Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice.

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Differential roles of macrophages in diverse phases of skin repair.

Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macrophages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events.

TGF-beta signaling in thymic epithelial cells regulates thymic involution and postirradiation reconstitution.

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.

TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves.

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.

Granulocyte function in grancalcin-deficient mice.

Grancalcin, one of the penta-EF-hand Ca(2+) binding proteins, is expressed at high levels in polymorphonuclear granulocytes (neutrophils). EF-hand proteins are implicated in the regulation of diverse processes including cell migration, apoptosis, and mobilization of neutrophil effector functions. To determine the role of grancalcin in vivo, we inactivated the gene encoding grancalcin (Gca) in embryonic stem cells and generated grancalcin-deficient mice. Homozygous Gca mutants appeared healthy and reproduced normally. Leukocyte recruitment into the peritoneal cavity upon induction of inflammation was not significantly affected by the absence of grancalcin. The mutants also resisted systemic fungal infection similarly to wild-type mice, and in vitro killing of Staphylococcus aureus by inflammatory cells was not significantly impaired. While marginally increased survival rates of mutants faced with endotoxic shock may indicate a contribution of grancalcin to immunopathogenesis, it is not essential for vital leukocyte effector functions required to control microbial infections.

Catalase negative Staphylococcus aureus retain virulence in mouse model of chronic granulomatous disease.

Myeloperoxidase-mediated chlorination is thought to be a necessary microbicidal mechanism. The H2O2 required for this process is generated by the NADPH oxidase. Staphylococcus aureus can also produce H2O2, which is not broken down by catalase negative organisms. It has been thought that this bacterial H2O2 can substitute for cellular H2O2 in the halogenation reaction in chronic granulomatous disease (CGD) where neutrophils are lacking the NADPH oxidase. We have readdressed this issue in a mouse model of CGD using clinical isolates of catalase positive and negative strains of S. aureus. The results showed these organisms to be equally virulent and that the H2O2 they produced is insufficient to cause significant iodination, a marker for chlorination, thereby contradicting the accepted views on this subject.

Key roles for transforming growth factor beta in melanocyte stem cell maintenance.

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor beta (TGF-beta) signaling plays important roles in this process. In vitro, TGF-beta not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-beta signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-beta type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-beta signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.

Mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fibrosis.

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins.

The gastrointestinal tract is constantly challenged by foreign antigens and commensal bacteria but nonetheless is able to maintain a state of immunological quiescence. Recent advances have highlighted the importance of active suppression by regulatory lymphocytes and immunosuppressive cytokines in controlling mucosal immunity. Failures of these mechanisms contribute to the development of inflammatory bowel disease, but how these regulatory networks are established remains unclear. Here, we demonstrate key roles for alphav integrins in regulation of mucosal immunity. We report that deletion of alphav in the immune system causes severe colitis, autoimmunity, and cancer. Mice lacking immune cell alphav have fewer regulatory T (Treg) cells in the colon and corresponding increases in activated T cells and T cell cytokine production, leading to colitis. Using conditional gene targeting, we demonstrate that this is specifically attributable to loss of alphav from myeloid cells. Furthermore, we show that gut-associated macrophages and dendritic cells fail both to remove apoptotic cells efficiently and to induce Treg cells. Our results identify a vital role for myeloid alphav integrins in generating mucosal Treg cells and emphasize the importance of antigen-presenting cells in establishing immune tolerance.

The role of grancalcin in adhesion of neutrophils.

Grancalcin is a protein specifically expressed in neutrophils and monocytes/macrophages. The function of grancalcin has not been identified. Grancalcin-deficient neutrophils were previously demonstrated to exert normal recruitment to the inflamed site, NADPH oxidase activation, extracellular release of secondary granules, apoptosis and activation-induced Ca2+ flux. In this study we analyzed granule numbers in resting and activated grancalcin-deficient neutrophils, their phagocytic activity and adherence to extracellular matrix proteins. Results revealed normal phagocytosis and degranulation of grancalcin-deficient neutrophils, while their adhesion to fibronectin was decreased by 60%. Consistently, the processes associated with neutrophil adhesion, such as formation of focal adhesion complexes and spreading, were also impaired in grancalcin-deficient neutrophils by 89 and 38%, respectively. In contrast, adherence to other extracellular matrix proteins: collagen, laminin and vitronectin, was not significantly altered. We thus report for the first time a function of grancalcin.

Expression and alternative processing of IL-18 in human neutrophils.

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species. Using specific protease inhibitors, and serine protease gene-targeted mice, we demonstrate that these IL-18-derived products arose through caspase-independent cleavage events mediated by the serine proteases, elastase and cathepsin G. Moreover, we report that the net effect of elastase treatment of mature recombinant IL-18 was to reduce its IFN-gamma-inducing activity. Thus, human neutrophils contain IL-18 and IL-18-derived molecular species that can arise through novel enzymatic processing pathways. Through cytosolic, membrane or secretory expression of such processing enzymes, together with generation of IL-18 itself, neutrophils likely play a critical role in regulating IL-18 activities during early innate immune responses.

Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death.

Mouse embryos genetically null for all alphav integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating alphav expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of alphav expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of alphav integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that alphav integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for alphav integrins expressed on axons in the postnatal central nervous system.

Patterned variation in murine MHC promoters.

To compare variation in regulatory and coding DNA, promoter sequences have been obtained from wild-derived mice and laboratory rats. The sequences are from the proximal promoter of the H2Aa, H2Ab, H2Eb, and H2K genes of 24 wild-derived inbred strains and a sample of the corresponding exon 2 sequences and of the RT1.Ba gene of six strains of laboratory rat. They reveal a high level of variation in the mouse MHC class II promoters (H2A and H2E), a low level in MHC class I (H2K), and none in the rat. The variation is pronounced in and around the cAMP response element, a major binding site for modulating promoter activity in response to external stimulation. This finding, together with the different levels of variation in MHC classes I and II, is suggestive of natural selection. However, selection operating via the MHC coding sequences must also contribute, as indicated by the minimal variation in both the MHC class II promoter and coding sequences of the rat. Furthermore CIITA (trans-activator of class II) of the mouse has been reported to have minimal variation in its promoter and none in its coding sequence. Taken together these data suggest that the regulatory and coding sequences undergo coselection. Each of the mouse class II promoters has a pattern of variation that appears to be basically dimorphic, with further variation added by recombination/mutation. The dimorphic allelic lineages are in marginally detectable linkage disequilibrium with the exon 2 sequences, particularly in H2Aa, thus lending further support to the coevolution hypothesis.

Redirection of B cell responsiveness by transforming growth factor beta receptor.

The multifunctional transforming growth factor beta receptor (TbetaR) ligand pair plays a central role in the regulation of lymphocyte homeostasis and prevention of autoimmunity. Although the mechanisms underlying the induction of transcriptional modulators by TbetaR have been studied in considerable detail, relatively little is known about the regulatory pathways targeted. To shed light on the mechanisms involved in negative regulation of B cell responses we identified TbetaR-dependent transcriptome changes by comparative gene expression profiling of normal and TbetaR-deficient primary B cells. The data reveal TbetaR-mediated induction of inhibitors of antigen receptor signaling (Ship-1, CD72) as well as inhibitors of the Jak/Stat pathway and signaling by means of Toll-like receptors (SOCS1,3). These inhibitory effects are complemented by induction of antiproliferative transcription factors. In contrast to this inhibition, G protein-coupled receptors such as CXCR4 and agonists mediating Ca2+ flux (inositol trisphosphate receptor subtype 2) are induced by TbetaR, indicating enhancement of the Ca2+ storage/ release system and chemotactic responses. Suppression of proapoptotic genes suggests support of cell survival. Confirming the shift in B cell responsiveness, antigen-receptor-mediated activation of Syk and phospholipase C-gamma2, as well as Stat6 phosphorylation, is inhibited, whereas chemotaxis, Ca2+ release, and cell survival are enhanced in transforming growth factor-beta-sensitive B cells. The data provide a molecular basis for TbetaR-mediated inhibition of B cell responsiveness and indicate that TbetaR maintains homeostasis not only through inhibition of the cell cycle but also by delivering a coherent instructive signal that redirects responsiveness to microenvironmental cues.

TGF-beta receptor signaling is critical for mucosal IgA responses.

TGF-beta receptor (TbetaR) signaling is important for systemic IgA production; however, its contribution to IgA secretion at mucosal sites remained uncertain. This important question was addressed using mice lacking the TbetaR in B cells (TbetaRII-B). Although reduced, IgA-secreting cells and IgA were still present in the systemic and mucosal compartments. The adaptive immune response was investigated after oral or nasal immunization using adjuvants acting on different molecular targets, namely, the cholera toxin B subunit and the macrophage-activating lipopeptide-2. Efficient Ag-specific cellular and humoral responses were triggered both in controls and TbetaRII-B mice. However, a significant reduction in Ag-specific IgG2b and increased levels of IgG3 were observed in sera from TbetaRII-B mice. Furthermore, Ag-specific IgA-secreting cells, serum IgA, and secretory IgA were undetectable in TbetaRII-B mice. These results demonstrate the critical role played by TbetaR in Ag-driven stimulation of secretory IgA responses in vivo.

Enhancer-deleted retroviral vectors restore high levels of superoxide generation in a mouse model of CGD.

BACKGROUND: Retroviral vectors possess many advantages for use in gene therapy protocols, especially within the haematopoietic system. A number of attendant problems, however, still limit their safety in clinical application. The effects of the enhancer present in the retroviral long terminal repeat (LTR) are a major concern for the clinical usage of such vectors, as they can exert a powerful regulatory influence on the genes that surround them. METHODS: To improve the safety and widen the applicability of retroviral vectors for use in gene therapy we have developed an enhancer-deleted (Delta-LTR) retroviral vector that retained high titre and demonstrated transcriptional activity in myeloid cells. RESULTS: When used to correct a mouse model of autosomal recessive chronic granulomatous disease, the Delta-LTR vectors gave acceptable levels of gene transfer to mouse bone marrow cells. Evidence for a slight preferential expression in myeloid cells was obtained with all the vectors studied. Nitroblue tetrazolium assay of superoxide generation in mouse bone marrow derived haematopoietic colonies revealed that transduction with Delta-LTR vectors could restore functional NADPH oxidase to cells from these animals. Superoxide assay of peripheral blood confirmed that, although relatively low numbers of cells were transduced, the Delta-LTR vector was capable of reconstituting very high levels of oxidase activity, comparable to that obtained from normal cells. CONCLUSIONS: The Delta-LTR vector described here could provide the basis for a new generation of retroviral vectors with improved safety.

C-terminal SRC kinase controls acute inflammation and granulocyte adhesion.

To establish whether the widely expressed regulator of Src family kinases Csk contributes to the control of acute inflammation in vivo, we inactivated csk in granulocytes by conditional mutagenesis (Cre/loxP). Mutant mice (Csk-GEcre) developed acute multifocal inflammation in skin and lung. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation with hyperinduction of integrins. This hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Hyperphosphorylation of key signaling proteins such as Syk and Paxillin in mutant granulocytes further supported breakdown of the activation threshold set by Csk. By enforcing the need for ligand engagement Csk thus prevents premature granulocyte recruitment while supporting the motility of stimulated cells through negative regulation of cell adhesion.

Ym1 is a neutrophil granule protein that crystallizes in p47phox-deficient mice.

Crystals were discovered within the aged lung and at sites of chronic inflammation in a mouse model of chronic granulomatous disease. Following re-crystallization at neutral pH, the crystals were identified as the chitinase-like protein Ym1, expressed in organs of the lymphoreticular system, the lung, and distal stomach. Ym1 was shown to be a neutrophil granule protein and to have weak beta-N-acetylglucosaminidase activity, indicating that it might contribute to the digestion of glycosaminoglycans. Crystal formation is likely to be a function of excess neutrophil turnover at sites of inflammation in the chronic granulomatous disease mouse. Failure to remove subcutaneous Ym1 crystals injected into knockout mice indicates that a failure of digestion may also contribute to crystallization.

Killing activity of neutrophils is mediated through activation of proteases by K+ flux.

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.