Selective fusion of azurophilic granules with Leishmania-containing phagosomes in human neutrophils.

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.

Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells.

Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating "pro(C)"-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

The tetraspanin CD63 is involved in granule targeting of neutrophil elastase.

Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)-positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63, with an adaptor protein-3-dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady-state level was similar to wild type in CD63-depleted clones, making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63-depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention, and granule targeting of proNE before storage as mature NE.

Rab27a regulates exocytosis of tertiary and specific granules in human neutrophils.

The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinase-enriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca(2+) and GTP-gamma-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation.

Granule targeting of soluble tumor necrosis factor (TNF) receptor expressed during granulopoietic maturation in murine bone marrow cells.

In this experiment, we explored the potential of secretory lysosomes of hematopoietic cells to act as vehicles for immunomodulatory protein delivery at an inflammation site. We investigated whether exogenous soluble TNF-receptor 1 (sTNFR1) could be expressed in primary hematopoietic progenitor cells and become targeted for storage and secretion during granulopoietic differentiation. An sTNFR1 construct with a transmembrane domain (tm) and a cytosol sorting signal (Y) taken from CD63, was retrovirally transduced to lineage-negative murine hematopoietic bone marrow stem/progenitor cells. This process was followed by cytokine-driven granulopoietic maturation. The sTNFR1-tm-Y was found to be synthesized in precursor cells and to persist in mature granulocytes and monocytes/macrophages. Immunofluorescence-localization studies showed a granule pattern of sTNFR1-tm-Y in both precursor and mature granulocytes and secretion to phagosomes after ingestion of bacteria. Immunoelectron microscopy revealed co-localization between the sTNFR1-tm-Y and the primary (azurophil) granule marker myeloperoxidase. Collectively, our results demonstrated granule targeting, storage, and secretion of exogenous sTNFR1-tm-Y constitutively expressed during normal granulopoietic differentiation. These findings support the concept of using storage organelles of circulating hematopoietic cells as vehicles for targeting sites of inflammation with immunoregulatory agents.

Prodefensins are matrix proteins of specific granules in human neutrophils.

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1-3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.

Activated human PMN synthesize and release a strongly fucosylated glycoform of alpha1-acid glycoprotein, which is transiently deposited in human myocardial infarction.

Alpha1-acid glycoprotein (AGP) is a major acute-phase protein present in human plasma as well as in polymorphonuclear leukocytes (PMN). In this report, we show that PMN synthesize a specific glycoform of AGP, which is stored in the specific and azurophilic granules. Activation of PMN results in the rapid release of soluble AGP. PMN AGP exhibits a substantially higher apparent molecular weight than plasma AGP (50-60 kD vs. 40-43 kD), owing to the presence of strongly fucosylated and sialylated polylactosamine units on its five N-linked glycans. PMN AGP is also released in vivo from activated PMN, as appeared from studies using well-characterized myocard slices of patients that had died within 2 weeks after an acute myocardial infarction. AGP was found deposited transiently on damaged cardiomyocytes in areas with infiltrating PMN only. It is interesting that this was inversely related to the deposition of activated complement C3. Strongly fucosylated and sialylated AGP glycoforms have the ability to bind to E-selectin and to inhibit complement activation. We suggest that AGP glycoforms in PMN provide an endogenous feedback-inhibitory response to excessive inflammation.

Highly glycosylated alpha1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils.

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer-binding protein (C/EBP)-epsilon in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

Compound exocytosis of granules in human neutrophils.

Human neutrophils are of prime importance for the immune defense. Recent data from eosinophils and pancreatic beta cells have indicated that granules, upon exocytosis, occasionally fuse with each other in the cytosol prior to their subsequent fusion with the plasma membrane. This is termed compound exocytosis. We therefore studied exocytosis of single granules from human neutrophils by the high-resolution cell-attached patch-clamp capacitance technique. We found that 1.5% of the capacitance steps was greater than 5 fF, i.e., significantly larger than steps expected for exocytosis of single granules. The mean step size of these events was 20.5 fF, corresponding to compounds formed by at least five granules. The capacitance input from compound steps contributed more than 20% of the total capacitance increase. Electron microscopy captured morphological manifestations of transient exocytic events, confirming the functional results obtained by capacitance measurements. Compound exocytosis may be a mechanism for efficient targeting of release during exocytosis.

Sorting soluble tumor necrosis factor (TNF) receptor for storage and regulated secretion in hematopoietic cells.

Hematopoietic cells contain secretory lysosomes that degranulate at sites of inflammation. We envisage that secretory granules can act as vehicles for targeting inflammatory sites, including malignancies, and thereafter, locally release therapeutically active agents to these sites. Exogenous proteins, such as the soluble tumor necrosis factor receptor 1 (sTNFR1), have been shown previously to be targeted to secretory lysosomes [1]. In this work, we asked whether exogenous, secretory lysosome-targeted proteins were subject to regulated secretion. sTNFR1-transmembrane (tm)-cytosol-sorting signal (Y) and sTNFR1-tm-Y-enhanced green fluorescent protein (egfp) were expressed in rat basophilic leukemia cell clones having different secretory capacities. sTNFR1-tm-Y was targeted directly from the Golgi to secretory lysosomes, followed by generation of membrane-free sTNFR1, whose secretion could be triggered by a Ca2+ ionophore or immunoglobulin E receptor activation. In contrast, sTNFR1-tm-Y-egfp was targeted to the plasma membrane and then subjected to endocytosis and presumably, secretory lysosome targeting, as judged by results from antibody ligation and cell-surface biotinylation. Activation of protein kinase C with phorbol ester promoted ectodomain shedding at the cell surface, resulting in sTNFR1 release from sTNFR1-tm-Y-egfp. These results support a concept for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.

Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils.

Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense.

Sorting of neutrophil-specific granule protein human cathelicidin, hCAP-18, when constitutively expressed in myeloid cells.

Neutrophil granulocytes carry storage organelles, e.g., azurophil and specific granules. Poorly understood are the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Therefore, we asked whether the specific granule protein human cathelicidin (hCAP-18) could be sorted for storage in other granules when the biosynthetic window is widened to allow this. We observed that hCAP-18 was targeted for storage in lysosome-related organelles when expressed constitutively in the rat basophilic leukemia and the mouse promyelocytic (MPRO) cell lines. In addition, premature release of the antibiotic C-terminal peptide LL-37 was observed. Retention of hCAP-18 was diminished by induction of differentiation of MPRO cells. In conclusion, a specific granule protein with native conformation may be sorted for storage in lysosome-related organelles of myeloid cells and converted prematurely to a supposedly biologically active form.

Localization of serglycin in human neutrophil granulocytes and their precursors.

Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neutrophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in humans during neutrophil differentiation. Immunocytochemistry revealed serglycin immunoreactivity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed serglycin immunoreactivity located to the Golgi apparatus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil granulocytes were obtained. Serglycin immunoreactivity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesicles by Western blotting and enzyme-linked immunosorbent assay. Serglycin was not detected in subcellular fractions containing primary, secondary, or tertiary granules. Together, these findings indicate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there.

Targeting proteins to secretory lysosomes of natural killer cells as a principle for immunoregulation.

Secretory lysosomes of natural killer (NK) cells combine storage, regulated secretion and lysosomal activity. We asked whether one could target exogenous proteins to the secretory lysosomes of NK-cells for final delivery into a tumor site upon degranulation. cDNAs for both soluble and transmembrane (tm) proteins were expressed in the human YT-Indy NK-cell line. Targeting of a soluble TNF receptor (sTNFR1) was achieved by expressing a cDNA construct with a transmembrane sequence to facilitate ER-export and by incorporating a cytosolic sorting signal (Y) from CD63 to overcome constitutive secretion. The resulting sTNFR1-tm-Y was targeted to secretory lysosomes as confirmed by results from biosynthetic radiolabeling in combination with subcellular fractionation, immunoelectron microscopy, and immunofluorescence microscopy. A soluble sTNFR1 form was generated in the secretory lysosome by endogenous proteolytic activity. Expression of exogenous normally secretory non-membrane proteins, such as alpha1-microglobulin (alpha1-m) and alpha1-antitrypsin (alpha1-at) resulted mostly in constitutive secretion although a small amount of alpha1-microglobulin was targeted to secretory lysosomes. Our results suggest a potential for delivery of pharmacologically active agents into tumor sites by use of the NK-cell secretory lysosome as a carrier.

Intracellular location of syntaxin 7 in human neutrophils.

Neutrophils are the first line of defense in the innate immune system. Neutrophils neutralize invading microorganisms mainly by phagocytosis, but the mechanism and molecules involved in this process are not well characterized. Because the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin 7 regulates vesicle trafficking events in phagocytosis, we investigated the expression and subcellular localization of syntaxin 7 in human neutrophils. Here we have found that human peripheral blood neutrophils and neutrophil-differentiated HL-60 cells express syntaxin 7 at both mRNA and protein levels. Using biochemical and ultrastructural approaches, we found that syntaxin 7 was broadly located in the membranes of the three major cytoplasmic granules of human neutrophils, with a major location in azurophilic granules, which are mainly involved in phagocytosis. A secondary, but extensive, location of syntaxin 7 was in specific and tertiary granules, which resulted translocated to the plasma membrane upon cell activation that promoted mobilization of these organelles. These data reveal the presence of syntaxin 7 in the membranes of exocytosis-prone granules (specific and tertiary granules) and phagocytosis-related granules (azurophilic granules) in human neutrophils, and therefore it might play a role in both exocytosis and phagocytosis in human neutrophils.

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils.

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.

Haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation.

Haptoglobin (Hp) is a plasma protein synthesized primarily by hepatocytes. It exerts a broad range of anti-inflammatory activities and acts indirectly as a bacteriostatic agent and an antioxidant by virtue of its ability to bind free hemoglobin (Hb) and to facilitate its immediate clearance by macrophages. We identified Hp as a novel specific granule protein of neutrophils by means of immunoelectron microscopy, subcellular fractionation, and exocytosis studies. Consistent with these findings, blood cells from a patient with specific granule deficiency (SGD) lacked neutrophil-derived Hp. Neutrophils contained a large amount of highly glycosylated Hp (beta-chain 45-65 kDa) synthesized in neutrophil precursors and stored in specific granules and a small amount of Hp (beta-chain 39 kDa) endocytosed from plasma and stored in secretory vesicles. Subsequent binding studies revealed that Hp from specific granules binds to Hb. Finally, the CCAAT enhancer binding protein-epsilon (C/EBPepsilon) induced Hp transcription in a myeloid cell line, suggesting that Hp expression in myeloid cells, as in hepatocytes, is at least partially regulated by members of the C/EBP transcription factor family. Collectively, these findings demonstrate that Hp is stored in specific granules and is released by neutrophils in response to activation. Hence, neutrophil-derived Hp might reduce tissue damage and bacterial growth at sites of infection or injury by propagating anti-inflammatory activities and Hb clearance.

Keratinocytes display normal proliferation, survival and differentiation in conditional beta4-integrin knockout mice.

The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised.