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CHEMOKINES
From the Laboratory of Molecular Immunology, Rega
Institute for Medical Research, University of Leuven, Leuven, Belgium.
Chemokines are mediators in inflammatory and autoimmune disorders.
Aminoterminal truncation of chemokines results in altered specific
activities and receptor recognition patterns. Truncated forms of the
CXC chemokine interleukin (IL)-8 are more active than full-length IL-8
(1-77), provided the Glu-Leu-Arg (ELR) motif remains intact. Here, a
positive feedback loop is demonstrated between gelatinase B, a major
secreted matrix metalloproteinase (MMP-9) from neutrophils, and IL-8,
the prototype chemokine active on neutrophils. Natural human neutrophil
progelatinase B was purified to homogeneity and activated by
stromelysin-1. Gelatinase B truncated IL-8(1-77) into IL-8(7-77),
resulting in a 10- to 27-fold higher potency in neutrophil activation,
as measured by the increase in intracellular Ca++
concentration, secretion of gelatinase B, and neutrophil chemotaxis. This potentiation correlated with enhanced binding to neutrophils and
increased signaling through CXC chemokine receptor-1 (CXCR1), but it
was significantly less pronounced on a CXCR2-expressing cell line.
Three other CXC chemokines Gelatinase B, or MMP-9, belongs to the growing
family of matrix metalloproteinases (MMP). It is produced mainly by
neutrophils but is also produced by various other blood-derived cell
types Chemokines are low-molecular-mass proteins that exert potent
chemoattractant activities on leukocytes and that have effects on
angiogenesis and hematopoiesis. Based on the position of the 4 conserved cysteine residues, chemokines have been grouped into 2 major
families. CXC chemokines with one residue between the first 2 cysteines
are active on neutrophils, lymphocytes, or both. CC chemokines
with 2 adjacent cysteines are active on a variety of cell types,
including monocytes, eosinophils, basophils, and lymphocytes.12,13
Human interleukin-8 (IL-8) was identified as a potent neutrophil
chemoattractant and a granulocytosis-promoting protein,14 and it became one of the best-characterized CXC chemokines. In front of
the first cysteine residue, IL-8 contains the Glu-Leu-Arg (ELR,
amino-acid sequence in 1-letter code) motif, essential for its
neutrophil chemotactic activity. Besides the chemoattractant activity
on neutrophils, IL-8 triggers neutrophils to release the content of
some of their granules.1
Different aminoterminal variants of natural IL-8 have been identified.
For instance, IL-8(1-77) (AVLPRSAKELRCQC...), and IL-8(6-77) (SAKELRCQC...) were characterized as the major forms derived from endothelial cells or fibroblasts and leukocytes, respectively. Additional natural forms are IL-8(-2-77) (EGAVLPR...), IL-8(7-77), IL-8(8-77), and IL-8(9-77).15 In general, the shorter
forms of IL-8 are more active than the full-length form.
Other members of the CXC chemokine family, containing the ELR-motif,
are granulocyte chemotactic protein-2 (GCP-2), epithelial-cell-derived neutrophil attractant-78 (ENA-78), GRO- By analogy with IL-8, different natural truncation variants of CC
chemokines have been isolated. A monocyte chemotactic protein-2 (MCP-2)
form, which lacks 6 aminoterminal residues, was identified as a
chemokine inhibitor.22 RANTES processed by dipeptidyl
peptidase IV/CD26 lacks 2 aminoterminal residues and is a chemokine
inhibitor with potent antihuman immunodeficiency virus
activity.23 We here describe the processing of several
chemokines by the secreted metalloproteinase gelatinase B, in contrast
to the proteolytic processing by the membrane-anchored serine protease CD26.
Purification of natural gelatinase B from human neutrophils
to homogeneity
Chemokines and chemokine receptors
Human embryonic kidney (HEK) cells, transfected with CXC chemokine receptor 1 (CXCR1) or CXCR2, were kindly supplied by Dr J. M. Wang (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD).28 HEK cells were cultured in DMEM (Life Technologies, Paisley, UK) with 10% fetal bovine serum and 800 µg/mL geneticin. Activation of gelatinase B by stromelysin-1 Stromelysin-1 (MMP-3; Biogenesis, Poole, UK) was activated with 2 mmol/L 4-aminophenylmercuric acetate (APMA) (from a 10× concentrated solution in dimethyl sulfoxide) for 5 hours at 37 °C, according to the instructions of the manufacturer. APMA was removed from the activated enzyme by ultrafiltration on membranes with a 10-kd cutoff (Millipore, Milford, MA) or by dialysis using the Slyde-A-Lyzer units (Pierce, Rockford, IL). Activation of 1 µmol/L gelatinase B was performed with 0.01 µmol/L APMA-activated stromelysin-1 for 4.5 hours in assay buffer at 37°C. The gelatinase B activity was controlled by gelatin zymography and by the conversion of the quenched fluorescent substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem, Bubendorf, Switzerland).29Digestion of chemokines with purified gelatinase B and detection of cleavage products Chemokines (4 µmol/L) were incubated with activated neutrophil gelatinase B (0.4 µmol/L) in assay buffer (see above) at 37°C for 20 hours (unless otherwise indicated). Control experiments were conducted under identical conditions without gelatinase B (but with 0.004 µmol/L activated MMP-3). Gelatinase B inhibition experiments were performed at 1.5 µmol/L chemokine and 0.2 to 0.5 µmol/L gelatinase B in the same buffer with the following individual reagents: 15 mmol/L EDTA, 2 mmol/L o-phenantrolin, 0.25 µmol/L recombinant human TIMP-1 (Calbiochem, La Jolla, CA), 2.6 µmol/L gelatinase B-inhibiting monoclonal antibody REGA-3G12,30 2 mmol/L pefabloc, 2 µg/mL E64, or 67 µg/mL aprotinin (Sigma, St Louis, MO). Chemokine cleavage products were analyzed by SDS-PAGE and separated by reverse-phase HPLC on a C8-column using a gradient of CH3CN; absorbance was recorded at 220 nm. Resultant peptides were identified by aminoterminal sequencing and mass spectrometry analysis (see below). Resultant peptide sequences are indicated by one-letter codes, and the number of the first residue derived from the mature proteinis indicated.
Mass spectrometry analysis of chemokines and chemokine cleavage products Chemokines and peptides were, if required, first desalted using the C18 ZIPTIP (Millipore) and were subsequently diluted in 50% acetonitrile/50% H2O/0.1% acetic acid. These peptide solutions were analyzed by electrospray mass spectrometry (MS) on an ion trap (Esquire-LC, Brucker, Germany) or on a quadrupole time-of-flight (QTOF-2; Micromass, Manchester, UK) apparatus. Sequencing of peptides was performed by tandem MS/MS on the ion trap apparatus. When C18 ZIPTIP was used for desalting chemokines, adducts were observed that were not present in preparations or samples prepared without the use of ZIPTIP; therefore, these adducts were not further considered. In addition, the aminoterminal sequence of specific chemokine peptides was further characterized by Edman degradation as described above.Competition for 125I-IL-8 binding to CXC receptors Purified granulocytes, HEK-CXCR1, or HEK-CXCR2 (2 × 106 cells in 200 µL) were incubated with 125I-IL-8(6-77) (10 nCi; Amersham, Uppsala, Sweden) and with various concentrations of unlabeled IL-8(1-77) or IL-8(7-77) for 2 hours at 4°C in phosphate-buffered saline with 20 g/L bovine serum albumin. After 3 subsequent washes with 1 mL phosphate-buffered saline and 20 g/L bovine serum albumin, the cell-bound radioactivity was measured with a Triathler -counter (PerkinElmer, Norwalk, CT).
Detection of intracellular Ca++ concentrations Intracellular Ca++ concentrations ([Ca++]i) were measured as described previously.18 Briefly, purified cells (107/mL) were loaded with the fluorescent indicator fura-2 (2.5 µmol/L fura-2/AM; Molecular Probes Europe BV, Leiden, The Netherlands) for 30 minutes at 37°C. After 2 washes, cells were stored on ice at 106 cells/mL for a maximum of 1.5 hours. After excitation at 340 and 380 nm, fura-2 fluorescence was detected at 510 nm in an LS50B luminescence spectrophotometer (PerkinElmer) and used for the calculation of the [Ca ++]i.In inhibition experiments, monoclonal anti-CXCR1 (R&D Systems, Minneapolis, MN) was added at a concentration of 15 µg/mL during loading of the cells with fura-2 and during subsequent storage on ice and analysis. Degranulation Aliquots of 5 × 106 purified granulocytes, derived from healthy human blood donors, were stimulated in heat-inactivated human plasma with various concentrations of IL-8(1-77) and IL-8(7-77) for 30 minutes at 37°C. Cells were removed by centrifugation, and gelatinase B in the supernatant was analyzed by gelatin substrate zymography and subsequently quantified by scanning densitometry.31 Background levels of gelatinase B-zymolysis were subtracted from the zymolysis data obtained after neutrophil stimulation with IL-8(1-77) or IL-8(7-77).Chemotaxis Chemotactic activities of IL-8(1-77) and IL-8(7-77) were compared in modified Boyden chemotaxis chambers as detailed previously.18 The chemotactic index was defined as the ratio of cells migrated toward the chemokine versus the cell numbers obtained with the excipiens.
Preparation of natural gelatinase B Human peripheral blood neutrophils were used to obtain natural gelatinase B devoid of gelatinase A contamination. Indeed, this cell type does not synthesize gelatinase A or TIMP, and it stores gelatinase B in its proenzyme form in granules. By degranulation, 3 forms of gelatinase B were released into the extracellular space: monomers, disulfide-linked homodimers, and heterodimers with NGAL.32 With the use of an NGAL monoclonal antibody, the NGAL complex was removed and an electrophoretically pure preparation of gelatinase B was obtained (Figure 1) and used for all chemokine-processing experiments.
Processing of chemokines by gelatinase B To investigate whether chemokines are processed by gelatinase B, different CXC and CC chemokines were incubated with pure activated neutrophil gelatinase B at 37°C for several time intervals (enzyme-substrate molar ratio, 1:10). Subsequently, the digestion products were identified by SDS-PAGE, aminoterminal sequencing, and mass spectrometry analysis. Two members of the CC chemokine family, RANTES and MCP-2, were not cleaved by gelatinase B (Figure 2). PF-4, an ELR-negative CXC chemokine, and GRO- , an ELR motif-containing CXC chemokine, were slowly
degraded by gelatinase B; the conversions were still incomplete after a
24-hour incubation period (Figure 2). By aminoterminal sequence
analysis, at least 2 gelatinase B cleavage sites could be identified in
GRO-, namely in front of Leu at position 7 (L7RCQCLQ...) and in front of Val at position 28 (V28KSPG...). CTAP-III was also degraded by gelatinase B
(Figures 2, 3), and multiple cleavage
sites were identified (Figure 4). To
obtain this detailed information, the CTAP-III peptides were fractionated by RP-HPLC, and the individual fractions were analyzed by
mass spectrometry (MS and MS/MS), protein sequence analysis, or both.
Incubation of natural or recombinant IL-8(1-77) with gelatinase B
resulted in the efficient removal of only 6 aminoterminal residues and,
thus, in the formation of IL-8(7-77) with A7KELR... as
the aminoterminal sequence (Figure 5,
Table 1). Both IL-8 forms, intact and
truncated, were analyzed by mass spectrometry, confirming the identity
of IL-8(1-77) and IL-8(7-77) and excluding carboxyterminal processing
of IL-8 by gelatinase B (Figure 6). IL-8(6-77) (S6AKELR...), present in the natural IL-8
preparations in addition to IL-8(1-77), was not converted by gelatinase
B to IL-8(7-77), which is consistent with the fact that gelatinase B is
not an exopeptidase (Table 1).
The kinetics of the cleavage of IL-8 and CTAP-III were also compared,
and IL-8 was found to be processed more efficiently, with IL-8(7-77)
already appearing after 1 hour of incubation and reaching 100%
conversion after 24 hours. In contrast, the first cleavage products of
CTAP-III appeared only after 2 hours, and the degradation was only
completed after 48 hours (Figure 7).
To ascertain further that the observed cleavages were performed by the metalloproteinase gelatinase B, various types of inhibition studies were performed (Figures 2,3,5). For instance, the conversions of natural or recombinant IL-8 preparations were inhibited by the matrix metalloproteinase inhibitors EDTA, o-phenantrolin, TIMP-1, and by a specific gelatinase B-inhibiting monoclonal antibody (REGA-3G12),30 but not by the inhibitors for serine (pefabloc, aprotinin) or thiol proteases (E64) (Figure 5). The IL-8 conversion was not caused by the activated stromelysin-1 (used to convert progelatinase B into active gelatinase B), because control incubations with stromelysin-1 did not result in substrate cleavage. Finally, progelatinase B in its latent form did not process IL-8, which illustrates that the activation of progelatinase B is necessary for aminoterminal clipping of IL-8. Effect of the processing by gelatinase B on the biologic activity of IL-8 Recombinant human IL-8(1-77) was converted by activated gelatinase B to IL-8(7-77), and the latter was repurified by reverse-phase HPLC. First, binding experiments were performed to compare the relative affinities of both IL-8 forms for their receptors on the surfaces of neutrophilic granulocytes. In comparison with intact IL-8(1-77), half-maximal binding of IL-8(7-77) on neutrophils was observed at concentrations more than 10 times lower (Figure 8). In addition, the biologic activities of both IL-8 forms, IL-8(1-77) and IL-8(7-77), were compared on neutrophilic granulocytes. Signal transduction by IL-8(1-77) and IL-8(7-77) through neutrophil receptors was analyzed by measuring the increase of [Ca++]i. It was found that IL-8(7-77) mobilized intracellular Ca++ at concentrations more than 20 times lower (minimal effective concentration, 0.018 nmol/L) than IL-8(1-77) (minimal effective concentration, 0.5 nmol/L) (Figure 8). Finally, degranulation and chemotaxis assays were performed on neutrophils (Figure 9). A significant release of gelatinase B was induced by IL-8(7-77) at 1 nmol/L, whereas 10 nmol/L IL-8(1-77) was required to obtain a similar effect. This difference in potency was even more pronounced in neutrophil chemotaxis assays. IL-8(7-77) induced neutrophil chemotaxis from 0.1 nmol/L onward, whereas the minimal effective concentration of IL-8(1-77) was 2.7 nmol/L. In all 4 assays (binding, [Ca++]i-increase, gelatinase B release, and chemotaxis), it was reproducibly shown that IL-8(7-77) was 14, 27, 10, and 27 times, respectively, more active on neutrophils than IL-8(1-77).
Because neutrophils have 2 IL-8 receptors, CXCR1 and
CXCR2,33 we analyzed the receptor-binding and
intracellular Ca++-mobilizing capacities of IL-8(1-77) and
IL-8(7-77) on both receptors separately. An anti-CXCR1 monoclonal
antibody was found to inhibit both IL-8 forms in the intracellular
Ca++-mobilization assay with neutrophils, showing that both
IL-8 forms use at least CXCR1 (data not shown). Binding of IL-8(1-77)
and IL-8(7-77) to CXCR1 and CXCR2 were compared on cell lines
separately transfected with either receptor (Figure
10), whereas the effects of both
ligands on intracellular Ca++-mobilization in CXCR
transfectants are documented in Figure
11. Our data on the receptor
transfectants are in line with the data on human neutrophils and
clearly show that both IL-8 forms use both receptor types. However, the
difference in binding and intracellular Ca++-mobilizing
capacity between IL-8(7-77) and IL-8(1-77) was 9-fold for CXCR1
transfectants, whereas for CXCR2 transfectants the difference (2.5- to
4-fold) between IL-8(7-77) and IL-8(1-77) was significantly less
pronounced (P < .05, Student t test) than
for CXCR1.
IL-8, the most potent neutrophil-activating chemokine, stimulates
the release of gelatinase B from neutrophils.1 Here it is
demonstrated that gelatinase B processes IL-8 and other CXC chemokines
and alters the specific activities and receptor usage. The best-known
substrates for gelatinase B are denatured collagens (gelatins).
Collagen types V and XI can also be cleaved by gelatinase B; however,
it is unclear whether gelatinase B can cleave native full-length type
IV collagen.34,35 Other extracellular matrix substrates
include aggrecan,36 link protein,37 and
elastin.38 Gelatinase B was also shown to degrade myelin
basic protein, resulting in the release of encephalitogenic
peptides.39 In addition to these structural components,
other gelatinase B substrates are functional proteins. These include
the serine-protease inhibitors We report for the first time that several CXC chemokines, but not the CC chemokines RANTES and MCP-2, are substrates for activated gelatinase B. CTAP-III was degraded by gelatinase B, and 7 different cleavage sites were identified. In contrast, IL-8(1-77) was aminoterminally truncated by gelatinase B to IL-8(7-77). The effects of this selective cleavage were reflected by the enhanced biologic activities of IL-8 and mediated more through CXCR1 and less through CXCR2. We propose that a more than 10-fold increase in neutrophil activation by truncated IL-8 has consequences not only for chemotaxis but for other processes mediated by IL-8, such as leukocytosis regulation, stem cell mobilization, and (septic) shock syndromes characterized by high circulating levels of IL-846 and gelatinase B.47 Different aminoterminally processed forms of IL-8 from natural sources have been previously described. Endothelial cells and fibroblasts produce mainly IL-8(1-77), whereas mononuclear leukocytes produce mainly IL-8(6-77).48-51 Other natural forms of IL-8 also exist, and these include IL-8(2-77), IL-8(7-77), IL-8(8-77), and IL-8(9-77).15,48 Thrombin50 and plasmin52 have been shown to convert IL-8(1-77) to IL-8(6-77), whereas proteinase-3 from neutrophils cleaves IL-8(1-77) to produce IL-8(8-77).53 In general, shorter forms of IL-8 have been shown to be more active than longer forms, provided that the ELR motif remains intact. Compared to IL-8(1-77), IL-8(6-77) was shown to be 2 to 10 times more active in binding to neutrophils and in inducing the adhesion of neutrophils, chemotaxis, and degranulation.50,53-55 IL-8(8-77) and IL-8(9-77) were shown to be even more active than IL-8(6-77), but shorter forms, lacking an intact ELR motif, are inactive.54 Here it is shown that natural and recombinant IL-8(1-77) can be
converted efficiently to IL-8(7-77) by activated neutrophil gelatinase
B. This conversion is physiologically relevant because IL-8(7-77) has
been recovered from leukocytes and fibroblasts15,48 and
because IL-8 is able to induce degranulation of the specific and
gelatinase B-containing granules from neutrophils.1,56 In
contrast, azurophilic granules In addition, IL-8(7-77) was found to be 10 to 27 times more
active on human neutrophils than IL-8(1-77), as shown in binding experiments, [Ca++]i measurements,
degranulation, and chemotaxis assays. This difference is more
pronounced than that found by Nourshargh et al55 with IL-8(6-77) in comparison with IL-8(1-77) on rabbit neutrophils, but it
is similar to the difference described by Schröder et al51 with IL-8(6-77) and IL-8(1-77) on human neutrophils.
Because neutrophils contain both IL-8 receptors, CXCR1 and CXCR2, and because it was not yet known on which receptor the shorter IL-8 forms
are more active, we also studied the Ca++-mobilizing
activities of IL-8(1-77) and IL-8(7-77) in cell lines transfected with
CXCR1 or CXCR2. In CXCR1 transfectants, the difference (9-fold) in
binding and Ca++-mobilization between IL-8(1-77) and
IL-8(7-77) is in the same order as the difference observed in
neutrophils. However, on CXCR2-transfected cells, IL-8(7-77) is only
about 2.5 to 4 times more active than IL-8(1-77). Although it is
uncertain that the binding of the chemokine to the receptors and the
signal transduction are the same in normal cells as in transfectants,
our results suggest that the aminoterminal truncation of IL-8 mainly
affects binding and signaling through CXCR1. The biologic effect of the
cleavage of IL-8 by gelatinase B may, therefore, be different for
various target cell types. Indeed, some cell types are shown to contain
predominantly one of either of the IL-8 receptors. For instance, the
chemotaxis of mast cells in response to IL-8 is mediated through CXCR2
and not through CXCR1.57 Moreover, different functions
have been ascribed to the 2 CXC chemokine-receptors on neutrophils.
This implies that the effect of the truncation of IL-8 on its activity may vary, depending on the IL-8-induced function. For instance, though
chemotaxis in response to GRO- Proteolytic activity of gelatinase B toward other chemokine substrates
was also investigated. The CXC chemokines PF-4, GRO- The neutrophil is a terminally differentiated phagocyte that contains enzymes for target cell killing and for degradation of extracellular matrix components. Activation of neutrophils by CXC chemokines such as IL-8 leads to the secretion of proteases including gelatinase B. Our results show that the predominant member of the matrix metalloproteinases in neutrophils, gelatinase B, also contributes to enhanced IL-8 activity by a positive feedback loop. This finding is not only relevant to better understand physiopathological processes such as pyogenic infections and sepsis, it is relevant for clinical applications such as stem cell mobilization, in which both gelatinase B65 and IL-866,67 are involved.
We thank Sofie Struyf for her scientific contributions and René Conings, Jean-Pierre Lenaerts, and Willy Put for experimental help. We thank the F.W.O.-Vlaanderen for its support in acquiring the mass spectrometry equipment.
Submitted October 11, 1999; accepted June 6, 2000.
Supported by the Fortis Insurances AB, the Charcot Foundation, the "Geconcerteerde OnderzoeksActies," and the National Fund for Scientific Research (F.W.O.-Vlaanderen), Belgium. P.V.d.S. is a research assistant and P.P. is a postdoctoral fellow of the F.W.O.-Vlaanderen.
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: Ghislain Opdenakker, Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
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TNF- |
and leaves RANTES and MCP-2 intact
Top
Abstract
Introduction
, GRO-
, incubation with stromelysin-1 only; +, incubation with
activated gelatinase B. Respective inhibitors are indicated at the top
of each lane.
