equally distributed between lysate pellet and supernatant. The soluble chemokines were refolded in 50 mM Tris at pH 8.5 by air oxidation and purified on an anti-DET-1 mAb affinity column, cleaved with enterokinase, and rechromatographed on DET-1 mAb to remove unclipped proteins. Enterokinase cleaved authentic N-terminal chemokines were further purified using a Vydac C18 RP-HPLC column. N-terminal amino acid sequencing and MALD-MS analysis confirmed the homogeneity of the preparation of all chemokine isoforms constructed.
Preparation of polyclonal affinity purified anti-KC antiserum
New Zealand White rabbits were immunized with KC peptide aa 31–52 with CFA. KC-specific IgG was purified from the serum of boosted rabbits on an immobilized KC affinity column. Control rabbit IgG was purchased from Accurate Chemical Scientific (Westbury, NY).
Effector cell enrichment/functional analysis
Resident murine peritoneal macrophages. Female C57BL/6 mice were sacrificed by CO2 asphyxiation. Following sacrifice, mice were injected with 10 ml of cold calcium and magnesium-free HBSS containing 10 U/ml heparin. The peritoneal cavity was gently massaged and the lavage fluid withdrawn and pooled. Peritoneal cell suspensions were adjusted to 4 x 106/ml in RPMI 1640 with 1% heat-inactivated (HI-) FCS (HyClone, Logan, UT) and 50 µl added to microtiter wells and incubated for 2 h at 37°C, 5% CO2 to allow for adherence. Following incubation, nonadherent cells were removed by washing with warm HBSS and fresh RPMI 1640 with 10% FCS added to the cell monolayers (>95% macrophages as determined by microscopic morphology).
Human peripheral blood leukocytes. Peripheral blood was obtained from normal healthy volunteers by venipuncture, following informed consent. Suspensions of purified human neutrophils were prepared by dextran sedimentation followed by lysis of residual RBCs with 10 mM Tris/0.83% NH4Cl buffer (pH 7.4) for 10 min. Mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Cell suspensions were washed resuspended in HBSS, and cell counts and differentials were obtained using a Technicon H1 Hematology Analyzer (Miles Diagnostics, Tarrytown, NY). Viability was >97% as determined by trypan blue dye exclusion. Monocytes (>80%) were prepared by incubating mononuclear cells in RPMI 1640 with 1% HI-FCS in microtiter wells followed by the removal of nonadherent cells as described above.
Neutrophil chemotaxis. PMNs were washed twice with PBS and resuspended in PBS containing 1 mM MgCl2 and 1 mM CaCl2. Cell motility was determined using a modified Boyden chamber procedure as described (15). For measurement of chemotaxis, lower chambers were filled with 30 µl of IL-8, GROß, or GROß-T (0.3–300 nM), the empty upper chambers were lowered into place and 50 µl of a PMN suspension (5 x 106 cells/ml). PMN migration proceeded for 60 min at 37°C in the cell incubator after which the chamber was disassembled. Following fixation (75% methanol) and staining (Diff-Quick; Baxter, Miami, FL) migrated cells were counted in four successive high power fields.
Ca2+ mobilization in neutrophils. Human polymorphonuclear leukocytes (PMNs) were separated from whole blood of healthy volunteers by the one-step Hypaque-Ficoll method (16). Cells were loaded with fura-2AM as described previously (17). Agonists were added at the designated concentrations to 106 cells/ml in Krebs-Ringer-Henseleit buffer. The maximal calcium concentration attained after agonist stimulation was quantitated as previously described (17).
Superoxide anion release. The superoxide dismutase (SOD)-inhibitable superoxide released by 2 x 105 mouse peritoneal cells, human neutrophils, or human monocytes in response to in vitro stimulation by KC proteins was quantitated in a microtiter ferricytochrome c reduction assay. Chemokines prepared in PBS were added at various concentrations and tested in quadruplicate. The assay was performed in HBSS with 1% gelatin (Sigma), containing 80 µM cytochrome c (Sigma) in a final volume of 200 µl/well. The OD550 of each well was determined at 10-min intervals for up to 1-h incubation at 37°C and 5% CO2, and the amount of cytochrome c reduced/well was quantitated. Wells containing all of the reagents plus SOD (200 U/well; Sigma) were run concurrently to determine the maximal amount of SOD-inhibitable cytochrome c reduction. Control superoxide activity was determined in the presence of PBS alone. Data are expressed as a percentage of the control PBS response at peak response times.
CD11b expression. Blood cells from PBS or SK&F 107647-treated mice were adjusted to 4 x 106/ml and stained with either FITC-labeled rat anti-mouse (Boehringer Mannheim, Indianapolis, IN) CD11b mAb or isotype control Ig for 30 min at 4°C. Following incubation, the cells were washed and resuspended in 2% paraformaldehyde for flow cytometry. Sample populations were analyzed for fluorescence intensity of the individual cell populations (e.g., monocytes and neutrophils) (50,000 cells) on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Preparation of C. albicans. C. albicans was prepared as previously described (10). Strain B311 (serotype A; ATCC 32354) was animal passed in mice and recovered from kidney homogenates. Recovered organisms were inoculated into Sabouroud dextrose broth (SDB) and grown overnight at 37°C, and the resultant culture aliquoted and frozen at -70°C. For use in studies, organisms from thawed stock aliquots were grown overnight on Sabouroud dextrose agar (SDA) and then inoculated into SDB and incubated for 18 h at 37°C on a rotary shaker. The cells were harvested by centrifugation, washed, resuspended in normal saline, and quantified by hemocytometer. Resulting suspensions were >95% viable as determined by methylene blue dye exclusion. All inoculum sizes reported are based on viable counts as determined by CFU analysis on SDA.
Candidacidal assay. Two hundred C. albicans blastospores were added to quadruplicate flat-bottom microtiter wells containing 2 x 103 human neutrophils (E:T = 10:1) in HBSS plus 10% autologous human sera. Various concentrations of KC proteins were added (quadruplicate wells/concentration) and following a 2-h incubation at 37°C and 5% CO2, the plates were centrifuged, the supernatant discarded, and the phagocytic cells lysed with three washes with cold sterile water. Following the last wash, molten SDA was added to each well and the plates were incubated at 37°C, 5% CO2 overnight. The numbers of microcolonies per well were then enumerated microscopically and the percent reduction from CFU in wells containing yeast only was determined. The data are expressed as a percentage of the control (PBS) group response. Data are expressed as the mean of at least two individual experiments, and statistical analyses were performed using a Student’s t test.
Murine C. albicans infection model
Animals. Strictly defined flora BALB/c mice bred in an in house laminar flow barrier facility were utilized (18). The mice were housed in sterile cages and fed sterilized lab chow and water ad libitum. The Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals approved procedures involving the use of laboratory animals.
Dosing schedule. Various concentrations of recombinant KC-T prepared in PBS were administered s.c. 2 h before infection. All mice were subsequently infected i.v. with a lethal dose of C. albicans (1.2 x 105 yeasts/mouse). After infection, the animals were followed for survival. Mann-Whitney U test determined differences in survival times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The degree
of synergy ranged from 130 to 200% of control CSF levels. The
synergistic activity of SK&F 107647-induced stromal cell supernatants
was best observed at suboptimal CSF concentrations, with only minor
increases in CFU-GM proliferation observed in combination with optimal
CSF concentrations (data not shown). Similar results were observed when
supernatants were tested on human CFU-GM (data not shown).
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The purification of SK&F 107647-induced synergistic activity was tracked by the CFU-GM bioassay. Due to the lack of signal at 280 nM in any of the chromatography steps, no purification figures have been included.
To identify the SK&F 107647-induced synergistic factor, supernatants from peptide-treated C6 cells (40 liters) were filtered using an Omega 30-kDa m.w. cut-off cassette assembled in a Filtron Centrasette (Filtron, Northborough, MA). The filtrate was concentrated to 500 ml using a 10-kDa cassette. The retentate, which contained 100% of the synergistic biological activity, was applied to a heparin agarose column (Bio-Rad, Richmond, CA). A solution of 0.5 M NaCl was used to elute nonactive proteins from the heparin column. Bound HSF was eluted with 1 M NaCl and diafiltered in PBS before application to an Aquapore C8 RP-HPLC column (Beckman). Fractions were collected using an acetonitrile (ACN) gradient and analyzed for the presence of HSF activity on mouse CFU-GM, over an extended dilution range. The fractions containing HSF activity (41% ACN) and adjacent peaks were analyzed by amino acid sequence and mass spectrometry.
Amino acid sequence analysis of the active 41% ACN fraction identified
a N-terminal sequence consistent with a four amino acid N-terminal
truncated form of the murine C-X-C chemokine KC (Fig. 1). Mass spectrometry analysis of this
same fraction resulted in a mass of 7459 Da, consistent with a
truncated 68-residue form of KC, i.e., KC5–72.
In an adjacent peak, full-length KC1–72 was also
purified and characterized (7811 Da), but did not demonstrate any
detectable hematopoietic synergistic activity on either murine or human
CFU-GM in vitro.
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Human SK&F 107647-induced synergistic activity was purified in a similar manner to murine HSF. Supernatants from SK&F 107647-treated TF274 cells (185 liters) were filtered with a Centrasette tangential flow concentrator (Filtron, Northborough MA) equipped with a 100K MWCO Omega-type membrane. The filtrate, 170 liters, was concentrated to 4 liters using the same apparatus equipped with a 3K MWCO Omega-type membrane. The retentate was diafiltered with PBS and adjusted to 0.5 M NaCl in PBS and applied to heparin agarose column (Bio-Rad). Bound HSF was eluted with 1.5 M NaCl in PBS (450 ml). The heparin column 1.5 M NaCl pool was concentrated to 100 ml and applied directly to an immunoaffinity column (10 ml) prepared by immobilizing anti-GROß mAb (Austral Biologicals, San Roman CA) to Affigel 10 (Bio-Rad) at 0.5 mg/ml of wet gel. The affinity column was developed by washing with buffer A (50 mM NaPO4/0.15 M NaCl, pH 7.2), buffer B (5 mM NaPO4/5 mM NaCl, pH 7.2), and buffer C (2.5 mM H3PO4/5 mM NaCl, pH 2.7), then re-equilibrated in buffer A. Fractions containing HSF activity determined by bioactivity on CFU-GM were pooled, reapplied to the same column, and rechromatographed. HSF activity was eluted with buffer A (50 mM NaPO4/0.15 mM NaCl, pH 7.2) upon column re-equilibration. Fractions containing HSF activity were pooled and analyzed by N-terminal amino acid sequencing and MALDI-MS.
The observed sequence in the eluted immunoaffinity column fraction
containing the HSF bioactivity was consistent with a 4-residue
N-terminal truncated form of the human C-X-C chemokine GROß (Fig. 1
).
Mass spectrometry indicated that this fraction contained a unique mass
species of 7550 Da compared with adjacent fractions. Estimated total
protein content in this fraction was 30 pmol, therefore no further
purification was attempted. No full-length GROß (expected molecular
mass of 7887 Da) was found in any adjacent fractions. GROß-T identity
was distinguished from GRO and GRO homologues by residues 20 (L
in GROß while P in GRO), 27 (K in GROß while N in GRO and
GRO), and 29 (K in GROß while R in GRO). Western blotting of
the GROß affinity column acid eluate revealed a protein band
migrating with recombinant GROß (data not shown).
Hematopoietic synergistic activity of recombinant KC and GROß proteins
KC-T and GROß-T plasmids were constructed and proteins were
expressed in E. coli and purified to confirm biological
activity. Recombinant KC-T (data not shown) and GROß-T enhanced the
proliferation of murine CFU-GM stimulated by a suboptimal concentration
of M-CSF (Fig. 2). Addition of
recombinant GROß-T resulted in a bell-shaped response curve similar
to that observed with unpurified SK&F 107647-induced stromal cell
supernatants. Significant enhancement of CFU-GM by GROß-T was
observed at concentrations between 5 pg/ml and 500 attograms/ml
(p < 0.01), with 50 fg and 5 fg/ml
concentrations of GROß-T consistently resulting in a statistically
significant enhancement of CFU-GM proliferation
(p < 0.01; n = 6 experiments).
Similar results were observed for recombinant KC-T tested on either
murine or human CFU-GM (data not shown).
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2 x
104 U/mg protein. In contrast, both KC-T and
GROß-T chemokines demonstrated dramatic increases in specific
activity. In six experiments, the specific activity of GROß-T in the
CFU-GM synergy assay was
1012–1013 U/mg protein,
which was >10 million times more potent than full-length GROß. A
similar specific activity of 1 x 1013 U/mg
protein was observed for recombinant KC-T. Effect of anti-KC Abs on SK&F 107647 induced serum HSF activity in the mouse
C57BL mice were injected i.p. with 100 ng/kg SK&F 107647. A group
of mice were pretreated 24 h previously with 10 ng of
affinity-purified anti-KC antiserum or rabbit IgG control followed
by administration of SK&F 107647. Six hours after injection, animals
were sacrificed and serum prepared. Serum samples from three mice per
group were fractionated on 30K MW membranes to remove serum CSF and
diluted in the HSF bioassay with suboptimal concentrations of M-CSF.
The serum from mice treated with anti-KC or the IgG control Ab did
not contain any synergistic activity detectable in these assays (Fig. 3). Serum collected from IgG pretreated
mice 6 h after a single injection of SK&F 107647 (100 ng/kg)
contained detectable synergistic activity = 428 U/ml. Mice
pretreated with anti-KC Ab followed by SK&F 107647 did not contain
any detectable serum synergistic activity. In addition, the SK&F
107647-induced serum synergistic activity was completely neutralized by
in vitro treatment with anti-KC Ab. Therefore, both SK&F
107647-induced production of synergistic activity in vitro (C6 cell
supernatants) and in vivo (C57 mouse serum) was completely neutralized
by the addition of anti-KC Abs.
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Activity of GROß forms on human PMN function.
To assess the activity of both truncated and full-length forms of
GROß, we tested the response of these two isoforms on calcium
mobilization and chemotaxis using freshly isolated human neutrophils.
Both GROß and GROß-T both promoted substantial calcium mobilization
in PMN over the dose-response range 0.03–100 nM with
EC50s = 0.2 and 2.2 nM for GROß-T and GROß,
respectively (Fig. 4A). The
same difference in potency was also noted for GROß-T and GROß in
the human neutrophil chemotaxis assay, which like the calcium assay
identified both ligands as full agonists. In the chemotaxis assay
GROß and GROß-T both promoted chemotaxis with
EC50 values of 2 and 0.3 nM, respectively (Fig. 4B). IL-8 was used for comparison and had an
EC50 similar to GROß-T. Thus GROß, GROß-T,
and IL-8 were found to be chemotactic rather than chemokinetic by
checkerboard analysis (data not shown).
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. Maximal
responses occurred at 0.1–1 and 100 nM with truncated and full-length
KC proteins, respectively (p < 0.01). KC-T was
1,000- to 10,000-fold more potent than KC in stimulating superoxide
anion release. Of note is the bell-shape dose-response curve to KC-T,
similar to the dose-response curve for CFU-GM synergistic activity.
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A).
Enhancement of human monocyte superoxide response has generally not
been observed as a major function of KC and GRO protein
(1). Consistent with this observation, human blood
monocytes responded weakly to KC at high concentrations (150% of
control at 100 nM). However, KC-T activated monocytes at 0.2 nM,
representing a 500-fold increase in potency.
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B). GROß-T was 
10,000-fold more potent than
GROß. Human blood monocytes responded weakly to GROß at high
concentrations (150% of control response at 30 nM). However, GROß-T
activated monocytes at 0.1 nM, representing a 300-fold increase in
potency.
Candidacidal activity.
The increased production of antimicrobial superoxide anion is a good
indicator for enhanced phagocytic cell killing of organisms such as
fungi. We determined the effects of recombinant KC proteins on the
capacity of human neutrophils to kill C. albicans in vitro
(Fig. 7). Candidacidal activity was
measured following a 2-h coincubation of neutrophils and fungi (E:T
ratio = 10:1). Treatment of neutrophils with 0.001–100 nM KC-T
resulted in significantly increased levels of candidacidal activity
(1.5- to 3-fold increase, p < 0.01) with optimum
enhancement occurring at a dose of 1 nM KC-T (225% of control
response). There was a minimal but significant increase in candidacidal
activity as a result of treatment with KC at doses of 10 and 100 nM
(p < 0.05).
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, administration of SK&F 107647
increased both the percentage of neutrophils and monocytes expressing
CD11b (top) and also the MCF of CD11b expressing
cells (bottom). Pretreatment with control IgG was without
effect on SK&F 107647-induced CD11b up-regulation; however,
pretreatment with anti-KC Ab completely blocked the CD11b effects
of SK&F 107647 in vivo.
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The effect of KC-T in enhancing superoxide production and
candidacidal activity of neutrophils and monocytes in vitro suggested
that this chemokine may have biological activity in an animal fungal
infection model in which neutrophils and monocytes have been shown to
be the primary host cells responsible for anti-fungal activity. We
therefore investigated the effect of administration of KC-T on survival
in a lethal murine C. albicans infection model.
Administration of SK&F 107647 in this model effectively increases the
survival of treated mice (8). Mice were dosed s.c. with
various concentrations of KC-T or PBS 2 h before i.v. infection
with C. albicans (Fig. 9). At
15 days postinfection, no survivors were present in the control group.
In contrast, significantly enhanced survival was observed in mice
treated with a single injection of 63 fg or 125 fg/kg KC-T. The
response to KC-T showed a bell-shaped curve with higher (250 fg/kg) and
lower (32 fg/kg) doses failing to significantly protect mice from
lethal infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines have been reported to have both enhancing and suppressive
effects on hematopoietic progenitor cell proliferation (5, 6, 20). In these studies, GROß-T and KC-T are potent synergistic
factors for CFU-GM proliferation in vitro. In addition, administration
of SK&F 107647 to mice results in the production of HSF activity in the
serum that is neutralized by anti-KC Ab in vivo and in vitro,
further supporting the physiological relevance of these chemokine
forms. The molecular signaling mechanisms for this effect is currently
unknown. Chemokines in the GRO family are known to bind the CXCR2
receptor (1). It has yet to be determined whether CXCR2
receptors are present on marrow hematopoietic progenitors (3, 21, 22); however, the growth of single cell
CD34+ progenitors is clearly affected by
chemokines (23). The in vitro effect of C-X-C chemokines
such as human IL-8 and GROß (macrophage-inflammatory protein-2) on
progenitor cells is growth suppression (4, 24) rather than
the enhanced growth observed with GROß-T and KC-T. Full-length GROß
has CFU-GM enhancing activity, whereas full-length KC had no enhancing
effect at the concentrations tested. It has been reported that the
suppressive effect of IL-8 and melanoma growth stimulatory activity
(GRO) on the growth of the IL-3-dependent murine myeloid progenitor
cell line 32D was via the murine CXCR2 (25). In addition,
GROß-T has 10 times the affinity for the CXCR2 receptor
than full-length GROß and the CXCR2 receptor as defined by human
neutrophil chemotaxis and Ca2+ mobilization
assays. In part, the apparent increases in receptor affinity for
GROß-T may account for some of the differences in potency seen in the
hematopoietic synergistic assay. The hematopoietic synergistic activity
of GROß-T is antagonized by the CXCR2 specific antagonist SB-220052
with an IC50 of 500 nM providing further evidence
that the CXCR2 receptor is involved GROß-T-mediated hematopoietic
activities (data not shown and Ref. 26). Alternatively,
the increased potency of GROß-T and KC-T may indicate an indirect
mechanism of action involving multiple marrow cell types whose
amplified signals result in enhanced CFU-GM proliferation. The cellular
mechanisms involved in the enhanced potency of truncated GROß and KC
is under investigation.
Identification of truncated chemokines has been reported; however, the
magnitude of enhanced response observed with GROß-T and KC-T on
proliferation of CFU-GM and effector cell function are more dramatic
than reported for truncations of any other chemokines. An N-terminal
5-aa truncated form of GROß has been reported to be 1 log more
potent than full-length GROß in activating neutrophil elastase
release (27). A mixture of N-terminal short forms of GCP-2
has higher specific activity than longer forms as measured by
neutrophil activation in vitro (28, 29). The 77-aa form of
IL-8 is less potent than the shorter 72-aa form in effector cell
activation and receptor binding (30). In contrast, IL-8
truncated by 4 N-terminal residues is less potent in binding to the
IL-8 receptor and stimulating chemotaxis and effector cell activation
(31). Recently, N-terminal truncated forms of ENA-78,
GRO, and GRO produced by tumor cells and leukocytes have
demonstrated increased potency in chemotactic assays and
Ca2+ signaling relative to full-length forms
(32). N-terminal truncations of RANTES (33)
and MCP-1 (34, 35) have also been described having
equivalent or less activity compared with their respective full-length
chemokines. Antagonistic N-terminally truncated forms of RANTES with no
chemotactic activity, but retaining anti-HIV activity have also
been described (36, 37). In many of these studies multiple
N-terminal truncations of various lengths were observed during
purification indicating possible nonspecific aminopeptidase
cleavage.
Specific N-terminal cleavage of chemokines such as RANTES and
stromal-derived factor-1 (SDF-1) and SDF-1ß can be mediated by
dipeptidyl peptidase IV (CD26). CD26-mediated cleavage of RANTES
reduces activity on human monocytes (38), whereas cleavage
of SDF-1 and SDF-1ß abolishes both chemotactic and anti-viral
activity (39, 40). Many chemokines including KC and GROß
have a proline in the second amino acid position from the N terminus.
CD26 cleaves 2 aa at a time with preference for post-proline and
post-alanine cleavage (41). Full-length KC and GROß have
proline at position 2 and alanine at position 4. During purification of
SK&F 107647-stimulated stromal cell supernatants, an N-terminal
2-residue truncated form of KC2–72 was isolated;
however, it lacked CFU-GM synergistic activity (data not shown). It is
unclear whether the production of specific 4-aa truncated chemokines in
SK&F 107647-stimulated stromal cells is mediated by CD26. It is
tempting to speculate that the specific enzymatic truncation of GROß
and KC at the same amino acid position (4 N-terminal amino acids)
represents an added level of specificity related to the mechanism of
action of SK&F 107647.
Chemokines in the CC and CXC classes have been shown to enhance effector cell function (1). The CC chemokines, in general, have more effects on monocytes and T cells, while CXC chemokines act mainly on neutrophils. Addition of KC-T or GROß-T to isolated neutrophils enhanced function as defined by increased expression of CD11b, chemotaxis, superoxide production, and killing of C. albicans in vitro. Increased monocyte superoxide production was noted after incubation with KC-T and GROß-T, while only modest changes in activity were observed with full-length KC and GROß.
The concentrations of KC-T and GROß-T that activated effector cell populations and those that enhanced CFU-GM synergistic activities were very different. Enhanced CFU-GM activity of KC-T or GROß-T occurred at concentrations of 0.01–0.1 pM (50–500 fg/ml). Full-length KC was inactive in the CFU-GM assay, whereas GROß was 10 million-fold less active. In human PMN studies, KC-T and GROß-T enhanced superoxide activity at 0.1–0.2 pM compared with 10 nM for the full-length forms (10,000- to 50,000-fold increase in potency). In human monocytes, KC-T and GROß-T enhanced superoxide activity at 0.1–0.2 nM, whereas the full-length forms were active at 30–100 nM (300- to 500-fold increase in potency). The complex mechanisms involved in differential enhanced potency with the truncated chemokines are unknown. All of the assay systems used contain different functional cell types, which may have differential sensitivities to the truncated chemokines. In addition, the response of the same cell type was significantly different depending on the functional assay. In isolated human PMN chemotaxis and Ca2+ mobilization studies, a clear 10-fold increase in potency was observed with GROß-T compared with GROß, whereas a 10,000-fold increase was observed in the superoxide assay. The biochemical and cellular nature of these activities of KC-T and GROß-T are currently under investigation.
Agents that can increase host defense effector cell activity, such as bacillus Calmette-Guerin (42, 43) M-CSF (44), GM-CSF (45), and SK&F 107647 (9) have all demonstrated protection in experimental models of C. albicans fungal infection. Mechanistically, we have demonstrated that the in vitro and in vivo effects of SK&F 107647 are mediated by KC-T production in the mouse. Studies were performed to determine whether enhanced effector cell function (enhanced superoxide and candidacidal activity) elicited by KC-T in vitro could result in protection of mice challenged with lethal doses of C. albicans. KC-T demonstrated potent anti-infective properties in this model in the 60- to 120-fg/kg dose range. KC-T mimicked the effectiveness of SK&F 107647 in vivo and also demonstrated a bell-shaped dose response that is characteristic of hematoregulatory compounds (9, 10, 44). The mechanism for this bell-shaped dose response is presently unknown. This study is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious disease model.
To our knowledge, this report represents the first example where any form of KC or GROß were purified from marrow stromal cells. The discovery that truncated forms of chemokines can demonstrate dramatically enhanced potency and activity has significant implications in defining the role of chemokines in physiological and pathological conditions.
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2 Abbreviations used in this paper: SK&F 107647, (s)-5-oxo-L-prolyl-L-glutamyl-L-aspartyl-N8-(5-amino-L-carboxypentyl)-8-oxo-N7-[N-[N-(5-oxo-L-prolyl)-L-glutamyl]-L-aspartyl]-2,7,8-triaminooctanoyl-L-lysine; HSF, hematopoietic synergistic factor; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PMN, polymorphonuclear leukocytes.
Received for publication July 12, 1999. Accepted for publication January 31, 2000.
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) on calcium
mobilization in human neutrophils. Freshly isolated neutrophils where
loaded with fura-2 and stimulated with varying concentrations of either
GROß-T or GRO-ß. Maximal calcium mobilization was monitored and
noted. The figure depicts that amount of calcium mobilized in nM at
each concentration of chemokine. Each point is the average of four
separate determinations ± SEM. B, Dose-dependent
chemotaxis of human neutrophils to GROß-T (•), GROß (
). Each point represent the average of three determinations counted
in triplicate. The chemotactic index is derived from the number of
cells that migrated to the chemokine divided by the number of cells
which migrated in the absence of added chemokine.
