Jack B. Cowland

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.

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.

hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules

A 19 kDa protein was identified in specific granules of human neutrophils. A full‐length cDNA clone was isolated from a human CML cDNA library, based on amino‐acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL‐39/CAP‐18. Aminoacid sequences of proteolytic fragments derived both from the conserved N‐terminal cathelin‐like region and the highly variable C‐terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP‐18, stored as a ‘pro‐peptide’ in specific granules.

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.

The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

We show here that megalin, a member of the low‐density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown by surface plasmon resonance analysis. Furthermore, a rat yolk sac cell line known to express high levels of megalin, endocytosed NGAL by a mechanism completely blocked by an antibody against megalin.

The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules

The in vivo mRNA levels for 16 granule proteins during neutrophil differentiation were determined to address the question of whether the synthesis of granule proteins is regulated individually or blockwise. RNA was extracted from peripheral blood granulocytes and three different populations of neutrophil precursors isolated from human bone marrow by Percoll density centrifugation. The mRNA levels in relation to the maturation of the cells were determined by Northern blot for the 12 matrix proteins myeloperoxidase, proteinase‐3, elastase, defensin, lactoferrin, NGAL, hCAP‐18, transcobalamin‐I, SGP28, gelatinase, lysozyme, and serglycin and the 4 membrane proteins CD68, CD11b, N‐formyl‐methionyl‐leucyl‐phenylalanine receptor, and CD35. This panel of transcripts ensured that markers for all exocytosable organelles of the neutrophil were included in the study. A highly differentiated distribution of mRNAs for granule proteins was demonstrated that can explain the heterogeneity of the intracellular storage granules and secretory vesicles of the neutrophil. Furthermore, the individual distribution of these transcripts provides the basis for a more detailed assessment of neutrophil maturation than that obtained by morphological studies or the use of a single marker protein for azurophil, specific, and gelatinase granules. J. Leukoc. Biol. 66: 989–995; 1999.

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.

MicroRNAs and cancer

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non‐protein‐encoding pri‐miRNAs are synthesized by RNA polymerase II and post‐transcriptionally modified by addition of a 5′‐cap and a 3′‐poly (A) tail. Subsequently, the pri‐miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature ∼22 nt miRNA, which can exert its function by binding to the 3′‐untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi‐RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor‐ and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target‐interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco‐miRs or tumor suppressor‐miRs, and may turn out to be potential targets for cancer therapy.

Neutrophil gelatinase-associated lipocalin, a siderophore-binding eukaryotic protein

NGAL (neutrophil gelatinase-associated lipocalin) also known as lcn2 or siderochalin is constitutively expressed in myelocytes and stored in specific granules of neutrophils. It is highly induced in a variety of epithelial cells during inflammation. Analysis of the crystal structure of NGAL expressed in E.coli showed that NGAL has the ability to bind catecholate type siderophores and in this way prevent bacteria from acquisition of siderophore-bound iron. NGAL (or 24p3 as the highly homologous murine orthologue is named) knock out mice have a profound defect in defense against E.coli after intraperitoneal injection. This defect can be mimicked in wild-type mice by providing siderophore iron, which cannot be sequestered by NGAL, testifying to the specific role of NGAL as a siderophore binding protein in innate immunity. Megalin, a scavenger receptor functions as a receptor for NGAL and mediates uptake into endosomes, but other NGAL receptors are likely to exist.

IL-1β-Specific Up-Regulation of Neutrophil Gelatinase-Associated Lipocalin Is Controlled by IκB-ζ

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding protein that exerts a bacteriostatic effect by sequestering iron. Strong induction of NGAL synthesis has been observed in inflamed epithelium of the lungs and colon. Expression of NGAL is up-regulated in the lung epithelial cell line A549 by IL-1β, but not by TNF-α, despite an induction of NF-κB binding to the NGAL promoter by both cytokines. In this study, we present evidence that the IL-1β specificity is caused by a requirement of the NGAL promoter for the NF-κB-binding cofactor IκB-ζ for transcriptional activation. Up-regulation of NGAL expression in A549 cells following IL-1β stimulation was dependent on de novo protein synthesis and was greatly diminished by a small interfering against IκB-ζ mRNA. Cotransfection of A549 cells with a plasmid expressing IκB-ζ made TNF-α capable of inducing NGAL transcription, indicating that IκB-ζ induction is the only factor discriminating between IL-1β and TNF-α in their ability to induce NGAL expression. Coexpression of the cofactor Bcl-3, which is closely related to IκB-ζ, did not enable TNF-α to induce NGAL transcription. A functional NF-κB site of the NGAL promoter was required for IκB-ζ to exert its effect. The human β defensin 2 gene also required IκB-ζ for its IL-1β-specific induction in A549 cells. Our findings indicate that a common regulatory mechanism has evolved to control expression of a subset of antimicrobial proteins expressed in epithelial cells.