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Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans. [ABSTRACT FROM AUTHOR]

A disintegrin and a metalloproteinase domain (ADAM) 9 is known to be expressed by monocytes and macrophages. In this study, we report that ADAM9 is also a product of human and murine polymorphonuclear neutrophils (PMNs). ADAM9 is not synthesized de novo by circulating PMNs. Rather, ADAM9 protein is stored in the gelatinase and specific granules and the secretory vesicles of human PMNs. Unstimulated PMNs express minimal quantities of surface ADAM9, but activation of PMNs with degranulating agonists rapidly (within 15 min) increases PMN surface ADAM9 levels. Human PMNs produce small quantities of soluble forms of ADAM9. Surprisingly, ADAM9 degrades several extracellular matrix (ECM) proteins, including fibronectin, entactin, laminin, and insoluble elastin, as potently as matrix metalloproteinase-9. However, ADAM9 does not degrade types I, III, or IV collagen or denatured collagens in vitro. To determine whether Adam9 regulates PMN recruitment or ECM protein turnover during inflammatory responses, we compared wild-type and Adam9-/- mice in bacterial LPS- and bleomycin-mediated acute lung injury (ALI). Adam9 lung levels increase 10-fold during LPS-mediated ALI in wild-type mice (due to increases in leukocyte-derived Adam9), but Adam9 does not regulate lung PMN (or macrophage) counts during ALI. Adam9 increases mortality, promotes lung injury, reduces lung compliance, and increases degradation of lung elastin during LPS- and/or bleomycin-mediated ALI. Adam9 does not regulate collagen accumulation in the bleomycin-treated lung. Thus, ADAM9 is expressed in an inducible fashion on PMN surfaces where it degrades some ECM proteins, and it promotes alveolar-capillary barrier injury during ALI in mice. [ABSTRACT FROM AUTHOR]

Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer–based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1′, P2′, P3, P4, and P2 positions were tested. This enabled us to construct an α¹-proteinase inhibitor variant (Ile-Lys-Pro-Arg−/−Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils. [ABSTRACT FROM AUTHOR]

Various states of inflammation, including sepsis, are associated with hypoferremia, which limits iron availability to pathogens and reduces iron-mediated oxidative stress. Lipocalin 2 (Lcn2; siderocalin, 24p3) plays a central role in iron transport. Accordingly, Lcn2-deficient (Lcn2KO) mice exhibit elevated intracellular labile iron. In this study, we report that LPS induced systemic Lcn2 by 150-fold in wild-type mice at 24 h. Relative to wild-type littermates, Lcn2KO mice were markedly more sensitive to endotoxemia, exhibiting elevated indices of organ damage (transaminasemia, lactate dehydrogenase) and increased mortality. Such exacerbated endotoxemia was associated with substantially increased caspase-3 cleavage and concomitantly elevated immune cell apoptosis. Furthermore, cells from Lcn2KO mice were hyperresponsive to LPS ex vivo, exhibiting elevated cytokine secretion. Additionally, Lcn2KO mice exhibited delayed LPS-induced hypoferremia despite normal hepatic hepcidin expression and displayed decreased levels of the tissue redox state indicators cysteine and glutathione in liver and plasma. Desferroxamine, an iron chelator, significantly protects Lcn2KO mice from LPS-induced toxicity, including mortality, suggesting that Lcn2 may act as an antioxidant in vivo by regulating iron homeostasis. Thus, Lcn2-mediated regulation of labile iron protects the host against sepsis. Its small size and simple structure may make Lcn2 a déployable treatment for sepsis. [ABSTRACT FROM AUTHOR]

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

Lysophosphatidylcholine has been shown to enhance neutrophil functions through a mechanism involving the G protein-coupled receptor G2A. Recent data support an indirect effect of lysophosphatidylcholine on G2A rather than direct ligand binding. These observations prompted the hypothesis that other lysophospholipids (lyso-PLs) may also signal for human neutrophil activation through G2A. To this end, 1-oleoyl-2-hydroxy-sn-glycero-3-[phospho-L-choline], but also C18:1/OH lyso-PLs bearing the phosphoserine and phosphoethanolamine head groups, presented on albumin, were shown to signal for calcium flux in a self- and cross-desensitizing manner, implicating a single receptor. Blocking Abs to G2A inhibited calcium signaling by all three lyso-PLs. Furthermore, inhibition by both pertussis toxin and U-73122 established signaling via the Galphai/phospholipase C pathway for calcium mobilization. Altered plasma membrane localization of G2A has been hypothesized to facilitate signaling. Accordingly, an increase in detectable G2A was demonstrated by 1 min after lyso-PL stimulation and was followed by visible patching of the receptor. Western blotting showed that G2A resides in the plasma membrane/secretory vesicle fraction and not in neutrophil primary, secondary, or tertiary granules. Enhanced detection of G2A induced by lyso-PLs was paralleled by enhanced detection of CD45, confirming mobilization of the labile secretory vesicle pool. Together, these data show that lyso-PLs bearing various head groups redundantly mobilize G2A latent within secretory vesicles and result in G2A receptor/Galphai/phospholipase C signaling for calcium flux in neutrophils.

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-1beta, but not by TNF-alpha, despite an induction of NF-kappaB binding to the NGAL promoter by both cytokines. In this study, we present evidence that the IL-1beta specificity is caused by a requirement of the NGAL promoter for the NF-kappaB-binding cofactor IkappaB-zeta for transcriptional activation. Up-regulation of NGAL expression in A549 cells following IL-1beta stimulation was dependent on de novo protein synthesis and was greatly diminished by a small interfering against IkappaB-zeta mRNA. Cotransfection of A549 cells with a plasmid expressing IkappaB-zeta made TNF-alpha capable of inducing NGAL transcription, indicating that IkappaB-zeta induction is the only factor discriminating between IL-1beta and TNF-alpha in their ability to induce NGAL expression. Coexpression of the cofactor Bcl-3, which is closely related to IkappaB-zeta, did not enable TNF-alpha to induce NGAL transcription. A functional NF-kappaB site of the NGAL promoter was required for IkappaB-zeta to exert its effect. The human beta defensin 2 gene also required IkappaB-zeta for its IL-1beta-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.

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.

To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene expression in keratinocytes. Notably, the up-regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing.

We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte beta(2) integrins. We studied here the complex formation between proMMP-9 and alpha(M)beta(2), the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/alpha(M)beta(2) complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and alpha(M)beta(2)-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a beta(2) integrin-dependent manner. Peptides that bound to the alpha(M) integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/alpha(M)beta(2) complex may be part of the cell surface machinery guiding neutrophil migration.

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-1beta induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-alpha, IL-6, and LPS had no effect. Similar IL-1beta 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-kappaB consensus site was found to control the IL-1beta-specific up-regulation. Involvement of the NF-kappaB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-kappaB pathway, and by EMSA. TNF-alpha activation of NF-kappaB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-kappaB to the NGAL promoter was observed in vitro. IL-1beta specificity was not contained within the NF-kappaB site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-kappaB-binding sequence with that of the IL-8 promoter or with the NF-kappaB consensus sequence and by testing the NF-kappaB-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.

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.

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-alpha, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human beta-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.