Marlene Wolf

Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase-3.

Activated neutrophils secrete two forms of IL‐8 with 77 and 72 amino acids, IL‐8(77) and IL‐8(72), along with proteinases that could process these cytokines. Significant conversion of IL‐8(77) to more potent, N‐terminally truncated forms was observed upon incubation with neutrophil granule lysates and purified proteinase‐3. IL‐8(72) was considerably more resistant to proteolytic processing than IL‐8(77). The present observations indicate that neutrophil proteinases released in inflamed tissues convert IL‐8 to more active forms and therefore tend to conserve or enhance, rather than decrease IL‐8 activity.

Rapid inactivation of stromal cell-derived factor-1 by cathepsin G associated with lymphocytes.

The CXC chemokine stromal cell‐derived factor (SDF)‐1 is produced constitutively in different tissues. It is the only known ligand for CXCR4, which is widely expressed in leukocytes and in some tissue cells, and acts as coreceptor for X4 HIV strains. Because of the general interest in the mechanisms that regulate the activity of constitutively expressed chemokines, we have studied the inactivation of SDF‐1 in cells that bear CXCR4. Here we show that B lymphocytes, NK cells and, to a lesser extent, T lymphocytes inactivate SDF‐1 by N‐terminal processing. Inactivation is due to cathepsin G which is associated with the membrane of lymphocytes and rapidly cleaves off five N‐terminal residues by acting on the Leu5‐Ser6 bond yielding SDF‐1(6–67). Processing was observed with intact cells, cell membrane preparations and soluble cathepsin G obtained by extraction of the membranes with Triton X‐100. Cathepsin G is released by neutrophils and monocytes and binds on the surface of lymphocytes by an apparently saturable process. Analysis of the product obtained, the time course and the sensitivity to inhibitors shows that cathepsin G is the only protease involved. Conversion of SDF‐1 to SDF‐1(6–67) was complete within minutes to 1–2 h depending on the enzyme source, and was abrogated by inhibitors of serine proteases and chymostatin. Diprotin A, an inhibitor of dipeptidyl peptidase IV, was without effect. Owing to its availability on the surface of SDF‐1‐responsive cells and its rapid effect, cathepsin G is likely to play a significant role in down‐regulating SDF‐1 activity.

Granulocyte chemotactic protein 2 acts via both IL‐ 8 receptors, CXCR1 and CXCR2

Interleukin‐8 (IL‐γ) acts on human neutrophils via two receptors, CXCR1 and CXCR2. It shares CXCR2 with all neutrophil‐activating chemokines, which like IL‐8 have a conserved Glu‐Leu‐Arg (ELR) N‐terminal motif, but is generally considered to be the only relevant agonist for CXCR1. IL‐8 has a basic residue at the sixth position after the second cysteine, which was suggested to contribute to CXCR1 specificity. Among the other ELR chemokines, only granulocyte chemotactic protein 2 (GCP‐2) has such a basic determinant. Using Jurkat cells that stably express either CXCR1 or CXCR2, we studied receptor activation by IL‐8, GCP‐2 epithelial neutrophil‐activating protein 2 (ENA‐78) (which shares 77 % identical amino acids with GCP‐2) and growth‐regulated oncogene α (GROα). At 10 nM and higher concentrations, GCP‐2 and IL‐8 induced significant activation of CXCR1‐expressing cells, but no activity was found with GROα and ENA‐78. As expected, however, all four chemokines had similar activities on CXCR2‐expressing cells. A variant of GCP‐2 in which the basic residue, Arg20, was replaced by a glycine was synthesized. This derivative was ineffective on CXCR1, but was as active as wild‐type GCP‐2 in CXCR2‐expressing cells. GCP‐2 displaced radiolabeled IL‐8 from both receptors with low affinity, and in this respect resembled ENA‐78 and GROα. Our data show that GCP‐2 acts via both IL‐8 receptors and thus appears to be functionally more similar to IL‐8 than to the other ELR chemokines. Activation of CXCR1 appears to depend significantly on the presence of a basic binding determinant close to the second cysteine.

Proteolytic processing of chemokines: implications in physiological and pathological conditions

Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and fine-tunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing.

Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes.

Staurosporine induced the association of purified protein kinase C (PKC) with inside-out vesicles from erythrocyte membranes. This effect was Ca2+ and concentration dependent, and maximum PKC translocation was observed at 50 nM staurosporine and 0.5 microM Ca2+, or higher. A significant effect of staurosporine was already obtained at free Ca2+ concentrations in the range found in resting cells. Under these conditions, the PKC activator 4-phorbol 12,13-dibutyrate was by itself inactive, but enhanced translocation by staurosporine. Protein phosphorylation by staurosporine-translocated PKC was inhibited in the presence or absence of phorbol esters. Translocation and inhibition of PKC occurred in the same staurosporine concentration range.

Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous Gram-positive bacteria and Candida albicans as well as the Gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human β-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Antimicrobial Activities of Chemokines: Not Just a Side-Effect?

The large family of chemoattractant cytokines (chemokines) embraces multiple, in part unrelated functions that go well beyond chemotaxis. Undoubtedly, the control of immune cell migration (chemotaxis) is the single, unifying response mediated by all chemokines, which involves the sequential engagement of chemokine receptors on migrating target cells. However, numerous additional cellular responses are mediated by some (but not all) chemokines, including angiogenesis, tumor cell growth, T-cell co-stimulation, and control of HIV-1 infection. The recently described antimicrobial activity of several chemokines is of particular interest because antimicrobial peptides are thought to provide an essential first-line defense against invading microbes at the extremely large body surfaces of the skin, lungs, and gastrointestinal-urinary tract. Here we summarize the current knowledge about chemokines with antimicrobial activity and discuss their potential contribution to the control of bacterial infections that may take place at the earliest stage of antimicrobial immunity. In the case of homeostatic chemokines with antimicrobial function, such as CXCL14, we propose an immune surveillance function in healthy epithelial tissues characterized by low-level exposure to environmental microbes. Inflammatory chemokines, i.e., chemokines that are produced in tissue cells in response to microbial antigens (such as pathogen-associated molecular patterns) may be more important in orchestrating the cellular arm in antimicrobial immunity.

Neutrophil-activating Peptide-2 and Melanoma Growth-stimulatory Activity Are Functional as Monomers for Neutrophil Activation

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH → NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 μM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of ∼5 μM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing.

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL201–66 isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL201–55, CCL201–52, and a 12-aa C-terminal peptide CCL2059–70. Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1α and TNF-α in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.