Angiogenesis, the growth of new blood vessels, is a complex process involving several cell types and molecular signals. Excessive vascular growth is a problem in tumors, and insufficient vascularization hampers the function of transplanted insulin-producing pancreatic islets. Understanding the mechanisms behind blood vessel growth generates increased means to control angiogenesis. In this thesis a model of pancreatic islet transplantation to muscle has been used to study the involvement of leukocytes in the development of new vasculature. Transplantation of isolated islets of Langerhans into mouse muscle promoted revascularization of the grafts to a level comparable to native islets in the pancreas. The complete and functional vascular restoration resulted in improved blood glucose control compared to the clinical standard implantation site, the liver. This proved muscle as a transplantation site to be a clinically relevant option for the treatment of type 1 diabetes. The rapid islet revascularization process was found to be dependent on a distinct subset of neutrophils characterized by high expression of the chemokine receptor CXCR4 and the enzyme matrix metalloproteinase 9 (MMP-9). These cells were recruited to recently transplanted and hypoxic grafts by islet-secreted vascular endothelial growth factor A (VEGF-A). Leukocyte migration and interactions in the engraftment area were monitored using a high-speed confocal microscope followed by software tracking. New software was developed to visualize migration statistics. This tool revealed areas around the islet graft where neutrophil gathering coincided with sites of angiogenesis. Macrophages in the engraftment area positioned themselves close to the newly formed vasculature and were shown to have a stabilizing effect on the vessels. When macrophages were removed, no pericytes were recruited to the forming vasculature. The perivascular macrophages also began to express a pericyte marker when in the graft, suggesting a close relationship between these cell types or macrophage plasticity. In conclusion, this thesis presents muscle as a proangiogenic transplantation site for pancreatic islets for the treatment of type 1 diabetes, where the revascularization of the grafts was dependent on the recruitment and actions of specialized immune cells.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Τα ουδετερόφιλα κύτταρα του ανθρώπινου περιφερικού αίματος, ανήκουν σε μία κατηγορία κυττάρων, τα οποία ονομάζονται επαγγελματίες φαγοκύτταρα και τα οποία είναι υπεύθυνα για την πρόσληψη και θανάτωση των παθογόνων μικροοργανισμών (Bessler et al.2002). Από παλαιότερες έρευνες της ερευνητικής μας ομάδας, έχει δειχθεί η συμμετοχή των σηματοδοτικών μορίων FAK, Elk-1 και MAP κινασών στη ρύθμιση της κυτταροφαγίας από τα εξειδικευμένα φαγοκύτταρα (πλασματοκύτταρα), που βρίσκονται στην αιμολέμφο της μύγας της Μεσογείου, Ceratitis capitata. (Lamprou et al. 2007, Mamali et al. 2008). Είναι επίσης γνωστό ότι κατά την κυτταροφαγία παράγονται διάφορες μορφές δραστικού οξυγόνου (ROS) και αζώτου (RNS), με κύριες το Η2Ο2 και ΝΟ αντίστοιχα. Οι παραγόμενες ROS και RNS συμμετέχουν ως μόρια μεταγωγής σήματος σε διάφορα ενδοκυτταρικά σηματοδοτικά μονοπάτια που ρυθμίζουν την κυτταροφαγία από τα λευκοκύτταρα. Όμως τα βιβλιογραφικά δεδομένα στο πεδίο αυτό είναι λιγοστά. Στην παρούσα εργασία, μελετήθηκε η συμμετοχή των FAK, Elk-1, MAP κινασών και Η2Ο2 στην κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου καθώς και η αλληλεπίδραση μεταξύ των FAK, Elk-1, MAP κινασών και του Η2Ο2 που παράγεται από τα κύτταρα λόγω της παρουσίας των βακτηρίων E.coli και S.aureus. Με κυτταρομετρία ροής και τη χρήση του εξειδικευμένου φθοροχρώματος διυδροροδαμίνη (DHR), πιστοποιήθηκε η παραγωγή Η2Ο2 από τα ουδετερόφιλα κύτταρα του αίματος, παρουσία E.coli και S.aureus. Η ενεργός συμμετοχή του παραγόμενου Η2Ο2 αποδείχτηκε όταν η μείωση της παραγωγής υπεροξεικών ανιόντων με τη χρήση του εξειδικευμένου αναστολέα Ν-εθυλεν-μαλεϊμίδιο (NEM), μείωσε την πρόσληψη βακτηρίων από τα κύτταρα. Η συμμετοχή των FAK και Elk-1 στην κυτταροφαγία ελέγχθηκε με καταστολή της γονιδιακής τους έκφρασης με τη χρήση μορίων siRNA, ενώ αντίστοιχα για τις MAP κινάσες χρησιμοποιήθηκαν αναστολείς της φωσφορυλίωσης τους, οι οποίοι καταστέλλουν την ενεργότητα τους. Με τη χρήση των ίδιων μεθόδων αναστολής για τα μόρια FAK, Elk-1 και MAPKs, μελετήθηκε και ο ρόλος τους στην παραγωγή Η2Ο2 κατά την κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου. Τέλος, ερευνήθηκε και ο ρόλος του Η2Ο2, σαν σηματοδοτικό μόριο κατά τη διαδικασία της κυτταροφαγίας, μελετώντας με ανάλυση κατά western, την επίδραση της αναστολής της παραγωγής του Η2Ο2, στην φωσφορυλίωση (ενεργοποίηση) των MAP κινασών. / Neutrophil cells from human peripheral blood belong in a group of cells, that are called professional phagocytes and are responsible for the uptake and killing of pathogen microorganisms (Bessler et al.2002). Research data from our lab, has shown that the signaling molecules FAK, Elk-1 and MAP kinases participate in the regulation of phagocytosis from the phagocytes of the Mediterranean fly, C.capitata (Lamprou et al. 2007, Mamali et al. 2008). It is also known, that during phagocytosis phagocytes produce different forms of reactive oxygen species (ROS) and nitrogen species (RNS), mainly Η2Ο2 and ΝΟ, respectively. Produced ROS and RNS, act as signaling molecules in different intracellular signaling pathways that regulate phagocytosis in leykocytes. . In the present work, we examined the participation of FAK, Elk-1, MAPKs and Η2Ο2 in the phagocytosis of bacteria E.coli and S.aureus and also the interaction between FAK, Elk- 1, MAPKs and Η2Ο2 that is produced from the cells, due to the presence of the bacteria E.coli and S.aureus. Using flow cytometry and the specialized fluorescent dye Dihydrorodamine-123 (DHR), we certified the production of Η2Ο2 from the human neutrophil cells, in the presence of E.coli and S.aureus. The participation of the produced Η2Ο2 was shown, when the reduction of the produced Η2Ο2 with the use of the inhibitor Ν-etheylen-maleimide (NEM), reduced the uptake of bacteria from the cells. The participation of FAK and Elk-1 in phagocytosis was examined by suppressing their expression with the use of siRNA molecules, while for the MAP kinases we used specific activation inhibitors. By using the same inhibitng methods for the molecules FAK, Elk-1 and MAPKs we studied their role in the production of Η2Ο2 during the phagocytosis of bacteria E.coli and S.aureus from human neutrophil cells. Finally, by reducing the production of superoxide anions, thereby reducing the production of H2O2, by using the specific inhibitor Ν-etheylen-maleimide (NEM), we examined the expression of the phosphorylated forms of MAPKs, by western analysis, in human neutrophils in the presence of the bacteria E.coli and S.aureus.

The polymorphonuclear neutrophil granulocytes (neutrophils) are gaining renewed interest regarding their involvement in chronic inflammatory disorders, including rheumatoid arthritis (RA). Besides phagocytic and destructive capabilities, neutrophils have regulatory roles, e.g. by influencing responses from dendritic cells and lymphocytes. Several animal models have revealed that neutrophils are crucial for the initiation and maintenance of chronic inflammatory diseases. Neutrophil function is highly dependent on their ability to produce superoxide, an oxygen radical which can be further metabolized to other free radicals. Whether or not neutrophils are capable of producing the oxygen radical nitric oxide (NO˙) has been a matter of debate. In this thesis it was shown that freshly isolated neutrophils from the joint cavity of patients with RA, but not from other arthritis patients, had ongoing intracellular production of superoxide, indicating the processing of ingested material. The finding that joint neutrophils, but seemingly not circulating cells, expressed the NO-inducing enzyme iNOS, led to a series of experiments aimed to elucidate where in the exudative process this enzyme could first be detected. We could finally, for the first time, present evidence that human neutrophils actually express iNOS constitutively. Our data also suggest that neutrophil iNOS may be membrane associated, thus differing from the cytosolic location in other cell types. Since NOS activity was not demonstrated in isolated cells, the notion that neutrophil iNOS is regulated primarily at the transcriptional level must be questioned. NO production from iNOS requires the presence of its substrate, L-arginine. To test the hypothesis that neutrophil arginase prevents neutrophil NO-production, we investigated whether arginase inhibition affects neutrophil NO-dependent oxidative function. Initial data revealed a difference in the effect of arginase inhibition comparing neutrophil stimulus with a soluble formylated tri-peptide (fMLF) and integrin-mediated stimulation with particle-bound collagen type-1. This led to the hypothesis that integrin-ligation on neutrophils induces extracellular liberation of arginase, which was confirmed both by measuring arginase and its enzyme activity. The findings in this thesis may be important not only regarding the role of neutrophils in chronic joint inflammation, but also as a link in the accelerated atherosclerosis observed in chronic inflammatory disorders, e.g. RA. / Vid reumatiska ledinflammationer ansamlas mycket stora mängder polymorfkärniga neutrofila granulocyter (neutrofiler) inne i den vätskefyllda ledhålan. Neutrofiler har kraftfull destruktiv potential och anses kunna bidra till uppkomst av skada i leden. Då flera djurmodeller av ledinflammation har visat sig omöjliga att initiera i frånvaro av neutrofiler, har intresset för denna celltyp åter ökat efter att de under lång tid har stått i skuggan av andra typer av vita blodkroppar. En viktig del i avdödning av mikroorganismer och cellsignalering är förmågan att bilda fria syreradikaler, t.ex. superoxid (˙O2-) och kväveoxid (NO˙). Denna avhandling belyser aspekter kring produktionen av dessa reaktiva syreprodukter och mekanismer av potentiell betydelse vid ledinflammation. I det första arbetet visas att neutrofiler isolerade ur ledvätska från patienter med ledgångsreumatism (RA) har ett unikt beteende avseende superoxidproduktion jämfört med motsvarande celler från patienter med andra reumatiska sjukdomar. RA-neutrofiler från ledvätska (men inte från blod) producerar superoxid intracellulärt redan i vila och stimulering via vidhäftningsmolekyler ger en snabb ytterligare ökning av denna aktivitet. Fyndet antyder att cellerna är engagerade med hantering av endocyterade partiklar och/eller immunkomplex/immunaggregat. I de båda nästkommande arbetena undersöktes förekomst av det NO˙-producerande enzymet iNOS i neutrofiler. En rad tidigare publikationer har rapporterat motsägelsefulla resultat i denna fråga. Efter en serie experiment kunde vi konstatera att humana neutrofiler uttrycker iNOS konstitutivt, men att både dess cellulära lokalisation och reglering skiljer sig från andra celler. Neutrofiler har nyligen även visats innehålla arginas, ett enzym som konkurrerar med iNOS om bindningen till L-arginin och som därmed kan hämma NO˙-produktion. I det fjärde arbetet undersökte vi därför om hämning av arginas påverkade neutrofilernas funktion och produktion av superoxid. Vi fann att effekterna av arginashämning var större hos celler som stimulerats genom vidhäftning av kollagenklädda partiklar jämfört med en löslig formylerad tri-peptid (fMLF). Vidare, kunde vi visa att vidhäftning av kollagenklädda partiklar medför större extracellulär frisättning av arginas. Med stöd av dessa fynd kunde vi i påföljande försök bekräfta hypotesen att extracellulär frisättning av arginas är större efter vidhäftning av kollagen-partiklar än med fMLF-stimulering. Fysiologiskt är fyndet logiskt då det skulle medföra ökade vidhäftningsmöjligheter för neutrofilen inne i blodbanan genom att begränsa blodkärlets egen NO˙ produktion. Fyndet är också förenligt med den ökade frekvensen hjärt- och kärlsjukdomar vid RA, då en intensiv kontinuerlig utvandring av neutrofiler skulle medföra ökad arginas frisättning, sänkta argininnivåer och endotelial dysfunktion.

The healing of tympanic membrane (TM) perforations is a complex wound healing process including inflammation, migration of keratinocytes and tissue remodelling. Most TM perforations in human heal spontaneously, however some perforations become chronic, and the reason to why is still largely unknown. In cutaneous wound healing plasminogen (plg) has been shown to play an important role. Plg is converted into the protease plasmin regulated by two plasminogen activators (PA), urokinase type PA (uPA) and tissue-type PA (tPA). The aim of the present thesis was to evaluate the role of plg in healing of TM perforations, both in vivo and in vitro. The main objectives were to determine the healing capacity of the TM, the involvement of keratinocytes, fibrin(ogen) and inflammatory cells in the healing process. The studies were performed in plg deficient and uPA deficient mice, with littermate wild type (wt) mice as controls It was shown that myringotomies of the TMs in plg deficient mice still remained open 143 days following a perforation. The wound area was characterized by an abundant recruitment and accumulation of inflammatory cells; mainly macrophages and neutrophils, an arrested keratinocyte migration and a fibrin deposition covering the surface of the TM. The TM perforations in the wt mice all healed within 11 days. Interestingly, the myringotomies of the plg deficient mice could be closed by reconstitution with systemic injections of plg, whereas injections of PBS had no affect on the healing. To characterize mechanisms involved in the development of persistent TM perforations in plg deficient mice after a myringotomy the early inflammatory response during the first 48 hours was studied. The recruitment and accumulation of inflammatory cells in the perforated TMs was found to be similar between the plg deficient and the wt mice. Myringotomized TMs in uPA deficient mice healed similar to perforations of wt controls. Neither did the keratinocyte migration nor the occurrence of inflammatory cells differ between these genotypes. In the in vitro experiments TMs from plg deficient and wt mice, were dissected out, perforated and cultured in absence or surplus of plg. A decrease in perforation size was seen in all groups regardless of genotype or amount of plg in the medium. In conclusion, the present studies show: • Plg is essential for the healing of TM perforations in mice. • The altered healing process after a myringotomy in plg deficient mice involves a disturbed keratinocyte migration, a massive deposition of fibrin and an abundant accumulation of inflammatory cells in the wound area. • Plasminogen deficiency does not alter the early inflammatory response, following a myringotomy. • Deficiency of uPA does not influence the healing of TM perforations. • During in vitro conditions healing of TM perforations is initiated irrespectively of genotype of the explant (plg deficient or wt) or supply of plg. The increased knowledge of the involvement of plg in the healing of TM perforations may open therapeutical possibilities in the treatment of chronic TM perforations in humans.

Neutrophils (polymorphonuclear leukocytes) are short lived granulocytes that playa primordial role in host innate defense against invading pathogens. Freshly isolated neutrophils spontaneously undergo apoptosis when cultured, which is associated with oxidative stress. We found that there is a dramatic shift in the metabolism of the 5-lipoxygenase product 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from its biologically inactive o-oxidation product in freshly isolated neutrophils to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in neutrophils cultured for 24 h. o-oxidation of the chemoattractant leukotriene B4 (LTB4) was also reduced in aging neutrophils incubated with arachidonic acid, resulting in higher levels of LTB4. The reduced o-oxidation activity appeared to be due to a decrease in active LTB4 20-hydroxylase. In contrast, the increased 5-oxo-ETE formation was not associated with an increase in the amount of active 5-hydroxyeicosanoid dehydrogenase, which is required for its formation, but rather with a dramatic increase in its cofactor NADP +. NAD+ levels also increased, but NADPH levels remained unchanged after 24 h. There was also evidence for increased oxidative stress (high GSSG/GSH) in aging neutrophils. The changes in 5-HETE metabolism and pyridine nucleotides in cultured neutrophils could be inhibited by neutrophil survival factors and antioxidants. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB4, resulting in delayed resolution of inflammation. Similarly, we found that the NADPH oxidase activator PMA caused a very rapid and dramatic increase in NADP + levels in both freshly isolated and cultured neutrophils, accompanied by a rapid increase in 5-oxo-ETE synthesis and reduced o-oxidation activity. Surprisingly, this was not accompanied by a corresponding decline in NADPH levels, which instead initially increased, but rather by a precipitous reduction in NAD+, which mirrored the increase in NADP+. These results suggest that the phosphorylation of NAD+ by NAD kinase may be very important for providing both NADP+ for 5-oxo-ETE synthesis and NADPH for the respiratory burst.

Neutrofilai ir makrofagai – vienos svarbiausių uždegiminiame procese dalyvaujančių ląstelių. Lėtinės obstrukcinės plaučių ligos (LOPL) metu aktyvintos šios ląstelės sintetina biologiškai aktyvias medžiagas, skatinančias intensyvesnį sisteminį ir kvėpavimo takuose vykstantį uždegimą. Iki šiol nėra aiškūs neutrofilų ir makrofagų sintetinamų žymenų (citokinų, proteinazių) ypatumai sergant LOPL ir atsižvelgiant į rūkymo įprotį. Nėra žinoma, kaip kraujo neutrofilų funkcines savybes tiesiogiai įtakoja pats sergančiųjų LOPL kvėpavimo takų sekretas. Šio tyrimo tikslas buvo įvertinti neutrofilų ir makrofagų funkcinių savybių ypatumus sergant lėtine obstrukcine plaučių liga ir galimus jų skirtumus priklausomai nuo rūkymo įpročio. Ištirti sergančių LOPL rūkančiųjų ir neberūkančiųjų matrikso metaloproteinazės-12 raiškos kvėpavimo takų sekreto makrofaguose ypatumai. Įvertintos sergančiųjų LOPL biologiniais ir cheminiais veiksniais aktyvintų kraujo neutrofilų funkcinės savybės (chemotaksis, fagocitozė, reaktyviųjų deguonies formų susidarymas), analizuotas potencialus moduliacinis kvėpavimo takų sekreto poveikis šioms funkcinėms savybėms. Nustatyta, jog sergančiųjų lėtine obstrukcine plaučių liga neutrofilai ir makrofagai buvo labiau aktyvūs nei sveikų asmenų ląstelės; rūkymo įprotis įtakojo kai kurias uždegimines neutrofilų ir makrofagų savybes kvėpavimo takų sekrete ir kraujyje. / Neutrophils and macrophages play an important role in inflammatory process. Activated during chronic obstructive pulmonary disease (COPD), these cells synthesize a variety of biologically active compounds, which may further amplify both systemic and local airway inflammation. The features of biologically active compounds (cytokines, proteinases) released by neutrophils and macrophages during COPD and according to smoking status are unknown. Still, a direct influence of airway mucous from COPD patients on functional neutrophil features is unknown. The aim of this study was to evaluate the functional features of neutrophils and macrophages during chronic obstructive pulmonary disease and possible differences according to smoking status. An expression of matrix metalloproteinase-12 in airway mucous of COPD smokers and ex-smokers was evaluated. The functional features (chemotaxis, phagocytosis, production of reactive oxygen species) of biologically and chemically activated blood neutrophils and a modulator effect of airway mucous were also analysed. The neutrophils and macrophages of patients with chronic obstructive pulmonary disease were more activated than the cells of healthy individuals; smoking status has influenced some inflammatory features of neutrophils and macrophages in airway mucous and blood.

At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of this form remains unknown as does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Inflammation is the normal biological response to tissue injury, and is characterised by the interactive activation of multiple mediators and cell types. One response to tissue injury is the production of pain, not only by direct trauma to sensory fibres, but also through the release of mediators from sensory nerve terminals. One such mediator is kinin which is a vasoactive peptide considered to play a primary role in inflammation by causing constriction of venules, dilation of arterioles, increasing permeability of the capillary membrane, and interacting with sensory nerve terminal transmitters to evoke pain. The kinin forming enzymes (kallikreins) reach inflammation sites either on the surface of migrating neutrophils or by transudation from plasma. The kininogen molecule which contains the kinin moiety, has been localised on the external surface of the neutrophil, and provides the substrate from which kinins can be cleaved through enzymatic action. The cellular actions of kinins are mediated through B2 receptors, which are also located on the external surface of the neutrophils. In addition, the induced effects of kinins are regulated by B1 receptors. The formation of nitric oxide (NO) from arginine released from the kinin C terminus, and receptor membrane signal transduction by nitric oxide following kinin receptor activation is discussed. A molecular response to cell injury is the formation of chemotactic mediators that attract neutrophils to sites of inflammation. The question whether neutrophils contribute to circulating levels of kinins was examined in infections and inflammatory disorders. This novel hypothesis was tested using circulating neutrophils harvested from patients with tuberculosis meningitis and pneumonia. These neutrophils showed a distinct loss of only the kinin moiety from the kininogen molecule located on the external surface. The confocal images of fixed, permeabilised neutrophils provided multi-dimensional constructs, and the intensity of fluorescence reflected the relative amounts of the molecule present in both neutrophils harvested from healthy volunteers as well as patient blood. The immunocytochemical labelling experiments using colloidal gold as markers, confirmed, at the ultrastructural level, the presence or disappearance of the kinin moiety from the kininogen molecule on the neutrophil surface. The cell component of synovial fluid in rheumatoid athritis (RA) consists mainly of neutrophils. This study demonstrates the absence of the kinin moiety from circulating and synovial fluid neutrophils from patients with RA, as well as an increased signal from immunolabelled B2 receptors in synovial fluid neutrophils. These findings support the hypothesis that in RA, kinins are released during the inflammatory response in the joints, and suggests that there is an upregulation of the B2 receptor at the site of inflammation. Neutrophils chemotactically drawn to the site of inflammation become activated to release kinin from the kininogen molecule, and thereafter re-enter the circulation where they were harvested systemically. B2 receptors may be upregulated following activation by kinins or by other mediators present in the inflammatory milieu. Interleukin-1 has been shown to upregulate kinin receptors on human synovial cells. Anti-peptide antibodies to the loops of cloned B1 and B2 receptors have provided powerful probes for the cellular identification of the two kinin receptor families. Mapping of the B2 receptors showed upregulation on the neutrophils gathered from inflamed joints. However, no activation of the Br receptors was observed in normal blood neutrophils as well as those obtained from the different disease states. / Thesis (M.Med.)-University of Natal, 1996.

The purpose of this study was to investigate which type(s) of somatic cells release nitric oxide (NO) in response to Escherichia coli lipopolysaccharide (LPS) and cytokines in vitro and how NO affects superoxide anion (O2-) production by bovine neutrophils and blood monocytes. Mammary epithelial cell line (FbE) released NO after stimulation with recombinant bovine interleukin-1beta (rBoIL-1beta). Moreover, monocytes produced NO in response to recombinant bovine interferon gamma (rBoIFN-gamma) alone or in combination with LPS in a dose- and time-dependent manner. Nitric oxide production was diminished by addition of inducible nitric oxide synthase (iNOS) inhibitors L-N 6-(1-Iminiethyl)lysine or aminoguanidine. However, NO release could not be induced in freshly isolated bovine neutrophils under the experimental conditions used, even after 96 h of incubation. Interestingly, when reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers for iNOS was performed to study mRNA expression, iNOS expression was observed in both monocytes and neutrophils in response to LPS and rBoIFN-gamma. / Unlike neutrophils, monocytes were poor producers of superoxide anion under the experimental conditions. A neutrophil-monocyte co-culture system was set up to study the effect of monocyte derived-NO and iNOS inhibitors on superoxide anion production by neutrophils. Neither NO derived from activated monocytes nor iNOS inhibitors seemed to have an effect on bovine neutrophil ability to release O2-. These results suggest that mammary epithelial cells and mononuclear phagocytes are among the cell types responsible for the important quantities of NO released by somatic cells recovered from LPS-infused mammary quarters during endotoxin-induced bovine mastitis. In addition, NO or iNOS inhibitors have no effect on the ability of activated bovine neutrophils to produce superoxide anions.