| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,,||,
* Department of Immunology,
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland;
Stanford University Medical School, Stanford, CA 94305;
Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304;
¶ Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093; and
|| Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Chemerin, also known as tazarotene-induced gene 2, has been recently characterized as a ligand for the seven transmembrane, G protein-coupled receptor CMKLR1 (also known as ChemR23 in humans and DEZ in mice). Chemerin mRNA is present in most tissues, including liver, pancreas, and skin (7, 8). The liver, however, appears to be a primary source of chemerin, and is likely to be responsible for the high levels of this protein in plasma (8, 9). Chemerin circulates as an inactive precursor (prochemerin) in blood and requires proteolytic processing for activation. Recently, we and others have demonstrated that active chemerin is generated by serine proteases of the inflammatory, coagulation and fibrinolytic cascades (9, 10). These include neutrophil-derived elastase and cathepsin G, mast cell tryptase, as well as factors VIIa, XIIa, tissue plasminogen activator, urokinase-type plasminogen activator, and plasmin. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, such as immature plasmacytoid dendritic cells (pDCs)4 and macrophages that express CMKLR1 (7, 8, 9, 10, 11) providing a potential mechanism for controlling immune responses at sites of inflammation or tissue injury.
With the growing prevalence of bacteria-mediated chronic inflammatory diseases (12, 13, 14, 15), there is a need to define the mechanisms underlying recruitment of Ag presenting and immune regulatory cells to sites of bacteria infection. Staphylococcus aureus is among the most successful of human pathogens. It is a major cause of skin and soft tissue infections and has been implicated in sepsis as well as in several chronic inflammatory disorders, including atopic dermatitis and psoriasis (15). The success of S. aureus as a pathogen may reflect an ability to regulate the recruitment and modulate the function of specialized host cells.
In this work, we demonstrate that the secreted cysteine protease staphopain B (SspB), a potential virulence factor that is likely to contribute to the chronicity of S. aureus infections, is a potent activator of chemerin. Processing of chemerin by SspB may therefore contribute to the pathologic inflammatory response to staphylococcal invasion through recruitment of immunomodulatory pDC and/or macrophages to sites of infection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Wild-type S. aureus strains 8325-4 and COL (16, 17), and isogenic knockout mutants of individual protease genes in the 8325-4 genetic background were used in this study. Staphopain A (
ScpA), SspB (SspB), SspB and V8 protease (SspBV8), and aureolysin (Aur) deficient strains (18) were obtained from Dr. L. Shaw (Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA) while the Spl proteases (Spl) mutant (19) was a gift from Dr. K. Bayles (Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID). Collection of clinical isolates of S. aureus sampled from joint, blood, and lesional skin of patients suffering from bone infection, sepsis, or atopic dermatitis were donated by Dr. S. Eick (Institute of Medical Microbiology, University Hospital, Jena, Germany) and Dr. M. Kapinska-Mrowiecka (Zeromski General Hospital, Krakow, Poland). The S. aureus strains were grown in tryptic soy broth (Sigma-Aldrich) at 37°C with shaking at 200 rpm for 14 h. The bacterial density was measured spectrophotometrically and the cell number was calculated by using previously determined standard curves. Typically, the bacterial OD (OD600) was within 8–10. No major growth differences were observed among S. aureus strains as confirmed by serial dilution and CFU counting on tryptic soy agar. The bacteria were centrifuged at 5000 x g for 15 min and conditioned medium was collected, diluted with tryptic soy broth to OD600 = 7, and used in chemotaxis assays and zymography.
Recombinant prochemerin
Prochemerin with a C-terminal HIS6 tag was cloned into pACGP67 (BD Biosciences) and transfected into Sf-9 cells. The expressed protein has the sequence NH2-ADPELTEA... LPRSPHHHHHH-COOH, where the underlined residues are nonnative. After viral amplification, prochemerin was expressed by adding high titer virus to shaker flasks containing Hi-5 insect cells in Ex-cell 405 medium (JRH Biosciences). After incubation for 2–3 days at 27.5°C, the supernatant was harvested by centrifugation, filtered to 0.22 µm, and concentrated at 4°C using a tangential flow concentrator (Filtron) with a 3-kDa cutoff filter. After a >100-fold buffer exchange into 50 mM HEPES, 0.3 M NaCl (pH 8.0), prochemerin was purified by running the solution over nickel-nitrilotriacetic acid (Amersham Biosciences) and C-18 reverse-phase HPLC columns (Vydac). The protein was lyophilized and checked for purity using electrospray mass spectrometry (9).
Protease purification
S. aureus V8 protease (glutamylendopeptidase), Aur, and SspB were purified as previously described (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Proteolytically inactive SspB (generated by replacing Cys24 with Ala (23)) was a gift from Dr. R. Filipek (International Institute of Molecular and Cell Biology, Warsaw, Poland).
Cell culture
Murine pre-B lymphoma cell line L1.2 and L.1.2 cells stably transfected with human recombinant CMKLR1 (CMKLR1/L1.2) (8) were cultured in RPMI 1640 supplemented with 50 µg/ml gentamicin and 10% FBS. The medium for CMKLR1/L1.2 transfectants was also supplemented with 1 mg/ml geneticin.
Chemotaxis assay
Growth medium derived from S. aureus, or purified staphylococcal proteases, were incubated with recombinant human prochemerin or human plasma for 10 min at 37°C and then tested in an in vitro chemotaxis assay using 5-µm pore Transwell inserts (Costar). In each case, enzymatic digestion was stopped by placing samples on ice and diluting them 1/15 into chemotaxis medium (RPMI 1640 containing 10% FBS). A total of 100 µl of cells (2.5 x 105 cells/well) were added to the top well and tested samples were added to the bottom well in a 600 µl volume. Migration was assayed for 2 h at 37°C. The inserts were then removed and cells that had migrated through the filter to the lower chamber were collected and counted by flow cytometry (FACSCalibur; BD Biosciences). The results are presented as percent input migration.
Human blood pDC transendothelial migration
The Institutional Review Board (Stanford University) approved all human subject protocols and informed consent was obtained for all donations. Human blood was collected and PBMC were harvested following Histopaque 1077 gradient separation. Total PBMC were preincubated 1 h in chemotaxis medium (RPMI 1640 plus 10% FCS, supplemented with L-glutamine, penicillin/streptomycin, sodium pyruvate, and nonessential amino acids) at 107 cells/ml to allow for recovery of receptor expression. Transwell inserts (5-µm pure; Costar) were coated with 2% gelatin and seeded with 105 HUVECs (passage <5), and incubated for 24–48 h. Confluent HUVEC monolayers were confirmed by DiffQuick staining (IMEB). Monolayers were rinsed with chemotaxis medium before use. A total of 106 PBMC (100 µl) were added to the top wells, and various concentrations of chemoattractants were added to the bottom wells in a 600-µl volume. Migration was assayed for 2 h at 37°C, then the inserts were removed, and the cells that had migrated through the filter to the lower chamber were harvested, stained (Lin FITC (CD3, CD14, CD16, CD19, CD20, CD56), CD11c PE, CD123 CyChrome, HLA-DR allophycocyanin; mAbs from BD Pharmingen and eBioscience), and analyzed by flow cytometry. An equivalent number of beads were added to each tube to allow the cell count to be normalized.
Intracellular calcium mobilization
Chemoattractant-stimulated Ca2+ mobilization was performed following Alliance for Cell Signaling protocol ID PP00000210. CMKLR1/L1.2 transfectants (5 x 106/ml) were loaded with 4 µM Fluo4-AM, 0.16% pluronic acid F-127 (Invitrogen Life Technologies) in modified Iscove’s medium (Invitrogen Life Technologies) plus 1% FBS for 30 min at 37°C. The samples were mixed every 10 min during loading, washed once, resuspended at 2 x 106/ml in the same buffer, and allowed to rest at room temperature in the dark for 20 min. Changes in Fluo4 fluorescence in the labeled cells following treatment with chemoattractants were measured over real-time with a FACScan flow cytometer and CellQuest software (BD Biosciences) at room temperature under stirring conditions (500 rpm). Fluorescent data were acquired continuously up to 512 s at 1-s intervals. The samples were analyzed for 45 s to establish their basal state, then removed from the nozzle for addition of stimuli, then returned to the nozzle for continued data acquisition. Mean channel fluorescence over time was analyzed with FlowJo software (Tree Star).
SDS-PAGE
Recombinant prochemerin was incubated with pure SspB at a 2:1 molar ratio at 37°C for 10 min in a buffer containing 50 mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT. The enzymatic digestion was stopped by addition of the SDS gel loading buffer. Samples were resolved by SDS-PAGE (13%) under nonreducing conditions. Bands were then visualized using the Coomassie G250 stain (Bio-Rad).
Gelatin zymography
Bacteria growth medium were resolved by SDS-PAGE (8%) containing 0.1% gelatin (type I from porcine skin; Sigma-Aldrich). After electrophoresis, the gel was washed in 2.5% Triton X-100 for 30 min at room temperature, followed by 40-h incubation at 37°C in buffer containing 50 mM Tris (pH 7.7), 5 mM CaCl2 and 0.02% NaN3. The gel was then stained with Coomassie to allow the bands of proteolytic activity to be detected as clear bands of lysis against a stained background.
Purification of chemerin fragments by reverse-phase HPLC
For analytical applications, recombinant prochemerin was incubated with pure SspB at 2:1 molar ratio at 37°C for 10 min in a buffer containing 50 mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT. The reaction was stopped by addition of trifluoroacetic acid (TFA) to a final concentration of 0.5%. Chemerin cleavage products were isolated using a Waters 501 HPLC System on a µBondapak C18 column (3.9 x 300 mm) also obtained from Waters. The column was run for 40 min with a linear gradient from 0 to 80% acetonitrile in water containing 0.1% TFA at a flow rate of 1 ml/min. Absorbance at 220 nm was monitored to detect the eluting peptides. Fractions containing peptides were collected, flash-frozen in liquid nitrogen, and lyophilized.
For preparative applications, prochemerin was resuspended and mixed with SspB at a 100:1 prochemerin:active SspB molar ratio at 37°C. The final buffer conditions for cleavage were 50 mM HEPES, 100 mM NaCl (pH 7.2), and prochemerin samples were cleaved at a concentration of 1 mg/ml. Samples containing 2 mg of prochemerin were removed at 150 min and the components present were separated using a semipreparative C18 Reverse-Phase HPLC column (Grace Vydac), using a gradient of 25–90% acetonitrile in 0.1% TFA. Fractions from the HPLC purification were lyophilized and identified using electrospray mass spectrometry.
MALDI-TOF mass spectrometry
The HPLC-purified chemerin fragments were suspended in 10 µl of 0.1% TFA in water and mixed 1:1 (v/v) with saturated solutions of either 
-cyano-4-hydroxycinnaminic acid or 3,5-dimetoksy-4-hydroxycinnaminic acid, both in water- acetonitrile (2:1; v/v). Samples were then applied to a stainless steel target plate, allowed to dry, and analyzed on a MALDI-TOF Reflex IV mass spectrometer (Brüker Daltonics) operated in a positive-ion mode.
Serum and plasma collection
The Institutional Review Board at Jagiellonian University approved all human subject protocols. Plasma and serum were obtained from whole blood of healthy individuals. To prevent clotting when purifying plasma, blood was collected in the presence of sodium citrate. The blood was then centrifuged once at 400 x g for 10 min followed by two centrifugations at 2880 x g for 15 min to separate the plasma from leukocytes and platelets. Serum was obtained by allowing the blood to clot for 1 h at 37°C before centrifugation twice at 2880 x g for 15 min.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
To determine whether proteolytic enzymes produced by the human pathogen S. aureus generate active chemerin, bacteria growth medium was incubated with recombinant full-length (proform) chemerin. As demonstrated in Fig. 1A, CMKLR1-transfected L1.2 cells migrated significantly in response to prochemerin treated with S. aureus growth medium. No cell migration was detected when the growth medium was tested in the absence of prochemerin, or when prochemerin was tested alone, indicating that prochemerin is activated by factors secreted by S. aureus.
|
SspB) and in the double-deficient SspBV8 mutant. Generation of active chemerin was also significantly diminished in the absence of Aur (Aur). In contrast, deficiency of the Spl proteases (Spl), or ScpA (ScpA), did not have a significant effect on chemerin activation. These data indicate that SspB and to a lesser extent Aur are responsible for S. aureus-mediated chemerin activation. Because Aur contributes to the activation of the V8 protease, which in turn activates SspB (26, 27, 28), the lack of Aur or V8 protease may indirectly affect chemerin activation by reducing the levels of functional SspB produced by these mutants. To determine whether Aur and the V8 protease are capable of activating chemerin directly, we incubated recombinant prochemerin with purified Aur, the V8 protease, and SspB. As demonstrated in Fig. 2A, while SspB was able to activate prochemerin, neither Aur nor the V8 protease were able to activate the chemoattractant. Thus, among enzymes secreted by S. aureus, SspB is an exclusive prochemerin-activating agent. Furthermore, purified SspB restored the ability of SspB-deficient medium to trigger the chemerin activity (Fig. 2B). This finding supports the notion that prochemerin activation by S. aureus growth medium results from a direct effect mediated by SspB and not from clonal or growth differences among staphylococcal strains. Moreover, purified, enzymatically inactive SspB (inSspB) protein, (the catalytic Cys to Ala mutant) (23) was unable to trigger prochemerin activation, confirming that proteolytic activity of SspB is required for S. aureus-mediated chemoattractant activation (Fig. 2A).
|
To determine the SspB-mediated cleavage site(s), recombinant prochemerin was incubated with purified SspB and then subjected to a separation by HPLC (Fig. 3A) or SDS-PAGE (Fig. 3B). As expected, coincubation of prochemerin with SspB resulted in limited proteolysis of the chemoattractant (Fig. 3B). HPLC fractions (labeled I-III) were analyzed by MALDI-TOF mass spectrometry (Fig. 3A, inset). Fraction I (1590.8 Da) corresponds to the C-terminal fragment of chemerin encompassing residues from 141 to 146, plus the proline linker and C-terminal HIS6 tag (Fig. 3, C and D). The larger portion of chemerin (16,155.4 Da) corresponding to residues 1–140, was collected in fractions II and III. Identification of two matching chemerin fragments corresponding to the C- and N-terminal portion of the molecule (1,590.8 and 16,155.4 Da, respectively) revealed the SspB-mediated chemerin cleavage site to be NH2..AFS
KAL..COOH (Fig. 3, C and D). Fraction II also contained an additional peptide of molecular mass 14,963.1 Da, corresponding to residues 1–130, indicating an additional cleavage event. In this case, however, the matching C-terminal peptide or its further degradation products could not be recovered. The 16,155.4 Da cleavage product of SspB, further referred to as "chem16", was the primary product observed under conditions when prochemerin was treated with SspB at 100:1 molar ratio for 150 min, in a buffer containing 50 mM HEPES, 100 mM NaCl (pH 7.2), or at 1000:1 molar ratio for 90 min. in a buffer containing 50 mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT (data not shown). Longer incubation resulted in further truncation of prochemerin and the generation of the 14,963.1 Da product, as described above and referred to as "chem15". Similar SspB cleavage fragments were detected when recombinant prochemerin lacking five of the six histidines in the C-terminal HIS6 tag was used, indicating that SspB-mediated prochemerin cleavage is not affected by the presence of the HIS6 tag (data not shown).
|
SspB-activated chemerin triggers a functional response in CMKLR1-positive cells
We characterized the bioactivity of the two SspB-generated chemerin isoforms using in vitro chemotaxis and calcium mobilization assays. Chem16 is a potent chemoattractant, eliciting a robust, dose-dependent chemotactic response in CMKLR1/L1.2 transfectants. Just 4 pM of chem16 was sufficient to induce a detectable migratory response, with 1 nM eliciting the maximal amplitude of migration (Fig. 4A). Chem16 also triggers intracellular calcium mobilization (Fig. 4B) in a dose-dependent fashion. In contrast, the bioactivity profile of chem15 was similar to the relatively inactive prochemerin in both chemotaxis and calcium flux assays (Fig. 4). Chem15 is a poor chemoattractant, requiring concentrations over 6000 times higher than chem16 to elicit the same magnitude of response (Fig. 4A). Taken together, these data suggest that SspB-mediated prochemerin cleavage results in generation of the biologically active chem16 form, which is then further truncated to an inactive chem15 form. To characterize any inhibitory effect chem15 may have on chem16, CMKLR1/L1.2 transfectants were simultaneously exposed to both isoforms. Chem15 did not inhibit the chemotactic response to chem16, even at a molar ratio of 500:1, respectively (Fig. 4C). We conclude that chem16 exerts biological effects on CMKLR1-expressing cells despite the presence of the truncated 15 kDa form.
|
|
To determine whether SspB can serve as the pathophysiologic trigger of chemerin activity, we evaluated sets of clinical S. aureus isolates for their ability to activate prochemerin. These included isolates from patients suffering from sepsis, septic arthritis and atopic dermatitis. The selected bacterial strains displayed proteolytic activity as evidenced by zymographic analysis of the growth medium (Fig. 6A). The effect of clinical isolates on the generation of active chemerin was compared with the effects of the widely used laboratory strains 8325-4 and COL. As demonstrated in Fig. 6B, chemerin activity was triggered by all three clinical isolates tested, as well as by strain 8325-4, but not by the laboratory strain COL. Strain 8325-4 was the most potent in inducing prochemerin activation, which is consistent with its high-level of proteolytic activity (18). Likewise, the lack of prochemerin activation by the COL strain can be explained by the negligible SspB proteolytic activity (Fig. 6A). These data indicate that SspB may be involved in generating a chemotactic gradient of chemerin at sites of S. aureus infection in humans.
|
Plasma represents a significant reservoir for proform chemerin in vivo, where it circulates at low nanomolar concentrations (3). Because plasma contains a high concentration of protease inhibitors (10% of total protein content), to evaluate the pathophysiologic relevance of chemerin activation by SspB it was important to determine whether or not SspB can process prochemerin in the presence of plasma inhibitors. As shown in Fig. 7, incubation of purified SspB with human plasma resulted in prochemerin activation, suggesting that the chemerin-activating potential of SspB is not blocked by plasma protease inhibitors. Moreover, the chemotactic effect of SspB-treated plasma on CMKLR1-expressing cells was comparable to the effect of serum (Fig. 7), which contains high levels of active chemerin that results from processing by serine proteases activated during clotting/fibrinolysis (9).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Several lines of evidence from our present work indicate that SspB may have a physiologically relevant role in chemerin activation. First, endogenous SspB released into S. aureus growth medium activates chemerin, suggesting that SspB triggers the chemerin chemotactic activity in the context of other S. aureus secreted enzymes. This is important, because high levels of proteolytic activity often result in protein cleavage at multiple sites, leading to nonspecific protein degradation. Second, SspB generates active chemerin with a six amino acid C-terminal truncation. This particular isoform is identical with an endogenous active human chemerin isoform isolated from ascites fluid (7). Third, the concentration of host proteases known to activate chemerin, including factor XII and VII, as well as plasmin, are in the range of 10–7–10–5 M (9). Therefore, the effect of SspB appears to be pathophysiologically significant because at the concentration of 1 x 10–7 M, SspB is a very effective trigger of chemerin activation in plasma, yielding chemotactic activity comparable to that generated by all combined host proteases released during blood coagulation and fibrinolysis. Fourth, active chemerin accumulates in SspB-treated plasma despite an abundance of various inhibitors that keep host-derived proteases under the tight control. Importantly, host proteases implicated thus far as chemerin activators, such as neutrophil elastase, cathepsin G, plasmin, urokinase-type plasminogen activator, and tissue plasminogen activator (9, 10) would be expected to be opposed by plasma inhibitors, including 1-protease inhibitor, 1-antichymotrypsin, 2-antiplasmin, and plasminogen activator inhibitor (30, 31, 32). At early stages of acute inflammation, the protease-antiprotease balance likely shifts in favor of proteolysis. However, as the inflammatory response continues, the plasma concentration of protease inhibitors increases (30, 31). Therefore, at later stages of the inflammatory response or during ongoing inflammation, host proteases are neutralized by plasma inhibitors. In contrast to host proteases, SspB remains active in plasma, suggesting that it can serve as a chemerin activator at any stage of the inflammatory response. Furthermore, SspB may contribute to chemerin processing during skin and soft tissue infections associated with significant edema, where plasma infiltrate is likely to be a primary source of prochemerin.
Collectively, our findings support the view that S. aureus-derived SspB, through the generation of active chemerin, may play a role in the selective recruitment of immature pDC and/or macrophages to S. aureus-infected inflammatory sites. Although SspB activity at S. aureus-infected sites has yet to be quantified, functional SspB is likely to be present within these lesions. Notably, inactivation of SspB and its processing enzyme, the V8 protease, results in attenuation of S. aureus virulence (18, 33). Thus while chemerin processing by SspB may provide a host distress "SOS signal" to initiate pDC and/or macrophage-mediated immunologic reactions, it is more likely to be an ongoing contributing factor to the pathological outcome of staphylococcal infections. S. aureus infection could lead to the chronic and excessive generation of local levels of active chemerin, resulting in the accumulation of immature pDC and macrophages and establishing a microenvironment permissive for chronic inflammation.
Although several theories have been put forward, it remains unclear why the host defense response is ineffective in clearing chronic S. aureus infections and how the organism contributes to the maintenance of a chronic inflammatory state (34). In this context, it is relevant that the two major cell subtypes that express CMKLR1, immature pDC and macrophages, are both multifunctional cell types that can contribute both to the generation and suppression of pathologic inflammation. Although a comprehensive analysis of pDC function remains incomplete, current data suggest that pDC may occupy a central stage as regulators of immune responses, able to modulate the functions of cells involved in both innate and acquired immunity, including NK, myeloid dendritic cells as well as T and B cells (35). Moreover, immature pDC can differentiate either into proinflammatory APC, capable for example of producing large amounts of type I IFNs in response to viral and microbial stimuli (36), or into potent inducers of T regulatory cells, thus suppressing local immune responses (37, 38, 39). Immature pDC circulate normally in blood and lymphoid organs, and are recruited to peripheral tissues during inflammation. Indeed, recent findings point to pDC as important contributors to chronic immune-based disorders such as lupus erythematosus and psoriasis (40, 41, 42, 43). Similarly, macrophages are emerging as malleable immune regulatory cells, associated with chronic inflammatory conditions, including atopic dermatitis (44). Thus the cells recruited by active chemerin are capable of complex regulation of immune responses. One possibility therefore is that S. aureus recruits these cells and manipulates their immunoregulatory potential to its own survival advantage. This would be consistent with the ability of S. aureus to both initiate and maintain a state of chronic pathologic inflammation.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported in part by grants from the Polish Ministry of Scientific Research 2P04A07629 (to J.C.), 158/E-338/SPB/5.PR UE/DZ 19/2003 (to J.P.), and 2P04A01129 (to J.P. and G.D.), by a Fogarty International Research Collaborative Award R03TW007174-01 (to E.C.B. and J.C.), a grant from the Commission of the European Communities, Programme QLRT-2001-01250, the European Social Fund and National Budget in the Frame of The Integrated Regional Operational Programme (to G.D.). G.D. is a recipient of the Polish Science Foundation Scholarship for young scientists. B.A.Z., T.O., and E.C.B. were supported by National Institutes of Health Grants AI-59635 and GM-37734. S.J.A. was supported by a postdoctoral fellowship from the Cancer Research Institute, New York. T.M.H. was supported by Grant AI37113-09 from the National Institutes of Health. 
2 P.K. and B.A.Z. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Joanna Cichy, Faculty of Biochemistry, Biophysics and Biotechnology, Ulica. Gronostajowa 7, 30-387 Krakow, Poland. E-mail address: Cichy{at}mol.uj.edu.pl
4 Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; SspB, staphopain B; Spl, serine protease-like protein; ScpA, staphopain A; Aur, aureolysin; TFA, trifluoroacetic acid; MS, mass spectrometry.
Received for publication July 28, 2006. Accepted for publication January 2, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
and TLR ligands. Exp. Hematol. 8: 1106-1114. 1-antichymotrypsin expression in human lung-derived epithelial cells. J. Clin. Invest. 95: 2729-2733. [Medline]1-proteinase inhibitor by human lung-derived epithelial cells. J. Biol. Chem. 272: 8250-8255. production. J. Exp. Med. 202: 135-143. . J. Invest. Dermatol. 125: xiv-xv. [Medline] in autoimmune diseases. Proc. Natl. Acad. Sci. USA 102: 3372-3377. This article has been cited by other articles:
|
|
 |
|
  C. Albanesi, C. Scarponi, S. Pallotta, R. Daniele, D. Bosisio, S. Madonna, P. Fortugno, S. Gonzalvo-Feo, J.-D. Franssen, M. Parmentier, et al. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment J. Exp. Med., January 16, 2009; 206(1): 249 - 258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-Y. Du, B. A. Zabel, T. Myles, S. J. Allen, T. M. Handel, P. P. Lee, E. C. Butcher, and L. L. Leung Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets J. Biol. Chem., January 9, 2009; 284(2): 751 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Guillabert, V. Wittamer, B. Bondue, V. Godot, V. Imbault, M. Parmentier, and D. Communi Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation J. Leukoc. Biol., December 1, 2008; 84(6): 1530 - 1538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tsuru and I. Kobayashi Multiple Genome Comparison within a Bacterial Species Reveals a Unit of Evolution Spanning Two Adjacent Genes in a Tandem Paralog Cluster Mol. Biol. Evol., November 1, 2008; 25(11): 2457 - 2473. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) strains: ScpA; SspB; V8; Aur; Spl. After the samples were diluted 15 times, CMKLR1/L1.2 migration was assessed by in vitro transwell chemotaxis. Cell migration to prochemerin alone was used as a control. The mean from duplicate wells of three or four experiments ± SD is shown. *, p < 0.05; **, p < 0.001 by Student’s t test comparing (A) prochemerin alone vs prochemerin + S. aureus GM or (B) comparing various protease-deficient mutants with WT.
