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Eosinophil granulocytes are found in all vertebrates and they are usually associated with parasitic and immunological diseases. Much of the previous work on eosinophils has concentrated on their distribution and variations in number in blood and tissues. This has led to many suggestions as to their possible functions. Few experiments have been done to study the way in which they are produced, and little quantitative information is available either about the kinetics of eosinophil production or the ways in which they can be mobilised. Even less is known about these processes under pathological states. Without this knowledge it has not been possible to distinguish factors altering eosinophil production from those influencing their turnover. For these reasons, the first part of this thesis describes experiments which were designed to measure the kinetics of eosinophil production, mobilisation and turnover in normal and stimulated rats. Rats were given single injections of 10,000 Trichinella spiralis larvae and the extent of labelling of eosinophils in bone marrow and blood was followed on autoradiographs in these stimulated rats, and in normal rats. In stimulated rats the bone marrow content of eosinophils increased and this resulted in a peripheral blood eosinophilia 2-10 days later. Three sequential steps in rat eosinophil development in marrow were defined: primitive dividing eosinophils, more mature dividing eosinophils and non-proliferating eosinophils. The diameter of eosinophils was greatest just before they divided, and there was no relationship between eosinophil size and their maturity. Other experiments supported the concept that eosinophils leave the marrow and mature in the spleen before release into the blood. The percentage of eosinophils in bone marrow showed no diurnal variation in normal rats. In rats given larvae intravenously the percentage of marrow eosinophils increased 23 h after stimulation. The peripheral blood eosinophil count rose 1-2 days later, and was related in parallel to the bone marrow level. The eosinophil mitotic index rose by a factor of 2.6, and there appeared to be a maximum number of eosinophil mitoses 40 h after stimulation. The proportions of primitive and more mature dividing eosinophils did not alter during the first 3 days, showing that all the eosinophils in the proliferative compartment were stimulated equally. The eosinophil cell cycle parameters in normal and stimulated rats were calculated from measurements of the proportion of eosinophil mitoses labelled at intervals after single injections of 3H-thymidine. In normal rats there was a wide variation of eosinophil cell cycle times with a median of 30 h. In stimulated rats this was reduced to a median of 9 h with a narrow distribution of cell cycle times. The post-mitotic rest period (T_G₁) and D.N.A. synthesis period (T_S) were shortened. It was calculated that the earliest recognisable eosinophils divided a total of 4-5 times in normal rats, and in stimulated rats eosinophils divided a total of 9-10 times. Despite the additional number of divisions in the stimulated rats, marrow transit times were unaffected as the cell cycle times were shorter in the stimulated rats. It was concluded that increased eosinophil production came about by additional divisions in the proliferative compartment, associated with shortening of cell cycle times. Eosinophil production rates were calculated from measurements of the number and labelling indices of eosinophils in weighed quantities of bone marrow combined with the known eosinophil cell cycle times. o Production rates increased by a factor of 28, from 0.5 andtimes; 10³ to 14.4 andtimes; 10³ new eosinophils/mg bone marrow/h. Eosinophil kinetics in the blood were studied by giving single injections of 3H-thymidine to normal or stimulated rats, and the number of labelled eosinophils in blood was measured subsequently. The interval between injections of 3H-thymidine and the appearance of labelled eosinophils in the blood (emergence time) was 40 h in normal rats and 17 h in stimulated rats, but neutrophil emergence time was not altered. This demonstrated that different factors regulated the emergence times of these two types of granulocyte. An eosinophil releasing factor was sought in plasma removed 12-24 h after the injection of larvae. Six hours after injection of 2 ml plasma the eosinophil count in the blood of normal recipient rats doubled. The eosinophil releasing effect of plasma was not removed by dialysis and was stable at -20°C. This eosinophil releasing effect was not associated with a neutrophil leucocytosis. Damaged eosinophils and homogenates of lungs containing larvae injected 16 h previously did not induce eosinophil release. Many stimuli which induce an eosinophilia 3-6 h after injection may act by producing 'eosinophil releasing factor' in blood as was demonstrated with larvae. Eosinophil half-life in the blood of normal rats was 6.6 h. Surprisingly, eosinophil half-life did not decrease 2 days after injection of the stimulus at a time when many eosinophils were moving from the blood into the tissues. It was concluded that there was an increase in the size of the intravascular eosinophil pool size. Blood eosinophil half-life was lengthened 5 days after injecting larvae at the height of the eosinophilia, and it was also increased in splenectomised rats 2 days after stimulation. In another section of the thesis, work is described which centered on the processes involved in the initiation of increased eosinophil production. Studies on rats injected with larvae proved to be very useful for studying the regulation of eosinophil production, as the tissues containing the stimulus were separate from the responding marrow. The initial response to the stimulus was studied in the tissues, lymph and blood. First, the inflammatory reactions induced by Trichinella spiralis larvae in lung, liver and muscle were examined histologically. Mast cell degranulation was followed by mononuclear cell and eosinophil accumulation. In other experiments, mast cell degranulation was not found to be essential for eosinophil production and intravenous injections of larvae coated with peritoneal macrophages produced a smaller eosinophilia than uncoated larvae. However, in lymph nodes stimulated by Trichinella spiralis larvae, large numbers of dividing large pyroninophilic lymphocytes were found which were labelled with 3H-thymidine. These lymphocytes were considered to be the progenitors of the thoracic duct lymphocytes which have previously been shown to induce eosinophils production. A study was mace of the distribution and fate of large pyroninophilic lymphocytes derived from the thoracic duct of rats with oral trichinosis. Large pyroninophilic lymphocytes, which were labelled with tritiated nucleic acid precursors, were distributed in a similar way in normal and stimulated rats. The majority of transferred labelled large pyroninophilic lymphocytes were found in the small intestine, spleen and lymph nodes, where they divided after 24 h. At 48 h they had the morphology of plasma cells. Only a few labelled large lymphocytes reached the bone marrow. When ⁵¹Chromium labelled lymphocytes were injected, less than 0.5% of the injected activity was found in each femur 22 h later. These results showed that if large pyroninophilic lymphocytes initiate eosinophil production by short range processes, then these lymphocytes may be a small subpopulation of the cells normally found in thoracic duct lymph. In related work, presented in the appendix, experiments were done to determine whether thoracic duct lymph, draining from rats with unilateral pyelonephritis, would stimulate neutrophil production when transferred to normal rats. No effect was found. This result, which agrees with other recently reported experiments, is evidence that neutrophil production is not initiated by lymphocytes. It was concluded that in rats given Trichinella spiralis larvae intravenously, alterations in the numbers of eosinophils in the blood take place in several ways; either through changes in the distribution of mature eosinophils, or by variations in eosinophil production. The measurements which were done on each of these processes has shown that they are regulated by separate mechanisms, each with a distinct time course involving both humoral and cellular mediators. In conditions associated with an eosinophilia these mechanisms act in unison to provide eosinophils in increased numbers in response to the stimulus. This work has shown that regulation of eosinophil production and blood eosinophil levels takes place by a number of inter-related processes, in ways which are as finely balanced as those known to control neutrophil or erythrocyte production and distribution.

The aim of this thesis was to develop the use of fluorescent probes for studying structural changes in biochemical systems. The investigation was carried out in two major ways:-</p> <ol type="a"> <li>chromophoric molecules with different structures and fluorescence properties were used in an attempt to discover the structural characteristics needed to make a fluorescent molecule behave as a probe.</li> <li>biochemical systems of differing complexity were studied using ANS (1-anilino-naphthalene-8-sulphonate) and other probes to see what sort of information this probe technique could give. The two main biochemical systems used were:- (i) the ligand induced conformational change of glutamate dehydrogenase (GDH) and, (ii) energy dependent structural changes in submitochondrial particles (SMP). (i.e. those changes induced in mitochondrial membrane fragments by oxidisable substrate which could be reversed by uncoupling agents).</li> </ol> <p>a) MNS (2-(N-methyl anilino)-naphthalene-6-sulphonate) and MNS-Cl were synthesised in an attempt to label GDH covalently with an ANS type molecule. MNS was shown to behave in the same way as ANS, but with a larger blue shift of fluorescence on binding, and a larger enhancement accompanying the conformational change (3 fold instead of 2 fold). MNS-Cl denatured the enzyme even under mild conditions of modification. A variety of dansyl (l-dimethylamino-naphthalene-5-sulphonyl) amino acids were used to probe the conformational change of GDH. The dansyl derivatives of glutamate and inhibitors of the enzyme had their fluorescence quenched by NADH + GTP. In contrast the dansyl derivatives of the monocarboxylic amino acids (whose oxidation is activated by the conformational change) had their fluorescence enhanced by NADH + GTP. Despite this specificity there were large numbers of dansyl amino acid binding sites on GDH (ca 35 per oligomer). Comparison of these responses with those of the MNS-amides showed that in order to detect the structural change in GDH, a probe needed to be negatively charged and to have the negative charge close to the aromatic part of the probe. In order to detect energy dependent changes in SMP a probe again needed to be negatively charged. However, in this case the negative charge did not have to be as close to the aromatic part of the probe as with the GDH system. Neutral, non-polar probes bind in the lipid phase of the membrane where they cannot sense energy dependent changes in SMP or ionic strength changes in erythrocyte stroma. The negative charge is important in directing the probe to the area of the membrane where it can sense structural changes. In order to test what type of negative charge was required, three 9-anilino acridine derivatives with different acidic groups in the aniline ring were synthesised. Only the sulphonate derivative responded to energy dependent changes in SMP, indicating a preference for this group rather than carboxylate or arsonate. b) (i) A limited amount of information was obtained concerning GDH. L-Glu enhanced the fluorescence of bound NADH, while D-Glu had no effect and αKg was a potent quencher. These sharp differences are difficult to explain since all three molecules bind at the same site on GDH. αKg also shifted the conformational equilibrium, making GTP much more effective at inducing the conformational change. The reason for this is as yet unclear. (ii) The responses of the polarity sensitive probes ANS and MNS, and of the excimer forming probe PS (pyrene-3-sulphonate) to the energy dependent changes in SMP were studied and compared. ANS and MNS bound rapidly to the membrane surface and slowly diffused to internal sites. The energy dependent response was a property of the internal sites only. On binding to SMP no excimer formation by PS was detectable. However, on energisation excimer fluorescence appeared, accompanied by a decrease in monomer fluorescence. This must be due to either an increase in PS concentration within the membrane or to decreased viscosity. Comparison of the rates of fluorescence decrease following uncoupling revealed a common fast phase (t_andfrac12; = 2 sec) with a slow phase characteristic of each probe (t_andfrac12; = 12 (ANS), 30 (PS), 60 (MNS) secs). The rates of the slow phases of ANS and PS correlated well with those measured for probe efflux by independent methods. This suggested that the fast phase was a rapid membrane change, followed by a efflux of probe from the membrane (slow phase). Binding parameters for ANS and MNS were measured by conventional fluorescence techniques. There was an increase in binding and in quantum yield of bound ANS and MNS on energisation of SMP. A novel filtration method was developed for measuring PS binding and showed a two fold increase in PS bound to SMP after energisation. However, this increase was not large enough to account for the observed excimer fluorescence. The technique was also used to verify the fluorescence results for ANS binding. By measuring fluorescence polarisation and life-times of ANS and MNS in resting and energised SMP, it was possible to rule out viscosity changes as a cause of the PS fluorescence changes. It was therefore proposed that all the probe effects could be explained if energisation involved expulsion of water from the membrane. This hypothesis was tested by examining the energy dependent responses of ANS and MNS in D₂O and H₂O. in free solution D₂O quenches ANS (and MNS) fluorescence less than H₂O giving an isotope effect of 2.8 (2.4). This effect was lowered for both probes on binding to SMP. On energisation, there was a further lowering of the isotope effect on MNS. This suggested that the internal sites at which ANS and MNS bind are partially excluded from water and that on energisation this exclusion increases. By comparing ANS with NPN (N-phenyl-1-naphthylamine) and other neutral probes, it was shown that the probe binding sites are close to the membrane protein, and are probably at interfaces between hydrophobic and aqueous regions within the membrane, i.e. in an area where the probe could easily sense water exclusion. A theory of the energised state of SMP was proposed, based on the suggestion that energisation involves water expulsion. The theory accounted for the observed probe responses, as well as for proton uptake, and provided a driving force for phosphorylation. One probe ABS (2-methoxy,6-chloro,9-acridinyl-p-amino benzene sulphonate) had an electron transport dependent response (as well as an energy dependent one), which appeared to follow the rate of oxidation of coenzyme Q.</p.

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