No abstract

Two monoclonal antibodies, anti-IL8Rl and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GROa, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 1251-labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8Rl in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GROa. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GROa was inhibited by anti-IL8R2 but was not affected by anti-IL8Rl. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GROca or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8Rl, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GROa or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL8Rl but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-SRi.Chemokines constitute a large family of structurally related proteins which activate and attract leukocytes. Interleukin 8 (IL-8) and its analogs, GROa, GRO,3, GROT, NAP-2, and ENA-78, act predominantly on neutrophils and are called CXC chemokines because their first two conserved cysteines are separated by one amino acid (1). CXC chemokines share two distinct receptors, IL-8 receptors 1 (IL-8R1) and IL-8 receptor 2 (IL-8R2), which are both expressed in high numbers on neutrophils (1). IL-8 binds with high affinity (Kd values between 0.5 to 3 nM) to both receptors, whereas all the other CXC chemokines have high affinity for IL-8R2 only (2, 3). The two receptors have considerable amino acid sequence identity within the seven transmembrane domains and connecting loops, but differ almost totally in their NH2-and COOHterminal regions. This suggests that they not only possess distinct ligand-binding properties but also signal differently. Because the receptors are expressed together on neutrophils, their individual properties were studied in Jurkat cells after transfection of the respective cDNA. IL-8R1 and IL-8R2 were found to function ind...

The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.

Activated neutrophils secrete two forms of IL-8 with 77 and 72 amino acids, and , 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 . 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.

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.

Interleukin-8 (IL-8) 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 alpha (GRO alpha). At 10 nM and higher concentrations, GCP-2 and IL-8 induced significant activation of CXCR1-expressing cells, but no activity was found with GRO alpha 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 GRO alpha. 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.

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 40C as well as at 30'C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase I, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase U by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.Phorbol esters influence cellular functions at various levels (1, 2), presumably by activating protein kinase C (3, 4). Some cellular effects can be detected rapidly after the addition of phorbol esters. These effects include the regulation of ionic transport, release of bioactive substances, and receptor down-regulation (1,2,5). Other effects of phorbol esters target the genome, resulting in the regulation of DNA replication or in the modulation of gene expression. Examples of genes that appear to be induced by phorbol esters include ornithine decarboxylase in epidermal cells (6,7), interleukin 2 and IL-2 receptor in mouse T cells and Tlymphoma cells (8-10), c-fos in U937 monocytes and in HL-60 cells (11), actin and vimentin in K562 erythroleukemia cells (12), and calcitonin in thyroid medullary carcinoma cells (13). Genes that seem to be repressed by phorbol esters include globin in Friend erythroleukemia cells (14), glycophorin in K562 erythroleukemia cells (15), and c-myc in thyroid medullary carcinoma cells (13). Moreover, phorbol esters increase the frequency of initiation of DNA replication for bacteriophage X injected into Xenopus eggs (16).The effects of phorbol esters on gene transcription generally require more time to become manifest than the effects on ionic fluxes or receptors. Thus, the regulation of c-fos mRNA in U937 monocytes is maximal after 30 min (11) and that of glycophorin and actin genes becomes evident after about 1 hr (12, 15), whereas the effect on c-myc mRNA is observed 4 hr after exposure of the cells to the tumor promoter (13). The relative delay in these responses may reflect an indirect effect ofphorbol esters on gene transcription, requiring one or more intermediary steps or a cascade of reactions; alternatively, phorbol esters may alter gene transcription more directly by stimulating protein kinase C, which then may phosphorylate a polypeptide(s) involved in transcriptional regu...

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141–13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.