A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies. The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice. In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Chemotaxis, the ability of a cell to migrate towards a directional stimulus, is a basic property of virtually all cells at some stage in their development. Chemotaxis is preceded by the development of a polarized cellular morphology. The region of the cell closest to the attractant forms a broad lamellipod. The contents of the cell flow forward into this lamellipod and the rear of the cell becomes constricted into a narrow uropod. These local differences in cell structure and function presumably reflect local differences in cell chemistry, but the chemical processes involved are poorly understood. Ca+2 is known to play a ubiquitous role as an essential second messenger in many cellular processes, but its role in chemotaxis is unclear. While many chemotactic stimuli cause Ca+2 to rise intracellularly, the relationship between this rise in Ca+2 and local changes in cell behavior has been difficult to understand. In my dissertation work, I directly tested the role of cellular Ca+2 changes in polarization and chemotaxis by simultaneously imaging intracellular Ca+2 and cell morphology. This work was carried out on single eosinophils isolated from the newt, Taricha granulosa, because of their large size (~100 um, when polarized) and rapid responsiveness (~20 um/min) to chemotactic stimuli present in newt serum. An imaging system was developed to simultaneously image cell behavior, and intracellular Ca+2 following microinjection of the Ca+2 sensitive fluorescent probe, Fura-2. Cell behavior was quantified from time lapse video images captured by a SIT video camera, stored on a video optical disk recorder, and later digitized for analysis. Quantitation was accomplished by interactively tracing the cell's outline and determining the position of the geometric centroid. Variation in the radius of the outline from the centroid was used to calculate a "polarization index", which could be monitored over time. Cell speed was calculated from the movement of the centroid over time. Agents which are known to interfere with Ca+2 signalling significantly inhibited both the polarization and the movement of cells in response to 10% newt serum. These treatments included: chelation of extracellular Ca+2 with EGTA, the organic Ca+2 channel antagonist, verapamil, the inorganic Ca+2 channel blocker, cobalt, the Ca+2 ionophore, ionomycin, and caffeine, an agent known to release Ca+2 from internal stores. In contrast, the K+ ionophore, valinomycin, and treatment of cells with dibutryl cAMP had no effect on cell behavior. The development of a polarized, motile morphology following stimulation of newt eosinophils with 10% serum was accompanied by a rise in intracellular Ca+2. In addition, Ca+2 in a polarized, moving cell was non-uniformly distributed and periodic elevations in intracellular Ca+2 were seen during changes in cell behavior. In turning cells, Ca+2 was significantly higher than in cells moving in a straight line and there was a clearly detectable gradient of Ca+2 within the cell. The region closest to the new direction of movement had the lowest Ca+2 and the rear of the cell was significantly higher. This gradient persisted following a turn, even though Ca+2 was much lower overall in cells moving in a straight line. A gradient of Ca+2 along the long axis of the cell might be important for the differential regulation of different regions of the moving cell. Loading cells with the cell-permeant, esterified form of Fura-2 revealed a region of high Ca+2 associated with the microtubule organizing center (MTOC). This region was surrounded by a membrane system labeled by the lipid soluble, membrane potential sensitive dye, DiOC6(3). This region of Ca+2 was depleted by caffeine treatment. These observations, coupled with the effects of caffeine on cell behavior, suggest that a Ca+2 storage site associated with the MTOC may play a role in regulating cell polarization and chemotaxis. The effects of releasing "caged calcium" on cell behavior and [Ca2+]i were examined as a means of directly testing the ability of changes in [Ca2+]i to regulate cell behavior. Although photolysis of the compound inhibited cell polarization and movement, technical problems made it difficult to attribute these effects entirely to the release of Ca2+. The results presented here, particularly the gradients of [Ca2+]i which were observed, suggest that local regulation of the cytoplasmic components involved in cell movement by local differences in [Ca2+]i could, in part, explain the regional specialization seen during this process. This form of regulation will be discussed in detail, as will potential mechanisms to test for its function during cell polarization and chemotaxis.

No description available.

In this thesis we have studied systems that driven by mechanisms broadly known as phoresis. More specifically, in the second chapter we calculate the excess noise in electrophoresis of a colloid due to microion fluctuations. In the next three chapters we study in detail a system of self-phoretic colloids, propelled by the energy released when an ambient fuel molecule makes contact with a catalytic region on the particle’s surface. We start with the behaviour of a single particle in a linear substrate gradient, then go on to study interactions between two particles due to their diffusion clouds, and finally obtain the collective equations of motion by a systematic coarse-graining of the microscopic Langevin dynamics. To understand the role of nonequilibrium fluctuations in an electrophoretic system we have theoretically analyzed the dynamics of a single colloidal particle in an externally applied electric field. We have studied the colloidal dynamics in two scenarios: a particle free to move in an unbounded fluid and a colloid near a wall which is stationary due to a balance between gravity and the electric field. The thermal motions of microions lead to an anisotropic, nonequilibrium noise, proportional to the field, in the effective Langevin equation for the colloid. The fluctuation-dissipation ratio depends strongly on frequency, in contrast to an equilibrium system, and the colloid if displaced from its steady-state position relaxes with a velocity not proportional to the gradient of the logarithm of the steady-state probability. Other measurable effects of this noise are a superdiffusive peak at short times and an enhanced diffusity at long times. We have then studied the effective potential and obtained a non-dimensional measure of the size of the excess noise. Possible extensions of this study to include the behaviour of the mean and fluctuation properties in the case of an applied alternating potential, and the effect of the excess noise on electrohydrodynamic aggregation of colloids. We next turn to a phoretic system that has been much studied in the recent years – active Janus colloids . On one hand these colloids are an important contribution to the general class of problems on self-propulsion at low Reynolds number. On the other hand since their behaviour can be tuned at the level of single particle we can ask how their collective behaviour depends on the swimmer design. This makes it a very rich field with lots of challenging questions. We first study the single particle behaviour of an active Janus colloid in an imposed substrate gradient, then build the two-particle interactions and ultimately the collective equations of motion by a generalisation of these results. Our work presents a new approach to active matter. We show theoretically how to design particles that are not only motile but can reorient in response to gradients, thus mimicking chemotaxis. We outline the collective behaviour emerging from these single-particle properties, including colloidal realisations of gravitational collapse, plasma oscillations and spontaneously ringing states, and present a phase diagram, in terms of single particle parameters, that can be tested in experiments. This provides a template to design collective behaviours of interest by tuning the surface properties of the colloids. We can also control the range of the interaction by varying the concentration of reactant. Our coarse-grained equations of motion for the polar orientation and number density fields for a collection of colloids propelled by and interacting through long-ranged dif-fusion fields are novel in a number of ways. This is the first example in active matter literature of a microscopic derivation of collective dynamics for particles interacting via long-ranged diffusion fields. The instabilities and possible phases that we predict are different from those in traditional flocking models, which consider only short-ranged aligning interactions. The long-ranged interactions of interest here cannot produce a globally polar ordered state, and we work in a concentration regime where steric and collisional interactions are not important. Instabilities towards flocking, and the advective nonlinearities of the Toner-Tu model, although not ruled out by the symmetries of our model, do not play a significant role in our system. The collective behaviour we predict will not be seen in purely locally interacting active-particle systems. The mechanisms at work in the “saturated” case where reactant is abundant cannot be viewed as totally generic features of collections of self-driven particles; they require interactions mediated by the production or consumption of long-ranged diffusing solute fields. Earlier work on saturated systems resolved neither interactions mediated by the polarity of the objects nor chemotactic effects. Their treatment truncated the equations at the level of the concentration [1]. In the “unsaturated” case more than one mechanism operates. One is related to the motility-induced phase separation discussed phenomenologically in refs. [2,3] (for which our system provides an important microscopic realisation). The other is due to chemo-taxis and phoresis which we report for the first time. Our expression of the various coefficients in the equaions of motion in terms of the single particle properties can also be used to design systems in which one or the other of these mechanisms dominate. We are now planning to study a collection of these particles in a fluid and examine the diffusion of a tracer particle as was done by Yeomans et al. [4] for hydrodynamic interactions. The Levy flights obtained in [4] is due to the long-ranged nature of the hydrodynamic fields, which cause effects like entrainment leading to interesting tracer dynamics. In this thesis we have considered colloids in which the symmetry axis of the colloid and the catalytic coat coincide. It might be of interest to consider cases when the axes are at an angle making the swimmer biaxial, or more complicated arrangements leading to chirality and thus rotation. Collective dynamics and two particle interaction between such swimmers can also be interesting. The formalism developed for the study of interaction between two active colloids through their diffusion fields and hydrodynamics can be extended to study their interaction with extended passive surfaces like walls or spheres. The collective dynamics of this class of active systems when it is confined between parallel walls is also of interest. Work in progress includes studies of the motion of the swimmer in a periodic array of passive colloids. In this study of collective dynamics, we have ignored the role of hydrodynamics, as the slowest decay of the field is 1/r3, which is subdominant to the decay of the chemical fields and in the dilute limit is expected to change things only qualitatively. However their role would be more important when we consider the stability of ordered structures like an aster in the saturated case. Another effect of hydrodynamics is to stir the fluid. It might be interesting to study the finite-P´eclet number regime [5, 6] of our system particularly in the unscreened region when advection of the scalar fields s and p by the velocity can affect clustering. We have derived the form of the nonlinear equations of motion in both the saturated and the unsaturated regimes. It will be interesting to investigate their relevance in the dynamics and phases that this extremely rich system can form. Even in the overdamped limit where we obtain an effective density equation it is not clear that the dynamics will resemble that of the Keller-Segel model due to the presence of the interesting nonlinear terms. Also, in this thesis, we have only looked at the fluid-like state of the system. We have just started exploring the high concentration regime where we can check the propensity of the system to develop crystalline order. In the screened limit where we obtain a condensation due a negative squared sound speed, it is posssible to study the condensation phenomenon in greater detail. In future we also plan to examine whether the tendency to condense at nonzero wavenumber (See Fig 5.1), i.e., microphase separation, can lead to liquid-crystalline phases like smectics. The systems described in this thesis are extremely rich and the few ideas mentioned above form just a small subset of the plethora of exciting theoretical and experimental explorations that can be performed with them. Since they can be “designed”, unlike biological substances, they can also become a test-bed for testing theoretical predictions of the nonequilibrium statistical mechanics of self-propelled systems.

Este estudo teve por objetivos: a) caracterizar o influxo de leucócitos (LC) induzido pelo veneno de Bothrops moojeni (VBm); b) avaliar o papel dos mastócitos (MC) e da histamina neste evento; c) analisar a liberação de TXA2, PGD2, LTB4, MCP-1 e KC e d) avaliar a capacidade do VBm desgranular MC in vivo e in vitro. A injeção intraperitoneal de VBm, em camundongos, causou o recrutamento de LC e neutrofilia. O tratamento dos animais com cromoglicato aboliu o influxo de LC enquanto a difenidramina, ranitidina e a tioperamida, antagonistas da histamina, reduziram o influxo de neutrófilos. Ainda, o veneno induziu a liberação de PGD2, TXA2, LTB4, MCP-1 e de KC e causou a desgranulação de MC in vivo e a liberação de -hexosaminidase de MC in vitro. Em conclusão, o VBm induz influxo de LC para o local de sua injeção. Este efeito depende da histamina, via receptores H1, H2 e H4 e da desgranulação de MC, que decorre de ação direta do veneno nestas células. A neutrofilia e o TXA2, LTB4, MCP-1 e KC devem contribuir para o influxo de leucócitos causado pelo VBm. / In this study the effects of Bothrops moojeni venom (BmV) on the cellular component of inflammatory responses and the mechanisms involved in this effect were investigated. The effects of venom on peritoneal and circulating leukocyte numbers and on the release of inflammatory mediators, such as LTB4, TXA2, PGD2, MIP-1 and KC, were assessed. The role of both mast cells and histamine receptors in leukocyte recruitment induced by BmV was assessed by selected pharmacological treatments. BmV caused a marked infiltration of leukocytes (3-24 h) when injected into the peritoneal cavity of mice. Neutrophils (PMN) were the predominant cell type in the early stages of response whereas macrophages (MN) were accumulated from 3 up to 24 h. Moreover, BmV increased blood neutrophil numbers at 3 h after injection. The BmV-induced leukocyte influx was abrogated by cromoglicate and significantly reduced either by difenidramine or ranitidine or tioperamide, histamine H1, H2 and H4 receptor antagonists, respectively, at 6 h after injection. Significant increments in peritoneal levels of LTB4, TXA2, PGD2, MIP-1 and KC were detected at distinct periods of time after venom injection. In addition, BmV induced mast cell degranulation both in vivo and in vitro. In conclusion, obtained data demonstrated the ability of BmV induce leukocyte recruitment into the site of its injection. This effect is dependent on mast cell activation and degranulation, which may be due to a direct effect of venom on these cells, and is mediated at least in part by histamine via H1, H2 and H4 receptors. Moreover, the ability of venom to mobilize leukocytes from bone marrow reserve compartments and to release the chemotactic mediators TXA2, LTB4, MCP-1 and KC may be relevant for leukocyte infiltration.

Este estudo teve por objetivos: a) caracterizar o influxo de leucócitos (LC) induzido pelo veneno de Bothrops moojeni (VBm); b) avaliar o papel dos mastócitos (MC) e da histamina neste evento; c) analisar a liberação de TXA2, PGD2, LTB4, MCP-1 e KC e d) avaliar a capacidade do VBm desgranular MC in vivo e in vitro. A injeção intraperitoneal de VBm, em camundongos, causou o recrutamento de LC e neutrofilia. O tratamento dos animais com cromoglicato aboliu o influxo de LC enquanto a difenidramina, ranitidina e a tioperamida, antagonistas da histamina, reduziram o influxo de neutrófilos. Ainda, o veneno induziu a liberação de PGD2, TXA2, LTB4, MCP-1 e de KC e causou a desgranulação de MC in vivo e a liberação de -hexosaminidase de MC in vitro. Em conclusão, o VBm induz influxo de LC para o local de sua injeção. Este efeito depende da histamina, via receptores H1, H2 e H4 e da desgranulação de MC, que decorre de ação direta do veneno nestas células. A neutrofilia e o TXA2, LTB4, MCP-1 e KC devem contribuir para o influxo de leucócitos causado pelo VBm. / In this study the effects of Bothrops moojeni venom (BmV) on the cellular component of inflammatory responses and the mechanisms involved in this effect were investigated. The effects of venom on peritoneal and circulating leukocyte numbers and on the release of inflammatory mediators, such as LTB4, TXA2, PGD2, MIP-1 and KC, were assessed. The role of both mast cells and histamine receptors in leukocyte recruitment induced by BmV was assessed by selected pharmacological treatments. BmV caused a marked infiltration of leukocytes (3-24 h) when injected into the peritoneal cavity of mice. Neutrophils (PMN) were the predominant cell type in the early stages of response whereas macrophages (MN) were accumulated from 3 up to 24 h. Moreover, BmV increased blood neutrophil numbers at 3 h after injection. The BmV-induced leukocyte influx was abrogated by cromoglicate and significantly reduced either by difenidramine or ranitidine or tioperamide, histamine H1, H2 and H4 receptor antagonists, respectively, at 6 h after injection. Significant increments in peritoneal levels of LTB4, TXA2, PGD2, MIP-1 and KC were detected at distinct periods of time after venom injection. In addition, BmV induced mast cell degranulation both in vivo and in vitro. In conclusion, obtained data demonstrated the ability of BmV induce leukocyte recruitment into the site of its injection. This effect is dependent on mast cell activation and degranulation, which may be due to a direct effect of venom on these cells, and is mediated at least in part by histamine via H1, H2 and H4 receptors. Moreover, the ability of venom to mobilize leukocytes from bone marrow reserve compartments and to release the chemotactic mediators TXA2, LTB4, MCP-1 and KC may be relevant for leukocyte infiltration.

Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-κB) nuclear binding activity. In some experiments, NF-κB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-κB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-κB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, characterized by progressive and largely irreversible airflow limitation due to alveolar destruction (emphysema), small airway narrowing, and chronic bronchitis. It is one of the leading causes of morbidity and mortality worldwide and in the UK, it may affect approximately 1.5 per cent of the population; and up to one in eight emergency admissions may be due to COPD,corresponding to over one million bed days, with some 24160 people in the UK dying as a result of COPD in 2005 (Burden of Lung Disease 2nd Edition,British Thoracic Society 2006). Most cases of COPD are triggered by chronic inhalation of cigarette smoke.However, some people do not suffer from COPD even if they smoke for many years. COPD cannot be cured, and patients usually live with poor life quality. Treatments include giving up smoking, medication and oxygen therapy. Genetic factors contribute to the development of COPD. In Northern Europe,Z-AT homozygotes (342Glu Lys) develop emphysema in their third or forth decade. One explanation is AT deficiency because they form inactive polymers. However, this cannot explain why bronchoalveolar lavage fluid (BALF) from Z-AT homozygotes with emphysema contains more neutrophils than BALF from individuals with emphysema and normal AT (M-AT). Inhaling pollutants which include smoking (cigarettes, pipes, cigars, etc.) and other fumes such as those found in many industrial work environments probably also plays a role in an individual’s development of COPD. Previously, it has been shown that the polymeric conformer of AT is present in BALF from Z-AT homozygotes and that it is a chemoattractant for neutrophils in vitro (Parmar JS, 2002). These findings have been confirmed by others (Mulgrew AT, 2004). However, it is unknown where the polymers form and if 4 they are chemotactic in vivo. My colleague Dr Carl Atkison† showed that polymers of Z 1-AT are present in the alveolar wall of Z-AT homozygotes with emphysema, which accounts for 20% of the total AT from lung homogenates.These Z-AT individuals also have an excess of neutrophils in the alveolar wall compared with M-AT homozygotes. Furthermore, neutrophils and polymeric AT co-localize in the alveolar wall (Mahadeva R, 2005). To investigate whether there was a direct relationship between polymers of Z-AT and the excess neutrophils, polymers of AT were instilled into the lungs of wild-type mice (Mahadeva R, 2005). This produced a significant increase in neutrophil influx into the lungs compared with instillation of the native protein.Examination of the time course demonstrated that the influx of neutrophils was closely linked to the presence of polymeric AT. The mechanism of neutrophil recruitment in this mouse model was subsequently shown to be a direct chemotactic effect rather than stimulation of IL-8 homologues or other CXC chemokines. Oxidized AT (Ox-AT) promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF- B and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the anti-elastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway † My colleagues’ contributions are acknowledged in future text where appropriate by the following superscripts: (a) Dr Sam Alam, (b) Dr Jichun Wang, (c) Dr Carl Atkinson, (d) Dr Sabina Janciauskiene. 5 interacts directly with epithelial cells to release chemokines IL-8 and MCP-1,which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells oxidizes AT, perpetuating the cycle, potentially contributing to the pathogenesis of COPD. Furthermore, this demonstrates that molecules such as oxidants, anti-proteinases, and chemokines, rather than acting independently, collectively interact to cause emphysema (Li Z, 2009). To investigate the molecular basis for the interaction between Z-AT and Ox-AT associated with cigarette smoking, female mice transgenic for normal (MAT)or Z-AT on CBA background were exposed to cigarette smoke (CS). Transgenic mice for Z-AT developed a significant increase in pulmonary polymers following acute CS exposure. Increased levels of neutrophils in CSZ lungs were tightly correlated with polymer concentrations. Oxidation of human plasma Z-AT by CS or -chlorosuccinimide greatly accelerated polymerization, which could be abrogated by antioxidants. The results showed that cigarette smoke accelerated polymerization of Z-AT by oxidative modification, which in so doing further reduced pulmonary defense and increased neutrophil influx into the lungs. These novel findings provided a molecular explanation for the striking observation of premature emphysema in ZZ homozygote smokers, and raised the prospect of anti-oxidant therapy in ZAT related COPD (Alam S, 2011).