Destruction of components of the extracellular matrix of the lung by neutrophil elastase is believed to be a critical event in the development of obstructive lung disease. The local synthesis of α1-proteinase inhibitor, the controlling inhibitor of this enzyme, may provide a partial mechanism for neutrophil elastase regulation, especially during inflammation, when proteolytic enzymes are released from phagocytes. In this study, we show that lung-derived epithelial cells not only have the capacity to synthesize functional α1-PI but also to increase the rate of its production when stimulated by specific inflammatory mediators, including oncostatin M, interleukin-1, and the glucocorticoid analogue, dexamethasone. Destruction of components of the extracellular matrix of the lung by neutrophil elastase is believed to be a critical event in the development of obstructive lung disease. The local synthesis of α1-proteinase inhibitor, the controlling inhibitor of this enzyme, may provide a partial mechanism for neutrophil elastase regulation, especially during inflammation, when proteolytic enzymes are released from phagocytes. In this study, we show that lung-derived epithelial cells not only have the capacity to synthesize functional α1-PI but also to increase the rate of its production when stimulated by specific inflammatory mediators, including oncostatin M, interleukin-1, and the glucocorticoid analogue, dexamethasone.
Plasmacytoid dendritic cells (pDCs) and neutrophils are detected in psoriatic skin lesions and implicated in the pathogenesis of psoriasis. pDCs specialize in the production of type I interferon (IFNI), a cytokine that plays an important role in chronic autoimmune-like inflammation, including psoriasis. Here, we demonstrate that IFNI production in pDCs is stimulated by DNA structures containing the neutrophil serine protease cathepsin G (CatG) and the secretory leukocyte protease inhibitor (SLPI), which is a controlling inhibitor of serine proteases. We also demonstrate the presence of neutrophil-derived DNA structures containing CatG and SLPI in lesional skin samples from psoriasis patients. These findings suggest a previously unappreciated role for CatG in psoriasis by linking CatG and its inhibitor SLPI to the IFNI-dependent regulation of immune responses by pDCs in psoriatic skin.
Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.
Congenital neutropenia, which refers to an inherited deficiency in neutrophils, is a rare pathologic condition that affects approximately 0.0001-0.0009% of the general population. While congenital neutropenia can result from mutations in approximately 30 genes, its leading cause is gain-of-function mutations in the ELANE gene, which encodes the neutrophil granule serine protease, neutrophil elastase. This review focuses on established and novel concepts in the genetic, molecular and cellular mechanisms underlying neutrophil elastase-dependent neutropenia, and discusses possible new avenues for neutropenia research as well as potential novel treatment options that target pathogenic elastase variants.
CD44 is a receptor for the matrix glycosaminoglycan hyaluronan. Proteoglycan forms of CD44 also exhibit affinity for fibronectin and collagen as well as chemokines and growth factors. CD44 plays a role in autoimmunity, inflammation, and tumor progression. Soluble CD44 (sCD44) is found in plasma, and the levels of sCD44 correlate with immune function and some malignancies. The mechanisms by which sCD44 is generated and its function are unknown. We demonstrate here that normal bronchial epithelial cells spontaneously release sCD44. Exposure to phagocyte- and bacterium-derived proteinases markedly increased the release of sCD44 from epithelial cells. The spontaneously released sCD44 was incorporated into high molecular mass complexes derived from the matrix that also contained chondroitin sulfate, fibronectin, hyaluronan, and collagens I and IV. Enzymatic digestion with proteinases liberated sCD44 from the high molecular mass complex. Consistent with the homology of CD44 to proteoglycan core and link proteins, these data suggest that CD44 spontaneously released from normal bronchial epithelial cells can accumulate as an integral component of the matrix, where it may play a role in the organization of matrices and in anchoring growth factors and chemokines to the matrix. Increases in plasma CD44 during immune activation and tumor progression therefore may be a manifestation of the matrix remodeling that occurs in the face of the enhanced proteolytic activity associated with infection, inflammation, and tumor metastasis, leading to alterations in cell-matrix interactions. CD44 is a receptor for the matrix glycosaminoglycan hyaluronan. Proteoglycan forms of CD44 also exhibit affinity for fibronectin and collagen as well as chemokines and growth factors. CD44 plays a role in autoimmunity, inflammation, and tumor progression. Soluble CD44 (sCD44) is found in plasma, and the levels of sCD44 correlate with immune function and some malignancies. The mechanisms by which sCD44 is generated and its function are unknown. We demonstrate here that normal bronchial epithelial cells spontaneously release sCD44. Exposure to phagocyte- and bacterium-derived proteinases markedly increased the release of sCD44 from epithelial cells. The spontaneously released sCD44 was incorporated into high molecular mass complexes derived from the matrix that also contained chondroitin sulfate, fibronectin, hyaluronan, and collagens I and IV. Enzymatic digestion with proteinases liberated sCD44 from the high molecular mass complex. Consistent with the homology of CD44 to proteoglycan core and link proteins, these data suggest that CD44 spontaneously released from normal bronchial epithelial cells can accumulate as an integral component of the matrix, where it may play a role in the organization of matrices and in anchoring growth factors and chemokines to the matrix. Increases in plasma CD44 during immune activation and tumor progression therefore may be a manifestation of the matrix remodeling that occurs in the face of the enhanced proteolytic activity associated with infection, inflammation, and tumor metastasis, leading to alterations in cell-matrix interactions. Cell-matrix interactions are critical for determination of cellular behavior. Some cells exhibit an extensive, highly organized pericellular matrix associated with the cell surface (1Knudson W. Aguiar D.J. Hua Q. Knudson C.B. Exp. Cell Res. 1996; 228: 216-228Crossref PubMed Scopus (169) Google Scholar). On the other hand, the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; sCD44, soluble CD44; NBEC, normal bronchial epithelial cell(s); FITC, fluorescein isothiocyanate; PE, phycoerythrin; SAMP, S. aureus metalloproteinase (aureolysin); PAMP, P. aeruginosa metalloproteinase (pseudolysin); mAb, monoclonal antibody; bPG, biotinylated bovine HA-binding protein; ELISA, enzyme-linked immunosorbent assay; ICAM-1, intercellular adhesion molecule-1; GAG, glycosaminoglycan; CatG, cathepsin G contains secreted molecules that are immobilized in the extracellular spaces and regulates the function of almost all cells. The interaction of cells with the matrix is mainly mediated by adhesion molecules. CD44 is a widely expressed family of adhesion receptors that range in molecular mass from 80 to 250 kDa and that represent alternatively spliced and post-translationally modified products of a single gene (2Schlossman S. Boumsell L. Gilks W. White Cell Differentiation Antigens. Oxford University Press, New York1995Google Scholar). The most common form, referred to as hematopoietic CD44, has an apparent molecular mass of 80–100 kDa and does not contain differentially spliced exons. CD44 isoforms expressing variant exons are mainly restricted to epithelia, activated lymphocytes, and tumor cells (3Droll A. Dougherty S.T. Chiu R.K. Dirks J.F. McBride W.H. Cooper D.L. Dougherty G.J. J. Biol. Chem. 1995; 270: 11567-11573Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). CD44 is a major receptor for hyaluronan (HA), a glycosaminoglycan that is ubiquitously distributed in the ECM (4Aruffo A. Stamenkovic I. Meinich M. Underhill C.B. Seed B. Cell. 1990; 61: 1301-1313Abstract Full Text PDF Scopus (2169) Google Scholar). In addition, proteoglycan forms of CD44 bind fibronectin (5Jalkanen S. Jalkanen M. J. Cell Biol. 1992; 116: 817-825Crossref PubMed Scopus (454) Google Scholar), collagen (6Ehnis T. Dieterich W. Bauer M. Lampe B. Schuppan D. Exp. Cell Res. 1996; 229: 388-397Crossref PubMed Scopus (70) Google Scholar), growth factors and cytokines (7Wolff E.A. Greenfield B. Taub D.D. Murphy W.J. Bennett K.L. Aruffo A. J. Biol. Chem. 1999; 274: 2518-2524Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), as well as matrix metalloproteinase-9 (8Yu Q. Stamenkovic I. Genes Dev. 1999; 13: 35-48Crossref PubMed Scopus (608) Google Scholar). In normal tissues, CD44 appears to play a role in regulating the metabolism of hyaluronan. However, data have accumulated that indicate a prominent role for CD44 in different pathologic states, including autoimmune and chronic inflammatory diseases (9Mikecz K. Brennan F.R. Kim J.H. Glant T.T. Nat. Med. 1995; 1: 558-563Crossref PubMed Scopus (259) Google Scholar, 10Estess P. DeGrendele H.C. Pascual V. Siegelman M.H. J. Clin. Invest. 1998; 102: 1173-1182Crossref PubMed Scopus (97) Google Scholar, 11Cuff C.A. Kothapalli D. Azonobi I. Chun S. Zhang Y. Belkin R. Yeh C. Secreto A. Assoian R.K. Rader D.J. Puré E. J. Clin. Invest. 2001; 108: 1031-1040Crossref PubMed Scopus (265) Google Scholar, 12Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Puré E. Henson P.M. Noble P.W. Science. 2002; 296: 155-158Crossref PubMed Scopus (581) Google Scholar, 13Blass S.L. Puré E. Hunter C.A. J. Immunol. 2001; 166: 5726-5732Crossref PubMed Scopus (34) Google Scholar) as well as tumor growth and dissemination (14Gunthert U. Hofmann M. Rudy W. Reber S. Zoller M. Haubmann I. Matzku S. Wenzel A. Ponta H. Herrlich P. Cell. 1991; 65: 13-24Abstract Full Text PDF PubMed Scopus (1602) Google Scholar,15Bartolazzi A. Jackson D. Bennett K. Aruffo A. Dickinson R. Shields J. Whittle N. Stamenkovic I. J. Cell Sci. 1995; 108: 1723-1733Crossref PubMed Google Scholar). Soluble CD44 (sCD44) is found in serum and lymph. Interestingly, immunodeficiency correlates with low plasma levels of sCD44, whereas malignant diseases, immune activation, and inflammation are often associated with increased plasma levels of CD44, indicating that immunological and malignant processes may promote the release of sCD44 (16Katoh S. McCarthy J.B. Kincade P.W. J. Immunol. 1994; 153: 3440-3449PubMed Google Scholar). However, the physiological inducers of CD44 release are unknown. The generation of sCD44 is believed to involve proteolytic cleavage of cell-surface CD44 (17Okamoto I. Kawano Y. Tsuiki H. Sasaki J. Nakao M. Matsumoto M. Suga M. Ando M. Nakajima M. Saya H. Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (218) Google Scholar). Potentially, all cells expressing CD44 are capable of releasing CD44. However, using cell lines stably transfected with different alternatively spliced isoforms of CD44, it has been demonstrated that release of CD44 variants is increased severalfold compared with that of hematopoietic CD44 (15Bartolazzi A. Jackson D. Bennett K. Aruffo A. Dickinson R. Shields J. Whittle N. Stamenkovic I. J. Cell Sci. 1995; 108: 1723-1733Crossref PubMed Google Scholar). Thus, in cells expressing variant forms of CD44, including epithelia, CD44 may be more vulnerable to proteolytic cleavage. The lung epithelium forms an important barrier between the external environment and the lung interstitium and is increasingly recognized as a participant in inflammatory processes both as a source of mediators and as a site of modulation of cell adhesion. In this study, we investigated the mechanism of release and characterized sCD44 from human lung-derived epithelial cells. Our results suggest that CD44 released from epithelial cells may accumulate as an integral component of the matrix, where it is physically associated with other ECM components, including fibronectin, HA, collagens I and IV, and chondroitin sulfate. Furthermore, bacterium- and phagocyte-derived proteases mediate the limited proteolysis and release of sCD44 from monolayers of normal bronchial epithelial cells (NBEC) into the fluid phase. Although previous studies have suggested a role for cell-surface CD44 in the assembly of hyaluronan-rich pericellular matrices, the function of sCD44 proteolytically cleaved from the cell surface is not known. Our results extend previous findings regarding cell-surface CD44 by demonstrating that CD44 released from cells can be incorporated as an integral component of cellular matrices and suggest a novel functional relationship between the release of sCD44 and tissue remodeling. The following anti-human CD44 monoclonal antibodies were used (2Schlossman S. Boumsell L. Gilks W. White Cell Differentiation Antigens. Oxford University Press, New York1995Google Scholar): 5F12 (a gift of Dr. B. F. Haynes), Hermes III (American Type Culture Collection, Manassas, VA), and G44-26 (Pharmingen). Rabbit anti-human fibronectin antibody, purified HA from rooster comb, chondroitinase ABC fromProteus vulgaris, heparinase III fromFlavobacterium heparinum, keratanase fromPseudomonas, hyaluronidase from Streptomyces, proteinase 14, and aprotinin were obtained from Sigma. Fluorescein isothiocyanate-conjugated rooster comb HA (FITC-HA) was prepared as described (18deBelder A.N. Wik K.O. Carbohydr. Res. 1975; 44: 251-257Crossref PubMed Scopus (174) Google Scholar). Purified HA from human umbilical cords was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). FITC- and phycoerythrin (PE)-labeled anti-human CD44 and PE-labeled anti-human CD3 antibodies were purchased from Pharmingen. Biotinylated hyaluronic acid-binding protein was obtained from Seikagaku Corp. (Tokyo, Japan). Biotinylated goat anti-type I and anti-type IV collagen antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL). Alkaline phosphatase-conjugated anti-FITC antibodies were obtained from Roche Molecular Biochemicals. Cathepsin G and α1-antichymotrypsin were a generous gift of Athens Research and Technology Inc. (Athens, GA). The Staphylococcus aureus metalloproteinase (SAMP) aureolysin and thePseudomonas aeruginosa metalloproteinase (PAMP) pseudolysin were isolated as described (19Potempa J. Travis J. Handbook on Proteinases. Academic Press, Inc., Orlando, FL1998: 1540-1542Google Scholar, 20Kessler E. Ohman D.E. Handbook on Proteinases. Academic Press, Inc., Orlando, FL1998: 1058-1064Google Scholar). p-Nitrophenyl β-d-xyloside was purchased from Calbiochem. CD44-2-Rg vector (4Aruffo A. Stamenkovic I. Meinich M. Underhill C.B. Seed B. Cell. 1990; 61: 1301-1313Abstract Full Text PDF Scopus (2169) Google Scholar) was a gift of Dr. I. Stamenkovic. The BCA kit was purchased from Pierce, and the enhanced chemiluminescence (ECL) system was obtained from Amersham Biosciences. NBEC were obtained from Clonetics Corp. (San Diego, CA) or isolated as follows. Cells were removed by digestion with proteinase 14 from main stem or lobular bronchi from excess donor tissue obtained at the time of lung transplantation under the auspices of the University of North Carolina Institutional Committee on the Protection of the Rights of Human Subjects. Cells at passages 3–10 from five individuals were used in this study. Cells were cultured in serum-free bronchial epithelial cell basal medium (Clonetics Corp.) containing 0.5 ng/ml epidermal growth factor, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.5 μg/ml epinephrine, 10 μg/ml transferrin, 0.5 ng/ml triiodothyronine, and 0.4% (v/v) bovine pituitary extract. To inhibit glycosaminoglycan attachment to the protein core, cells were cultured for 36 h in medium supplemented with 5 mm p-nitrophenyl β-d-xyloside. HTB55 human lung adenocarcinoma and HTB58 human lung squamous carcinoma cell lines were obtained from American Type Culture Collection. HTB55 and HTB58 cells were cultured in Eagle's minimal essential medium supplemented with 0.1 mmnonessential amino acids, 1 mm sodium pyruvate, 50 μg/ml gentamycin, and 10% heat-inactivated fetal bovine serum. Total RNA was isolated by SDS/phenol extraction and then reverse-transcribed with avian myeloblastosis virus reverse transcriptase (Promega) using oligo[d(T)15] primer (Roche Molecular Biochemicals). The product was amplified with Taq DNA polymerase (Promega) using the standard exon-specific primer C13 and variable exon-specific primers pV2–pV10 as 5′-primers and the standard exon-specific primer HS3′ as a 3′-primer (21Konig H. Moll J. Ponta H. Herrlich P. EMBO J. 1996; 15: 4030-4039Crossref PubMed Scopus (44) Google Scholar, 22Mackay C.R. Terpe H.-J. Stauder R. Marston W.L. Stark H. Gunthert U. J. Cell Biol. 1994; 124: 71-82Crossref PubMed Scopus (412) Google Scholar): C13, 5′-AAGACATCTACCCCAGCAAC-3′; pV2, 5′-GATGAGCACTAGTGCTACAG-3′; pV3I, 5′-GTAGGTCTTCAAATACCATC-3′; pV3II, 5′-TGGGAGCCAAATGAAGAAAA-3′; pV4, 5′-TCAACCACACCACGGGCTTT-3′; pV5, 5′-GTAGACAGAAATGGCACCAC-3′; pV6, 5′-TCCAGGCAACTCCTAGTAGT-3′; pV7, 5′-CAGCCTCAGCTCATACCAGC-3′; pV8, 5′-TCCAGTCATAGTACAACGCT-3′; pV9, 5′-AGCAGAGTAATTCTCAGAGC-3′; pV10, 5′-ATAGGAATGATGTCACAGGT-3′; and HS3′, 5′-TTTGCTCCACCTTCTTGACTCC-3′. Each cycle included 1 min at 94 °C, 1 min at 60 °C, and 1.5 min at 72 °C. Thirty cycles were performed. Cells were incubated for 48 h in methionine-free medium containing 200 μCi/ml [35S]methionine/cysteine (Tran35S-label) or in sulfate-free medium supplemented with 500 μCi/ml Na235SO4 (ICN Biomedicals, Inc.). Conditioned media were collected and centrifuged at 300 ×g for 10 min, followed by 600 × g for 15 min to remove cells and cellular debris. Cells were lysed in 1% Nonidet P-40, 0.1% sodium deoxycholate, and protease inhibitors (0.2 units/ml aprotinin, 100 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride). Conditioned media and cell lysates were normalized based on protein concentration as determined by BCA assay, and equal amounts of protein were precleared with preimmune serum and then precipitated with Hermes III conjugated to Sepharose or with G44-26, followed by protein A/G Plus-agarose. Immune complexes were washed sequentially with high salt buffer (0.6 m NaCl, 125 mm KPO4 (pH 7.4), and 0.02% NaN3), mixed detergent buffer (0.05% Nonidet P-40, 0.1% SDS, 0.3 m NaCl, and 10 mm Tris (pH 8.6)), and phosphate-buffered saline; boiled in Laemmli sample buffer; resolved by SDS-8% PAGE under nonreducing conditions; and detected by fluorography. CD44 was immunoprecipitated with Sepharose-conjugated anti-CD44 monoclonal antibody (mAb) Hermes III. Immune complexes were resolved by SDS-8% PAGE under nonreducing or (where indicated) reducing conditions. CD44 was then visualized by ECL after electrotransfer to polyvinylidene difluoride membranes (PerkinElmer Life Sciences) and incubation with mouse anti-CD44 mAb (Hermes III or G44-26) and peroxidase-conjugated donkey anti-mouse IgG antibody. Cells were treated on tissue culture dishes with 0.1 unit/ml chondroitinase ABC and/or 20 units/ml hyaluronidase for 90 min. In all other instances, cell lysates were prepared as described above, and CD44 was immunoprecipitated with Sepharose-conjugated anti-CD44 mAb Hermes III. Treatment with chondroitinase ABC (2 units/ml) as well as keratanase (30 milliunits/ml) and heparinase III (5 units/ml) was performed on anti-CD44 immune complexes bound to Sepharose-conjugated Hermes III in phosphate-buffered saline at pH 7.4 (chondroitinase ABC and keratanase) or in phosphate-buffered saline at pH 7.0 (heparinase III) for 2 h at 37 °C. Cells were harvested using 0.2% EDTA, stained with PE-conjugated mouse anti-human CD44 mAb G44-26 or PE-conjugated anti-human CD3 antibody, and analyzed on a FACScan (BD Biosciences). Soluble HA binding was assayed using saturating amounts of FITC-HA. Specific binding of FITC-HA to CD44 was determined using blocking anti-CD44 mAb 5F12. To determine the level of HA associated with the cell surface, NBEC were stained with biotinylated bovine HA-binding protein (bPG), followed by PE-conjugated streptavidin. Specificity was verified by pretreating the cells with 20 units/ml hyaluronidase at 37 °C for 2 h, followed by staining with bPG and PE-conjugated streptavidin. Confluent monolayers were fixed with 3.7% formaldehyde. After quenching endogenous peroxidase activity with 0.3% H2O2 and blocking with 1% bovine serum albumin, cells were incubated with 0.5 μg/ml bPG for 40 min at 4 °C. Controls for specificity of bPG binding included treatment with 20 units/ml hyaluronidase for 2 h at 37 °C, followed by incubation with bPG. Bound bPG was detected with avidin-biotin complex (VECTASTAIN ABC reagent, Vector Labs, Inc., Burlingame, CA) and diaminobenzidine tetrahydrochloride in 0.2 m Tris-HCl (pH 7.6) containing 0.01% H2O2 as substrate. Sections were counterstained with hematoxylin. The release of CD44 was quantified using a sandwich ELISA. Plates were coated with 2 μg/ml Hermes III in Tris-buffered saline (50 mm Tris-HCl (pH 9.5) and 150 mm NaCl). The plates were then washed with 0.05% Tween 20, and nonspecific protein-binding sites were blocked with 3% bovine serum albumin. Samples were added and incubated at room temperature for 2 h. Affinity-purified CD44-immunoglobulin fusion protein (CD44-2-Rg) containing the complete extracellular domain of hematopoietic CD44 was used as a standard (4Aruffo A. Stamenkovic I. Meinich M. Underhill C.B. Seed B. Cell. 1990; 61: 1301-1313Abstract Full Text PDF Scopus (2169) Google Scholar, 23Stamenkovic I. Aruffo A. Amiot M. Seed B. EMBO J. 1991; 10: 343-348Crossref PubMed Scopus (521) Google Scholar). Bound sCD44 was detected using FITC-conjugated anti-CD44 mAb G44-26 incubated at 37 °C for 30 min, followed by incubation with alkaline phosphatase-conjugated anti-FITC mAb and development withp-nitrophenyl phosphate as substrate. Soluble ICAM-1 was quantified using a soluble ICAM-1 ELISA kit (R&D Systems, Minneapolis, MN). To detect matrix components physically associated with sCD44, conditioned media were incubated on streptavidin plates (Pierce) coated with biotinylated HA-binding protein or biotinylated anti-type I or anti-type IV collagen antibody to capture HA or collagen I or IV, respectively, or with biotinylated anti-CD3 antibody as a negative control. After washing, bound CD44 was detected by FITC-conjugated anti-CD44 mAb G44-26 as described above. To determine the HA content in cell lysates and conditioned media, we developed a modified ELISA based on competitive binding of endogenous HA and FITC-labeled exogenous HA to microwell-bound bPG. Cells were incubated in culture media without bovine pituitary extract for 48 h. Cell lysates and conditioned media were then collected and incubated at room temperature for 30 min to capture endogenous HA. Unlabeled purified HA from human umbilical cords was used as a standard. Unsaturated bPG sites were detected by incubation with FITC-HA at 37 °C for 30 min, followed by incubation with alkaline phosphatase-conjugated anti-FITC mAb and p-nitrophenyl phosphate. CD44 and HA are both expressed on the surface of the bronchial epithelium (24Green S.J. Tarone G. Underhill C.B. J. Cell Sci. 1988; 89: 145-156Google Scholar). However, the molecular basis and functional significance of this potential CD44-HA interaction have not been elucidated. We characterized CD44 in cultured primary NBEC. NBEC were found to express CD44 on the cell surface (Fig.1 A, left panel) and to synthesize HA (Fig. 1 B). The HA appeared to be at least partially cell-associated (Fig. 1, A, right panel; and B). ELISA analysis of HA levels in lysates and conditioned media of NBEC incubated for 48 h demonstrated ∼2-fold higher levels of cell-associated HA than HA released into the conditioned media (1.93 versus 1 μg/1 × 106 cells). These data indicate that a majority of the HA that is synthesized at the cell surface is retained on the cell surface, possibly as a part of the pericellular matrix. Similar to most other primary cells studied, CD44 expressed on NBEC did not constitutively exhibit affinity for exogenous HA (Fig. 1 A,middle panel). These data might indicate that the receptor was occupied by endogenous ligand. However, pretreatment of NBEC with hyaluronidase to degrade endogenous HA only slightly increased the capacity of cells to bind exogenous HA (data not shown). Using exon-specific reverse transcription-PCR analysis, we found that NBEC transcribed all known CD44 exons (Fig.2). A predominant band indicating the presence of full-length transcripts expressing all intervening exons was detected with the C13/HS3′ primer set flanking the site of insertion of variant exons on the 5′- and 3′-ends, respectively. Several other bands indicating the presence of shorter alternatively spliced transcripts were also detected, but at significantly lower abundance with the same primer set. To examine the expression of CD44 at the protein level, we used mAb G44-26, which recognizes all forms of CD44 (2Schlossman S. Boumsell L. Gilks W. White Cell Differentiation Antigens. Oxford University Press, New York1995Google Scholar). Three major species of CD44 with average molecular masses of 90, 150, and 180 kDa were identified in lysates of [35S]methionine-labeled NBEC by immunoprecipitation. A higher molecular mass species of >210 kDa that exhibited extensive microheterogeneity was also evident (Fig.2 C). A similar profile of species was detected under nonreducing conditions by immunoprecipitation with Hermes III and then detection by immunoblotting with Hermes III (Fig. 2 D) or mAb G44-26 (data not shown). Four major species of CD44 were also identified in CD44 immunoprecipitates subjected to SDS-PAGE under reducing conditions, although in this case, the higher molecular mass species of >210 kDa appeared to be less intense (Fig. 2 D). The similar patterns obtained under reducing and nonreducing conditions suggest that the majority of the high molecular mass species containing CD44 are not the result of formation of disulfite-linked complexes. A band with average molecular mass of 50 kDa represents IgG heavy chains released from IgG-Sepharose conjugates as a result of boiling in the presence of reducing agent. CD44 can be modified by attachment of glycosaminoglycans (GAGs) (25Greenfield B. Wang W.C. Marquardt H. Piepkorn M. Wolff E.A. Aruffo A. Bennett K.L. J. Biol. Chem. 1999; 274: 2511-2517Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To detect CD44-associated sulfated GAGs, CD44 was immunoprecipitated from lysates of [35S]sulfate-labeled NBEC. Only the high molecular mass species with an average molecular mass of >210 kDa were sulfated (Fig. 2 E, left lane). To characterize the target of sulfation, CD44 was digested with enzymes that cleave specific GAGs. Heparinase III or keratanase had no effect on the sulfated species of CD44 (Fig. 2 E). In contrast, chondroitinase ABC digestion resulted in the degradation of the vast majority of the CD44-associated sulfated GAGs, indicating that the sulfated entity mainly represents chondroitin sulfate chains that are either covalently attached or otherwise tightly physically associated with CD44 in a high molecular mass complex. Elevated proteolytic activity related to infection and inflammation is associated with lung epithelial cell dysfunction. We examined the effect of pathogen- and phagocyte-derived proteinases on the release of sCD44 from NBEC. Neutrophil serine proteinases, including elastase and cathepsin G (CatG), have been identified in purulent bronchial secretions. On the other hand, P. aeruginosa and S. aureus, which can colonize airways in patients with a variety of chronic lung conditions, secrete metalloproteinases, including aureolysin (SAMP) and pseudolysin (PAMP) (26Stockley R.A. Am. J. Respir. Crit. Care Med. 1994; 150: S109-S113Crossref PubMed Google Scholar, 27Suter S. Am. J. Respir. Crit. Care Med. 1994; 150: S118-S122Crossref PubMed Google Scholar). Low levels of sCD44 were detected in the conditioned media of control cells at 90 min (Fig.3 A) and continued to accumulate in the conditioned media of control cells progressively over a 48-h culture period. However, treatment of NBEC with CatG, SAMP, and PAMP resulted in a marked increase in the release of sCD44 (Fig.3 A). Enzyme-induced release of CD44 was time- and dose-dependent, reaching a maximum with 2 μg/ml proteinases at 90 min. Neutrophil elastase at 2 μg/ml also induced the release of sCD44, but less efficiently compared with CatG (data not shown). Neither CatG nor PAMP induced release of ICAM-1 (Fig.3 B), suggesting that sCD44 is selectively released under these conditions. The SAMP- and PAMP-induced generation of sCD44 was completely blocked by the metalloproteinase inhibitor 1,10-phenanthroline, whereas CatG-induced release was inhibited by a specific inhibitor of CatG, α1-antichymotrypsin, and by an inhibitor of chymotrypsin- and trypsin-like proteinases, aprotinin (Fig. 3 A), thus confirming that enzymatic activity is required for the protease-induced release of sCD44. Because CD44 has been implicated in the growth and metastasis of epithelium-derived tumors, we also examined the capacity of lung-derived tumor cell lines to release CD44. CatG and SAMP had a significant effect on CD44 release in HTB55 human adenocarcinoma cells, but did not induce the generation of sCD44 in HTB58 human squamous carcinoma cells. Interestingly, enzyme-induced release of sCD44 appeared to correlate with the expression of variant forms of CD44 in the epithelial cell lines (Fig. 4) (28Cichy J. Puré E. J. Biol. Chem. 2000; 275: 18061-18069Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Epithelial cells express a proteoglycan form of CD44 decorated with chondroitin sulfate as well as isoforms of CD44 that are not associated with sulfated GAGs as demonstrated above. To investigate whether the association of CD44 with chondroitin sulfate affected susceptibility of the receptor to proteolysis, NBEC were treated with chondroitinase ABC. Surprisingly, treatment with chondroitinase alone resulted in release of sCD44 from NBEC (Fig. 3 A). The effect of chondroitinase was not inhibited by the metalloproteinase inhibitor 1,10phenanthroline or by the chymotrypsin- and trypsin-like proteinase inhibitor aprotinin, indicating the specificity of the chondroitinase activity (Fig. 3 A). Treatment with hyaluronidase did not induce the release of sCD44, but enhanced the release of sCD44 in combination with chondroitinase (Fig.3 C). These results suggest that in addition to cleavage of cell-surface CD44, sCD44 may be generated as the result of dissociation of a reservoir of sCD44 that accumulates as a component of the cell-associated matrix. We also tested whether inhibition of proteoglycan synthesis altered the susceptibility of CD44 to release by chondroitinase. NBEC were grown in the presence of β-d-xyloside, which acts as a primer of free GAG chains, diverting assembly of the chains from the core proteins and causing secretion of free GAGs from cells. Incubation of NBEC with β-d-xyloside resulted in an average 85% reduction of the sulfation of CD44, indicating extensive but not complete loss of chondroitin sulfate chains from CD44 (data not shown). As expected, the release of CD44 by chondroitinase from β-d-xyloside-treated NBEC cells was reduced compared with control cells. Treatment with β-d-xyloside also compromised CatG-induced release of CD44, but did not inhibit the capacity of SAMP- or PAMP-treated NBEC to generate sCD44 (Fig.3 A). In contrast to SAMP or PAMP, CatG is a strongly cationic protein (19Potempa J. Travis J. Handbook on Proteinases. Academic Press, Inc., Orlando, FL1998: 1540-1542Google Scholar, 20Kessler E. Ohman D.E. Handbook on Proteinases. Academic Press, Inc., Orlando, FL1998: 1058-1064Google Scholar, 29Salvesen G.S. Handbook on Proteinases. Academic Press, Inc., Orlando, FL1998: 60-62Google Scholar). Therefore, cationic sites on CatG might be important in the initial interaction with negatively charged chondroitin sulfate on the surface of epithelial cells, targeting CatG to a specific substrate and amplifying the effect of this enzyme on release of CD44. In any case, these results suggest that CD44 is subject to proteinase-mediated release to varying degrees depending on whether the receptor is or is not associated with proteoglycans. Immunoprecipitation of CD44 from lysates of [35S] methionine-labeled cells treated with CatG, SAMP, PAMP, and chondroitinase revealed that high molecular mass species of >210 kDa were mainly targeted by the enzymes (Fig.5 A), consistent with the evidence that the pro
Chemerin-derived peptide Val66-Pro85 (p4) restricts the growth of a variety of skin-associated bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To better understand the antimicrobial potential of chemerin peptide, we compared p4 activity against MRSA in vitro to cathelicidin LL-37, one of the key endogenous peptides implicated in controlling the growth of S. aureus. The efficacy of p4 was also validated in relevant experimental models of skin pathology, such as topical skin infection with community-acquired MRSA, and in the context of skin inflammatory diseases commonly associated with colonization with S. aureus, such as atopic dermatitis (AD). We showed that p4 collaborates additively with LL-37 in inhibiting the growth of S. aureus, including MRSA, and that p4 was effective in vivo in reducing MRSA burden. p4 was also effective in reducing levels of skin-infiltrating leukocytes in S. aureus-infected AD-like skin. Taken together, our data suggest that p4 is effective in limiting S. aureus and, in particular, MRSA skin infection.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
