Ole E. Sørensen

The Antimicrobial Peptide LL-37 Activates Innate Immunity at the Airway Epithelial Surface by Transactivation of the Epidermal Growth Factor Receptor

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.

Wound Healing and Expression of Antimicrobial Peptides/Polypeptides in Human Keratinocytes, a Consequence of Common Growth Factors

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-α, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human β-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.

IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37.

Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3), has pleiotropic immune effects. The mechanisms by which 1α,25(OH)2D3 protects against tuberculosis are incompletely understood. 1α,25(OH)2D3 reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1α,25(OH)2D3 reduced transcription and secretion of protective IFN-γ, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1α,25(OH)2D3 does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1α,25(OH)2D3, inhibition of NO formation marginally affected the suppressive effect of 1α,25(OH)2D3 on bacillus Calmette Guérin in infected cells. By contrast, 1α,25(OH)2D3 strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1α,25(OH)2D3 stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1α,25(OH)2D3-stimulated PBMC cultures. A total of 200 μg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by “nonclassical” mechanisms that include the induction of antimicrobial peptides.

Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha.

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1β induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-α, IL-6, and LPS had no effect. Similar IL-1β selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-κB consensus site was found to control the IL-1β-specific up-regulation. Involvement of the NF-κB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-κB pathway, and by EMSA. TNF-α activation of NF-κB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-κB to the NGAL promoter was observed in vitro. IL-1β specificity was not contained within the NF-κB site of the NGAL promoter, as determined by exchanging the NGAL promoter′s NF-κB-binding sequence with that of the IL-8 promoter or with the NF-κB consensus sequence and by testing the NF-κB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the β-defensin family, human β-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of β-defensins is mediated by at least three different mechanisms.

The Human Cationic Antimicrobial Protein (hCAP-18) Is Expressed in the Epithelium of Human Epididymis, Is Present in Seminal Plasma at High Concentrations, and Is Attached to Spermatozoa

ABSTRACT Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 ± 37.8 μg/ml (mean ± standard deviation; range, 41.8 to 142.8 μg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/106 spermatozoa, corresponding to 6.6 × 106 molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.

An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ng/ml. The amount of hCAP-18 in neutrophils is 0.627 microgram protein per 10(6) cells. The plasma level is 1.18 micrograms/ml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins.

Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor - Critical regulators of beta-defensins during helicobacter pylori infection

Host-pathogen interactions that allow Helicobacter pylori to survive and persist in the stomach of susceptible individuals remain unclear. Human β-defensins (hBDs), epithelial-derived antimicrobial peptides are critical components of host-defense at mucosal surfaces. The role of H. pylori-mediated NF-κB and epidermal growth factor receptor (EGFR) activation on β-defensin expression was investigated. Transient transfection studies utilizing β-defensin promoter constructs were conducted in gastric cells with contribution of individual signaling events evaluated by the addition of specific inhibitors, small interference nucleotide-binding oligomerization domain 1 (NOD1) RNA or plasmids encoding Vaccinia virus proteins that interrupt interleukin-1 and Toll-like receptor signaling. The role of individual MAPK pathways was further delineated in HEK-293 cells expressing conditional MAPK mutants. We found hBD2 expression exclusively dependent on the presence of the bacterial cag pathogenicity island, with NOD1 a critical host sensor. Impairment of murineβ-defensin 4 (an orthologue of hBD2) expression in NOD1-deficient mice 7-days post-infection further confirmed the role of this cytoplasmic pattern-recognition receptor in eliciting host innate immunity. In contrast to hBD2, hBD3 expression was NOD1-independent but EGFR and ERK pathway-dependent. Importantly, Toll-like receptor signaling was not implicated in H. pylori-mediated hBD2 and hBD3 gene expression. The divergent signaling events governing hBD2 and hBD3 expression suggest temporal functional variation, such that hBD2 may contribute to antimicrobial barrier function during the inflammatory phase with hBD3 playing a greater role during the repair, wound healing phase of infection.

TGF-α Regulates TLR Expression and Function on Epidermal Keratinocytes

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-α, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-α regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human β-defensins. These findings provide evidence that TGF-α up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.

Neutrophil primary granule proteins HBP and HNP1–3 boost bacterial phagocytosis by human and murine macrophages

In acute inflammation, infiltrating polymorphonuclear leukocytes (also known as PMNs) release preformed granule proteins having multitudinous effects on the surrounding environment. Here we present what we believe to be a novel role for PMN-derived proteins in bacterial phagocytosis by both human and murine macrophages. Exposure of macrophages to PMN secretion markedly enhanced phagocytosis of IgG-opsonized Staphylococcus aureus both in vitro and in murine models in vivo. PMN secretion activated macrophages, resulting in upregulation of the Fcgamma receptors CD32 and CD64, which then mediated the enhanced phagocytosis of IgG-opsonized bacteria. The phagocytosis-stimulating activity within the PMN secretion was found to be due to proteins released from PMN primary granules; thorough investigation revealed heparin-binding protein (HBP) and human neutrophil peptides 1-3 (HNP1-3) as the mediators of the macrophage response to PMN secretion. The use of blocking antibodies and knockout mice revealed that HBP acts via beta2 integrins, but the receptor for HNP1-3 remained unclear. Mechanistically, HBP and HNP1-3 triggered macrophage release of TNF-alpha and IFN-gamma, which acted in an autocrine loop to enhance expression of CD32 and CD64 and thereby enhance phagocytosis. Thus, we attribute what may be a novel role for PMN granule proteins in regulating the immune response to bacterial infections.