Novel findings on cellular trafficking and targeting for granule storage of neutrophil elastase, a multifunctional effector molecule of innate immunity

Neutrophil elastase (NE) has important roles in innate immunity, killing pathogens and controlling the immune response; but how NE is targeted to developing granules is not understood. Therefore, the aim of this thesis was to investigate the sorting of NE. Transfection experiments in a leukemic cell line, which were confirmed in normal hematopoietic cells, showed that a population of proNE was targeted to the plasma membrane and endocytosed. This targeting required an intact carboxy-terminal propeptide. Furthermore, modified proNE was not endocytosed, indicating structural requirements for endocytosis. An association was demonstrated between the tetraspanin CD63 and proNE upon coexpression in COS cells. Furthermore, depletion of CD63 in a promyelocytic cell line (achieved by RNA interference or a CD63 mutant) caused reduced processing of proNE into mature NE and reduced constitutive secretion of proNE. We therefore propose that CD63 may be a transmembrane linker that facilitates granule targeting of proNE. Results from a monocytic cell line indicated that the sorting of proNE was a multistage process including trafficking to the cell surface, endocytosis through coated vesicles and possibly lipid rafts, and possible conversion to mature NE in late endosomes. The inhibition of proNE’s activation into mature NE was accompanied by the accumulation of proNE, suggesting a requirement for activation before granule targeting. This research provided new perspectives on the cellular trafficking of NE. The thesis proposes that granule sorting of proNE is facilitated by a tetraspanin protein serving as a transmembrane linker and transporter. The hypothesis needs further testing in primary cells to acquire additional evidence of the interactions involved

Kinin System Activation in Vasculitis

The kinin system is activated when high-molecular-weight kininogen (HK) is cleaved by plasma kallikrein thus generating bradykinin. Bradykinin is a potent proinflammatory peptide that induces plasma leakage, blood pressure drop, liberation of inflammatory cytokines and pain. Vasculitis is an autoimmune systemic inflammatory disease, characterized by leukocyte inflammation in and around vessel walls leading to perturbed vessel patency and tissue damage. Many different organs may be afflicted but most commonly the kidney, respiratory tract and skin are involved. Some patients with severe vasculitis have circulating antibodies against neutrophil enzymes called anti-neutrophil-cytoplasmic antibodies (ANCAs). Theoretically kinin system activation may explain some of the inflammation seen during vasculitis. In this thesis we demonstrate, for the first time, activation of the kinin system in patients with vasculitis. Elevated kinin levels were demonstrated in the circulation and kinins were detected at sites of inflammation. We also found that neutrophil-derived proteinase 3 (PR3) cleaves HK liberating a novel vasoactive kinin, termed PR3-kinin. PR3-kinin binds to and activates kinin B1-receptors both in vitro and in vivo. In addition, PR3-ANCAs from patients with vasculitides inhibit PR3-induced HK proteolysis and subsequent PR3-kinin release. In the MRLlpr/lpr mouse, that develops systemic inflammation and vasculitis, we demonstrate B1-receptor upregulation both systemically, on circulating leukocyte-derived microparticles (MPs), and locally, in the renal vasculitic lesions. In conclusion, we demonstrate kinin system activation in vasculitis and suggest that it may partake in the pathogenesis of this inflammatory condition. We therefore propose that inhibiting kinin system activation by blockage of B1-receptors may prove to be effective by reducing the inflammatory response during vasculitis

Cross-presentation Is A Source of Tumor Antigens For Multiple Myeloma Immunotherapy

Cross-presentation is an essential bridge between the innate and adaptive arms of the immune system where antigen presenting cells (APCs) prime cytotoxic T cell responses. We have recently identified cross-presentation as a mechanism by which solid tumors present exogenous antigens. We therefore hypothesized that multiple myeloma would be capable of cross-presentation as these cells are derived from B cells, known APCs. We explored the capacity of multiple myeloma to cross-present PR1, a human leukocyte antigen (HLA)-A2 nonameric peptide that is derived from neutrophil elastase (NE) and proteinase 3 (P3), and the ability to treat multiple myeloma using PR1-targeting immunotherapies. Here we demonstrate that multiple myeloma cells lack endogenous NE and P3 expression, possess the ability to take up exogenous NE and P3 and cross-present PR1. This process employs the cytosolic antigen presentation machinery including the proteasome, Golgi, and TAP. Subsequent PR1 cross-presentation renders multiple myeloma cells susceptible to PR1-CTL and anti-PR1/HLA-A2 antibody, both in vitroand in vivo. To our knowledge, this is the first report of multiple myeloma cross-presenting tumor antigens. Collectively, our data demonstrate that PR1 is a novel tumor antigen in multiple myeloma and can be effectively targeted using PR1-targeting immunotherapies. Our study suggests that the multiple myeloma antigen repertoire is much larger than previously appreciated, and that there is a new catalogue of potential immunotherapeutic targets in multiple myeloma that can be derived from exogenous antigens

IMBALANCE BETWEEN NEUTROPHIL ELASTASE AND ELAFIN PROMOTES BREAST CANCER GROWTH AND PROGRESSION

Elafin, an endogenous serine protease inhibitor, is a critical component of the epithelial barrier against neutrophil elastase (NE) activity. The central hypothesis examined in this dissertation was that elafin has tumor suppressive properties in breast cancer. In support of this hypothesis, immunohistochemical (IHC) analysis revealed that elafin was downregulated in the majority of invasive breast tumors and a subset of pre-invasive ductal carcinoma in situ (DCIS) compared to elafin expression in the normal mammary epithelium. To understand the role of elafin in the mammary epithelium and the impetus for its downregulation during breast tumorigenesis, primary and immortalized human mammary epithelial cells (HMECs) were utilized as a model system. Elafin was highly expressed in G0-arrested HMECs, suggesting a previously unrecognized role for elafin in growth control. Stable knockdown (KD) of elafin compromised the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation. This effect was reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function, suggesting a role for deregulated protease activity. Elafin KD HMECs demonstrated enhanced sensitivity to NE-induced proliferation. Mechanistically, activation of the ERK signaling pathway downstream of toll-like receptor 4 (TLR4) was essential to the mitogenic effect of NE in this system. Compared to HMECs, the majority of breast cancer cell lines lack endogenous elafin expression. Adenoviral-mediated expression of elafin was utilized to evaluate the tumor suppressive properties of elafin in breast cancer cell lines. Rb-status was identified as the critical factor governing the anti-tumor effect of elafin in this system. In breast cancer cell lines expressing functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. However, in breast cancer cell lines lacking functional Rb, elafin expression induced caspase-3 dependent apoptotic cell death. Elafin is a critical counterbalance against NE-activity. IHC analysis revealed that high levels of NE-expressing tumor-associated neutrophils (TAN) were associated with reduced recurrence-free survival, high tumor grade, and triple-receptor negative breast cancer (TNBC). ERK-catalyzed phosphorylation of p90RSK (T359/S363) and Rb phosphorylation (S807/811) were significantly enriched in NE-positive breast tumors, suggesting that NE-induced ERK signaling and proliferation may be relevant to human breast cancer. The in vivo role of deregulated NE in breast tumorigenesis was examined in the C3(1)TAg mouse model of TNBC. Knockout of NE in C3(1)TAg mice significantly reduced tumor growth and proliferation. Elafin has tumor suppressive properties in the context of breast cancer and is a critical counterbalance against the growth promoting effect of NE in vitro and in vivo. Deregulated NE-activity is a viable therapeutic target in breast cancer

Modulation of dendritic cells by human neutrophil elastase and its inhibitors in pulmonary inflammation

Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs

Activation and inactivation of the complement anaphylatoxins during chronic neutrophilic inflammation of the cystic fibrosis airway

Cystic fibrosis (CF) is a fatal genetic disease that affects 1/2500 people in the UK. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause dehydration of mucosal membranes, leading to mucus obstruction of the small airways. The CF lungs are susceptible to recurrent infection promoting chronic neutrophilic inflammation. Neutrophils recruited to the CF lungs become dysfunctional and are ineffective at clearing pathogens, perpetuating inflammation. Neutrophil serine proteases (NSPs) released by neutrophils collaterally remodel the airway, reducing lung function and causing mortality. The complement anaphylatoxins (C5a and C3a) are important mediators of inflammation. C5a and C3a are chemotactic for monocytes and granulocytes, they also stimulate degranulation and the generation of reactive oxygen species (ROS). C5a is particularly potent towards neutrophils and is critical for orchestrating their response towards pathogens. C5a and C3a are elevated in the CF airway. Furthermore, in addition to complement activation these anaphylatoxins can be generated by non-complement proteases including NSPs. The mechanisms by which C5a and C3a promote chronic neutrophilic inflammation in the CF airways are not fully understood. In my study I show that C5a and C3a correlate with markers of neutrophilic inflammation (neutrophil count and CXCL8) in bronchoalveolar lavage (BAL) fluid from paediatric CF patients. I further characterise the generation of functionally active C5a-like and C3a-like forms by NSPs. Moreover, I demonstrate that atypical C5a production by NSPs cannot be prevented by therapeutic complement inhibitors. I show for the first time that NSP-generated C5a-like fragments are resistant to inactivation by carboxypeptidase B, an important regulator of C5a activity. I also further characterise the interaction between C5a and soluble glycosaminoglycans (GAGs), these are abundant during chronic neutrophilic inflammation of the CF airway. Additionally, I show that GAG interaction influences C5a activity. In conclusion, the CF airway environment modifies C5a function; these mechanisms could promote chronic neutrophilic inflammation

Detection of ligand-gated chloride ion channels on human lymphocytes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Origin and expansion of the serine protease repertoire in the myelomonocyte lineage.

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold