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PLENARY PAPER
From the Laboratory of Molecular Immunology, Rega
Institute for Medical Research, University of Leuven, Belgium.
Chemokines constitute a large family of chemotactic cytokines that
selectively attract different blood cell types. Although most
inflammatory chemoattractants are only induced and released in the
circulation during acute infection, a restricted number of CXC and CC
chemokines are constitutively present in normal plasma at high
concentrations. Here, such a chemotactic protein was purified to
homogeneity from serum and fully identified as a novel CC chemokine by
mass spectrometry and amino acid sequence analysis. The protein,
tentatively designated Regakine-1, shows less than 50% sequence
identity with any known chemokine. This novel CC chemokine
chemoattracts both neutrophils and lymphocytes but not monocytes or
eosinophils. Its modest chemotactic potency but high blood
concentration is similar to that of other chemokines present in the
circulation, such as hemofiltrate CC chemokine-1, platelet factor-4,
and Serum is a rich source of leukocyte
chemotactic factors that influence the migration of different
leukocytic cell types to and from the blood circulation. For example,
anaphylatoxin, or C5a, a cleavage product formed during complement
activation, chemoattracts both polymorphonuclear and mononuclear blood
cells. Other serum proteins such as platelet factor-4 and
neutrophil-activating protein-2 are thrombocyte-derived chemotactic
cytokines belonging to the chemokine family.1-4 In
contrast to C5a, these and other chemokines are each selectively
attracting a defined set of leukocytic cell types. The chemokine family
is subdivided into 2 major classes, ie, CXC and CC chemokines depending
on the positioning of conserved cysteine residues.1-5 The
spectrum of target cells for each chemokine depends on the expression
of one or more specific receptors on the different leukocyte subtypes.
The receptors of all chemokines as well as those of C5a and other
chemoattractants such as leukotriene B4 and bacterial
N-formylmethionyl-containing peptides belong to the family
of G protein-coupled 7-transmembrane domain
receptors.6
Addition of bovine or human serum is often essential for the growth or
maintenance of continuous and primary cell cultures. For example, we
and others have used in the past low serum concentrations to preserve
high viability of freshly isolated human leukocytes or to support the
growth of hematopoietic progenitor cells in well-defined
media.7 Conflicting or variable experimental results have
often been related to the presence or absence of serum or partially
purified plasma proteins in the test system, eg, in chemotaxis
assays.8-12 Furthermore, even after transfer of cells to
serum-free conditions, the biologic responses can still be influenced
by serum components sticking to the cultured cells. Indeed, because
chemokines have high affinity for heparin-like glycosaminoglycan
molecules, serum-derived chemokines are candidates to interfere in
migration assays.13 This is likely the case if more than
one cell type (cultured in serum) is implicated in the test system, eg,
to measure transendothelial migration of leukocytes.14
In an attempt to identify additional serum-derived chemotactic factors
that might influence standard chemotaxis assays, we have purified such
molecules from commercially available bovine serum routinely used to
grow or maintain cells in vitro. This study revealed the existence of
an unknown bovine CC chemokine for which no human homolog has yet been
described. Furthermore, this CC chemokine did not only attract
lymphocytes, but also neutrophilic granulocytes. The relatively high
abundancy of this chemokine compared with other CC chemokines indicates
a different physiologic role for this molecule.
Chemokine purification
After each purification step, fractions were analyzed for purity by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
under reducing conditions on Tris/tricine gels.15 Proteins
were visualized by silver staining. The relative molecular mass markers
included in the gels were carbonic anhydrase (Mr 31 000),
soybean trypsin inhibitor (Mr 21 500), and lysozyme
(Mr 14 400) (Bio-Rad) and the low molecular weight marker
aprotinin (Mr 6500) (Pierce Chemical, Rockford, IL).
Chemokine identification by amino acid sequence analysis and
mass spectrometry
The NH2-terminal amino acid sequences of homogeneous intact
or fragmented peptides were determined by Edman degradation using a
pulsed liquid phase 477A/120A protein sequencer (Applied Biosystems). Extended sequences were obtained by removing the background on the
sequencer with o-phtalaldehyde. Briefly, when a proline was present at the NH2-terminal position during the Edman
degradation, the peptides without NH2-terminal proline
(derived from incomplete chemical reactions) were
NH2-terminally blocked by incubation for 10 minutes at
43°C in o-phtalaldehyde solution (20 mg
o-phtalaldehyde and 50 µL For mass spectrometry, RP-HPLC-purified proteins were diluted in 50% acetonitrile/50% water/0.1% acetic acid to a concentration of 0.5 to 5 nM and injected at 5 µL/min (dry gas flow 3 L/min, dry temperature 300°C, nebulizer gas pressure of 7 psi, skimmer 1 voltage of 31 V, octopole lens at 3 V, and trap drive at 75.3) on an Esquire ion trap mass spectrometer (Bruker/Daltonic, Bremen, Germany). Relative molecular masses of peptides or proteins were calculated from 100 or more averaged spectra (accumulation time of ± 0.1 msec) to increase the accuracy of the mass/charge measurements. Chemical synthesis of Regakine-1 Regakine-1 was chemically synthesized (0.1 mM scale) using standard Fmoc programs on a solid-phase peptide synthesizer (Model 433A, Applied Biosystems) as described in detail elsewhere.15,16 Final deprotection and cleavage of the peptide from the resin was performed with TFA, and the synthetic chemokine was separated from the resin over a glass filter. Crude synthetic Regakine-1 was separated from incomplete fragments by RP-HPLC on a Resource RPC column (Amersham Pharmacia Biotech). After purification, disulfide bridges were formed by incubation (90 minutes, 20°C) of unfolded peptide in 150 mM Tris, pH 8.6; 2 M ureum, 3 mM EDTA, 0.3 mM oxidized glutathione, and 3 mM reduced glutathione. The folded peptide was purified by RP-HPLC. The molecular mass of unfolded and folded peptide was confirmed by mass spectrometry on an Esquire ion trap mass spectrometer.Isolation of peripheral blood cells and chemotaxis assay Polymorphonuclear and mononuclear cells from human peripheral blood were separated by density gradient centrifugation (30 minutes, 400g) on Ficoll-sodium diatrizoate (Lymphoprep, Nycomed Pharma, Oslo, Norway). The total mononuclear cell fraction (2 × 106 cells/mL) was used for chemotaxis as a source for monocytes. Lymphocytes were further enriched by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) after labeling with magnetic microbeads coated with monoclonal antibody against CD3 and used at 107 cells/mL in migration assays. Neutrophilic and eosinophilic granulocytes were isolated from the polymorphonuclear cell pellet obtained by density gradient centrifugation. This pellet was first suspended in hydroxyethyl starch (Plasmasteril, Fresenius, Bad Homburg, Germany) for 30 minutes to remove most erythrocytes by sedimentation. Residual erythrocytes were then lysed in bidistilled water (30 seconds). The total granulocytic cell fraction was used at 106 cells/mL in neutrophil chemotaxis tests. Finally, after tagging the neutrophils with anti-CD16 beads (Miltenyi Biotec), eosinophils were isolated by MACS as the negatively selected cell fraction. Eosinophils were seeded at a final concentration of 2 × 106 cells/mL for migration tests.For the isolation of bovine neutrophils, whole peripheral blood of adult cows was collected, diluted in PBS, and fractionated by density gradient centrifugation on Lymphoprep (Nycomed Pharma). The granulocyte pellet was resuspended and washed, and residual erythrocytes were lysed by hypotonic shock. Chemotaxis with bovine neutrophilic granulocytes was performed as described for human neutrophils. The chemotactic potency of chemokines was determined in the Boyden microchamber (Neuro Probe, Gaithersburg, MD). Cell fractions and samples were diluted in Hank's balanced salt solution (Life Technologies) supplemented with human serum albumin (Belgian Red Cross) at 1 mg/mL (dilution buffer) and tested in triplicate. For granulocytes, migration through 5-µm pore-size polycarbonate membranes (Nuclepore, Corning Costar, Acton, MA) was measured after 45 minutes at 37°C for neutrophils and after 1 hour for eosinophils. Lymphocyte chemotaxis (4 hours, 37°C) was performed using fibronectin-coated (25 µg/mL; 12 hours, 4°C) polycarbonate membranes (5-µm pore-size), and for monocyte chemotaxis (2 hours, 37°C) polyvinylpyrrolidone-treated polycarbonate membranes (5- µm pore-size) were used. In each chemotaxis experiment either N-formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma, St Louis, MO), purified natural interleukin 8 (IL-8) (neutrophils), or synthetic monocyte chemotactic protein-3 (MCP-3) (monocytes, lymphocytes, eosinophils) was included as a positive control.12,16 After incubation, the cells were fixed and stained using Hemacolor solutions (Merck, Darmstadt, Germany). Migrated cells were counted microscopically in 10 oil immersion fields at a 500 × magnification. The chemotactic potency of a sample was expressed as the chemotactic index (CI), ie, the number of cells migrated to the chemoattractant divided by the number of cells migrated to dilution buffer. Chemokinesis was measured by adding the chemokine to the cells at the time of transfer to the upper wells of the microchamber or by preincubation of the test cells with chemokine for 10 minutes at 37°C prior to transfer to the microchamber. The latter conditions were also used in experiments measuring the combined effect of Regakine-1 and the CXC chemokines IL-8 or granulocyte chemotactic protein-2 (GCP-2) in the migration assay; ie, neutrophils were preincubated with different concentrations of Regakine-1 (10 minutes, 37°C) and then added, without washing, to the upper compartment of the microchamber. Alternatively, Regakine-1 was added simultaneously with IL-8 to the lower wells of the microchamber to measure a synergistic effect in the chemotaxis assay. Statistical analysis of chemotaxis data was performed using the Mann-Whitney test.
Isolation and identification of a novel CC chemokine from bovine serum Tissue culture-grade newborn calf serum was processed according to our standard procedure for the isolation of chemokines from conditioned medium of in vitro-stimulated cell cultures.8,12,15 Due to its high protein content (about 50 mg/mL), the serum (2 L) was diluted 1:5 prior to adsorption of proteins to silicic acid. Because protein binding to this substrate was rather selective (99% unadsorbed), only 1 g of the initial amount of protein was recovered by elution from silicic acid. Subsequent heparin-Sepharose affinity chromatography allowed further enrichment of serum-derived chemotactic activity for neutrophils, which eluted at 0.5 M NaCl, after the bulk of protein showing low or no affinity for heparin (Figure 1A). Further purification to homogeneity of the biologic entity was achieved by cation-exchange chromatography (elution at 0.3 to 0.4 M NaCl) and finally by RP-HPLC. The neutrophil chemotactic activity was recovered from the RP-HPLC column (Figure 1B) over a rather broad range in the elution gradient (from 25% to 30% acetonitrile). However, SDS-PAGE analysis showed the presence of a single protein band of 7.5 kd corresponding to the neutrophil chemotactic activity (Figure 1C). None of the fractions containing the chemotactic protein corresponded to known bovine CXC chemokines. Indeed, IL-8 and GCP-2 derived from stimulated MDBK cells eluted at different positions upon cation-exchange chromatography and RP-HPLC.17 Surprisingly, NH2-terminal sequence analysis of this pure protein revealed the presence of a novel CC chemokine, whereas the CXC hallmark is typical for neutrophil chemoattractants. In view of the unusual source (serum) and target cell (neutrophils) for this CC chemokine, the molecule was tentatively designated Regakine-1.
The complete primary structure (70 residues) of Regakine-1 was obtained
by NH2- and COOH-terminal sequence analysis and by sequencing internal fragments obtained by proteolytic digestion with
the endoproteinases asparagine-C and lysine-C (Figure
2). In addition, mass spectrometry
allowed for the identification of the COOH-terminal serine that was
undetectable during the COOH-terminal sequence analysis. Both the
origin and the primary structure of Regakine-1 were confirmed by an
independent purification and sequencing run using fetal calf serum
instead of newborn calf serum. Furthermore, this same CC chemokine was
isolated and identified from serum obtained through coagulation of
blood from adult cows collected in a local slaughterhouse. This
confirmed the true bovine nature of this molecule and excluded possible
artifacts due to industrial processing of commercially available fetal
or newborn serum, ie, the admixture with serum from other species.
Furthermore, it demonstrated that the presence of this chemokine in
serum is not restricted to young animals. On average, 100 µg
Regakine-1 was isolated from 1 L bovine serum. This amount is
comparable to the production of IL-8 by in vitro-stimulated leukocytes
from 1 L human blood.12
The sequence of Regakine-1 was not picked up by a search in the
SWISS-PROT/TrEMBL protein database.18 Alignment of
the sequence of Regakine-1 with those of known human and bovine
chemokines (Figure 3) and other proteins
did not reveal a high structural similarity (< 50%). However,
residues other than the 4 cysteines that are conserved in most CC
chemokines are also selectively present in the amino acid sequence of
Regakine-1, such as Ile20, Pro21, Tyr28, Val40, Phe42, Ala52, Pro54,
Trp58, and Val59 (Figure 3). Regakine-1 was found to be most homologous
to human eotaxin (49% identical residues). However, murine, guinea
pig, rat, and human eotaxin share residues that are not present in the
sequence of Regakine-1. Because for other known bovine chemokines the
structural homology with their human counterparts is evidenced by more
than 65% identical residues (eg, 67% for
GCP-217), the human homolog of Regakine-1 remains
to be identified.
Neutrophil and lymphocyte chemotactic potency of natural Regakine-1 The bovine serum-derived CC chemokine (purified from different serum batches) was compared with human leukocyte-derived IL-8 in the standard microchamber migration assay using human and bovine neutrophils. On human neutrophils, IL-8 was still chemotactic at 10 ng/mL, whereas for Regakine-1, 300 ng/mL was necessary to obtain a significant chemotactic effect (Figure 4A). In addition to its lower potency (minimal effective concentration), the efficacy (maximal CI) of Regakine-1 was on average weaker than that of human IL-8 (Figure 4A and data not shown). Furthermore, Regakine-1 was tested on bovine neutrophils to confirm the chemotactic potency in the homologous species. Figure 4B shows that on bovine neutrophils comparable results were obtained, human IL-8 being a more potent chemoattractant than Regakine-1. The chemotactic effect of Regakine-1 on granulocytes remained restricted to neutrophils, because human eosinophils, responsive to MCP-3 at 30 ng/mL, were not attracted by this chemokine at 1000 ng/mL (data not shown). This indicates that the relatively higher structural identity of Regakine-1 with human eotaxin is probably not biologically relevant.
In view of the modest and unexpected chemotactic activity of Regakine-1 on neutrophils, this CC chemokine was further investigated on mononuclear cells. In contrast to MCP-3, which induced monocyte migration from 10 ng/mL onward, up to 1000 ng/mL natural Regakine-1 had no significant chemotactic effect on freshly isolated peripheral blood monocytes (Figure 4D). However, natural Regakine-1 was chemotactic for CD3+ lymphocytes at 300 ng/mL, whereas MCP-3 was active on these cells at 10 ng/mL (Figure 4C). These biologic data demonstrate that Regakine-1 has a modest but significant chemotactic activity for both neutrophils and lymphocytes. Chemotactic activity of synthetic Regakine-1 To exclude that the chemotactic effect of Regakine-1 was due to a minor contamination of this CC chemokine with other more potent chemokines, Regakine-1 was chemically synthesized by Fmoc chemistry. The synthetic protein was deprotected, folded, and purified to homogeneity according to a standard procedure used in our laboratory.15,16 Synthetic Regakine-1 was found to be biochemically and biologically identical to the natural product, as shown by mass spectrometry, amino acid sequence analysis, SDS-PAGE, and chemotaxis assays. The neutrophil chemotactic potency of both synthetic and natural Regakine-1 was inferior to that of human IL-8 and MCP-3 (Figure 5), another CC chemokine to which weak neutrophil chemotactic activity has been ascribed.19 However, Regakine-1 was equally efficacious on neutrophils when compared with MCP-3, as can be deduced from the maximal CIs (Figure 5). Notably, the concentration of MCP-3 required to maximally attract neutrophils (30 ng/mL) can only be reached in serum during pathologic conditions, eg, viral infection,20 whereas 100 ng/mL Regakine-1 is a physiologic plasma concentration. Additionally, the lymphocyte chemotactic activity of Regakine-1 was also confirmed with the synthetic protein (data not shown). Taken together, these data with synthetic Regakine-1 confirm the authentic chemotactic activity of the natural chemokine.
Regakine-1 enhances the chemotactic potency of IL-8 and GCP-2 In an attempt to further define the role of Regakine-1 in leukocyte migration, it was verified whether this chemokine exerts chemokinetic effects. When applied with the cells in the upper compartment of the microchamber, different concentrations of IL-8 and MCP-3 as well as synthetic or natural Regakine-1 failed to induce neutrophil chemokinesis (Table 1). Furthermore, preincubation of the neutrophils for 10 minutes with either MCP-3 or Regakine-1 did not induce chemokinesis (Table 1). However, under the same conditions neutrophils responded chemotactically to IL-8 (at 15 and 50 ng/mL) when added in the lower compartment of the chamber (data not shown).
In the next experimental setting, Regakine-1 and IL-8 were verified for
their cooperative effect in the chemotaxis assay (Figure 6). When applied together in the lower
compartment of the microchamber, the chemotactic response toward
suboptimal doses of IL-8 (1-10 ng/mL) was further enhanced by
physiologic concentrations (100-300 ng/mL) of Regakine-1 (Figure 6A).
For example, when 300 ng/mL Regakine-1 was combined with 3 or 10 ng/mL
IL-8, a 3-fold increase in chemotactic response was observed, ie, a
3-fold enhancement in the number of migrated cells above the additive
effect of both chemokines when tested separately
(P < .05). Similarly, a combination of 100 ng/mL
Regakine-1 and fMLP at 10
Furthermore, an enhanced chemotactic response toward IL-8 was
obtained when Regakine-1 was added together with the cells in the upper
compartment of the chamber. Indeed, when neutrophils were preincubated
for 10 minutes with 100 ng/mL Regakine-1 before transfer to the
chamber, the chemotactic response toward 10 or 30 ng/mL IL-8 was
increased 3-fold (Figure 6B). Comparable data (Table
2) were obtained when neutrophils were
treated with synthetic (300 ng/mL) instead of natural Regakine-1 to
enhance the chemotactic effect of IL-8 (5 or 15 ng/mL) as well as of
GCP-2 (15 or 50 ng/mL). On average, a 2.5-fold enhancement of the CI of
both GCP-2 and IL-8 was observed by preincubation of neutrophils with
Regakine-1 (Table 2). Addition of Regakine-1 to the cells did not
induce cell migration (CI 1.3 ± 0.2) toward dilution buffer in the
lower microchamber compartment. This indicates that, irrespective of the presence of a chemotactic gradient for Regakine-1, this chemokine is capable of enhancing the migration capacity of neutrophils toward
CXC chemokines.
Synergy between Regakine-1 and MCP-3 in lymphocyte chemotaxis To further explore the cooperation between Regakine-1 and other chemoattractants, the former CC chemokine was combined in the lower wells of the microchamber with MCP-3 to stimulate lymphocyte chemotaxis. Figure 7 illustrates that 100 to 300 ng/mL Regakine-1 together with a suboptimal concentration of MCP-3 (3 ng/mL) resulted in a 2-fold increase in lymphocyte chemotactic response above the additive effect of the individual chemokines. However, at an optimal concentration of MCP-3 (30 ng/mL), Regakine-1 failed to further enhance the efficacy of MCP-3 as a lymphocyte chemoattractant. Furthermore, at an inactive concentration (30 ng/mL), Regakine-1 failed to synergize with MCP-3. Taken together, these data demonstrate that the synergistic action of Regakine-1 is not restricted to neutrophil chemotaxis but is also effective between members of the same CC chemokine subfamily in lymphocyte chemotaxis. This indicates that different receptors or signal transduction pathways are implicated.
Chemotactic cytokines or chemokines form a large family of selective leukocyte chemoattractants. CXC chemokines predominantly stimulate the migration of neutrophils or lymphocytes, whereas CC chemokines attract one or more leukocytic cell types, including monocytes, dendritic cells, lymphocytes, natural killer cells, eosinophils, and basophils.1-5 For most chemokines their biologic selectivity can be explained by binding and signaling through cell-specific G protein-coupled 7-transmembrane domain receptors.6 This study describes the isolation and identification of a novel CC chemokine derived from serum, often used to support cell viability or proliferation. The 7.5-kd protein was purified to homogeneity from fetal and newborn calf serum, and its primary structure was elucidated by mass spectrometry and NH2- and COOH-terminal amino acid sequence analysis on peptide fragments. Because its amino acid sequence did not show more than 50% identity with any known human or bovine chemokine, this CC chemokine was tentatively designated Regakine-1. Natural Regakine-1 exerted chemotactic activity for neutrophils and lymphocytes, 300 ng/mL being the minimal effective concentration. However, Regakine-1 was found to be abundantly present (about 100 ng/mL) in fetal, newborn, and adult bovine serum. Contamination of natural Regakine-1 preparations with other neutrophil- or lymphocyte-attracting chemokines is excluded because the chemotactic activity of natural Regakine-1 was confirmed with chemically synthesized protein. Regakine-1 did not show chemotactic activity for monocytes or eosinophils at concentrations up to 1 µg/mL. The CC chemokine did not exert chemokinetic activity but enhanced the neutrophil and lymphocyte chemotactic response to CXC chemokines (IL-8 and GCP-2) and CC chemokines (MCP-3), respectively. Indeed, when Regakine-1 was combined with IL-8 or MCP-3, the number of migrated cells increased at least 2-fold compared with the cumulative effect of these individual chemokines. Other human plasma proteins such as platelet factor-4 (PF-4) and
The rather weak chemotactic activity of Regakine-1, a characteristic
feature shared with PF-4, The exact biologic significance of the known plasma chemokines remains
controversial. The weak neutrophil-degranulating activity of PF-4 was
significantly increased by preincubation or coincubation of neutrophils
with tumor necrosis factor- Post-translational modification of chemokines can enhance or
reduce their chemotactic potency. For some CC chemokines, cleavage of
the NH2-terminal dipeptide by the dipeptidyl peptidase
IV/CD26 resulted in reduced receptor recognition and hence impaired
chemotactic activity.39 In contrast, such processing of
the macrophage inflammatory protein-1 isoform LD78 In conclusion, the identification of a novel CC chemokine with low sequence identity to any known chemokine highlights the paradox of apparent redundancy within the family of chemotactic cytokines. This phenomenon, resulting in overlapping activities, provides robustness to the chemokine network and guarantees effective immune reactions during host defense.3 An efficient and successful immune response to infection depends on endogenous and exogenous mediators that strictly regulate the production of individual chemokines by multiple cellular sources. Enhanced expression and activity of inflammatory chemokines is well controlled to prevent tissue damage. Therefore, these chemokines are only detected at high concentrations in the circulation during severe acute infections, eg, septic shock.43 In contrast to these inducible chemokines, chemokines that are constitutively produced at low levels probably fulfill homeostatic functions, eg, the regulation of leukocyte traffic under physiologic conditions.5 In this context, the constant high concentration of Regakine-1 and HCC-1 in the circulation seems to be an exception. It can at present only be speculated that Regakine-1 has a function in hematopoiesis, leukocyte homing, or angiogenesis, complex processes in which the role for chemokines has only recently been investigated.
The authors thank the members of the Laboratory of Clinical Immunology of the University of Leuven and Dr F. Shyselinck and the members of the Blood Transfusion Center of Leuven and Antwerp for providing blood samples and buffy coats. The editorial help of D. Brabants and I. Aerts, the technical assistance of R. Conings and W. Put, and the critical reading of this manuscript by Prof G. Opdenakker are greatly appreciated.
Submitted June 23, 2000; accepted November 30, 2000.
Supported by the Fund for Scientific Research of Belgium (FWO-Vlaanderen), the Concerted Research Actions of the Regional Government of Flanders, the InterUniversity Attraction Pole Initiative of the Belgian Federal Government, and the Biomed and Biotech Program of the European Community.
P.P. and S.S. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jo Van Damme, Rega Institute, Minderbroedersstraat 10, B-3000 Leuven, Belgium; e-mail: jozef.vandamme{at}rega.kuleuven.ac.be.
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© 2001 by The American Society of Hematology.
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M. Gouwy, S. Struyf, F. Mahieu, W. Put, P. Proost, and J. Van Damme The Unique Property of the CC Chemokine Regakine-1 to Synergize with Other Plasma-Derived Inflammatory Mediators in Neutrophil Chemotaxis Does Not Reside in Its NH2-Terminal Structure Mol. Pharmacol., July 1, 2002; 62(1): 173 - 180. [Abstract] [Full Text] [PDF]
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L. Michalec, B. K. Choudhury, E. Postlethwait, J. S. Wild, R. Alam, M. Lett-Brown, and S. Sur CCL7 and CXCL10 Orchestrate Oxidative Stress-Induced Neutrophilic Lung Inflammation J. Immunol., January 15, 2002; 168(2): 846 - 852. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-thromboglobulin. Regakine-1 did not induce neutrophil
chemokinesis. However, it synergized with the CXC chemokines interleukin-8 and granulocyte chemotactic protein-2, and the CC chemokine monocyte chemotactic protein-3, resulting in an at least a
2-fold increase of the neutrophil and lymphocyte chemotactic response,
respectively. The biologic effects of homogeneous natural Regakine-1
were confirmed with chemically synthesized chemokine. Like other plasma
chemokines, it is expected that Regakine-1 plays a unique role in the
circulation during normal or pathologic conditions.
(Blood. 2001;97:2197-2204)
-
-
) were determined at dilution 1/50 and represent the mean CI ± SEM of 5 independent experiments. HPLC fractions containing
neutrophil chemotactic activity were analyzed by SDS-PAGE (C) under
reducing conditions (fractions 32 to 43, 4 µL/lane). The proteins
were visualized by silver staining. The left lane shows Mr
markers (see "Materials and methods").
9 M synergized in the neutrophil
chemotaxis assay, yielding a 5-fold increase in CI (89 ± 29,
n = 4, P < .05) compared with the additive effect of
Regakine-1 (5.6 ± 2.0, n = 4) and fMLP (11.3 ± 3.1, n = 4).
-granules of activated blood platelets, were structurally identified more than 20 years ago.